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Individuals with T1D must rely on exogenous insulin delivered subcutaneously (subq) either by periodic injection or by continuous pump-assisted infusion to control the hyperglycemia and ketonemia. Two types of therapeutic injection regimens are currently used, standard and intensive. [Note: Pump delivery is also considered intensive therapy.] 1. Standard versus intensive treatment: Standard treatment is typically two to three daily injections of recombinant human insulin. Mean blood glucose levels obtained are typically 225–275 mg/dl, with a glycated hemoglobin (HbA1c) level (see p. 33) of 8%–9% of the total hemoglobin (blue arrow in Fig. 25.4). [Note: The rate of formation of HbA1c is proportional to the average blood glucose concentration over the previous 3 months. Thus, HbA1c provides a measure of how well treatment has normalized blood glucose over that time in a patient with diabetes.] In contrast to standard therapy, intensive treatment seeks to more closely normalize blood glucose through more frequent monitoring and subsequent injections of insulin, typically ≥4 times a day. Mean blood glucose levels of 150 mg/dl can be achieved, with HbA1c ~7% of the total hemoglobin (see red arrow in Fig. 25.4). [Note: Normal mean blood glucose is ~100 mg/dl, and HbA1c is ≤6% (see black arrow in Fig. 25.4).] Therefore, normalization of glucose values (euglycemia) is not achieved even in intensively treated patients. Nonetheless, patients on intensive therapy show a ≥50% reduction in the long-term microvascular complications of diabetes (that is, retinopathy, nephropathy, and neuropathy) compared with patients receiving standard care. This confirms that the complications of diabetes are related to an elevation of plasma glucose.
therapy. [Note: Nondiabetic individuals are included for comparison.] 2. Hypoglycemia: One of the therapeutic goals in cases of diabetes is to decrease blood glucose levels in an effort to minimize the development of long-term complications of the disease (see p. 345 for a discussion of the chronic complications of diabetes). However, appropriate dosage of insulin is difficult to achieve. Hypoglycemia caused by excess insulin is the most common complication of insulin therapy, occurring in >90% of patients. The frequency of hypoglycemic episodes, seizures, and coma is particularly high with intensive treatment regimens designed to achieve tight control of blood glucose (Fig. 25.5). In normal individuals, hypoglycemia triggers a compensatory secretion of counterregulatory hormones, most notably glucagon and epinephrine, which promote hepatic production of glucose (see p. 315). However, patients with T1D also develop a deficiency of glucagon secretion. This defect occurs early in the disease and is almost universally present 4 years after diagnosis. Therefore, these patients rely on epinephrine secretion to prevent severe hypoglycemia. However, as the disease progresses, T1D patients show diabetic autonomic neuropathy and impaired ability to secrete epinephrine in response to hypoglycemia. The combined deficiency of glucagon and epinephrine secretion creates a symptom-free condition sometimes called “hypoglycemia unawareness.” Thus, patients with long-standing T1D are particularly vulnerable to hypoglycemia. Hypoglycemia can also be caused by strenuous exercise. Because exercise promotes glucose uptake into muscle and decreases the need for exogenous insulin, patients are advised to check blood glucose levels before or after intensive exercise to prevent or abort hypoglycemia.
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3. Contraindications for tight control: Children are not put on a program of tight control of blood glucose before age 8 years because of the risk that episodes of hypoglycemia may adversely affect brain development. Elderly people typically do not go on tight control because hypoglycemia can cause strokes and heart attacks in this population. Also, the major goal of tight control is to prevent complications many years later. Tight control, then, is most worthwhile for otherwise healthy people who can expect to live at least 10 more years. [Note: For most nonpregnant adults with diabetes, the individual treatment strategies and goals are based on the duration of diabetes, age/life expectancy, and known comorbid conditions.]
III. TYPE 2
T2D is the most common form of the disease, afflicting >90% of the U.S. population with diabetes. [Note: American Indians, Alaskan Natives, Hispanic and Latino Americans, African Americans, and Asian Americans have the highest prevalence.] Typically, T2D develops gradually without obvious symptoms. The disease is often detected by routine screening tests. However, many individuals with T2D have symptoms of polyuria and polydipsia of several weeks’ duration. Polyphagia may be present but is less common. Patients with T2D have a combination of insulin resistance and dysfunctional β cells (Fig.
25.6) but do not require insulin to sustain life. However, in >90% of these patients, insulin eventually will be required to control hyperglycemia and keep HbA1c <7%. The metabolic alterations observed in T2D are milder than those described for type 1, in part because insulin secretion in T2D, although inadequate, does restrain ketogenesis and blunts the development of DKA. [Note: Insulin suppresses the release of glucagon (see p. 314).] Diagnosis is based on the presence of hyperglycemia as described above. The pathogenesis does not involve viruses or autoimmune antibodies and is not completely understood. [Note: An acute complication of T2D in the elderly is a hyperosmolar hyperglycemic state characterized by severe hyperglycemia and dehydration and altered mental status.]
T2D is characterized by hyperglycemia, insulin resistance, impaired insulin secretion, and, ultimately, β-cell failure. The eventual need for insulin therapy has eliminated the designation of T2D as non–insulin-dependent diabetes.
A. Insulin resistance
Insulin resistance is the decreased ability of target tissues, such as the liver, white adipose, and skeletal muscle, to respond properly to normal (or elevated) circulating concentrations of insulin. For example, insulin resistance is characterized by increased hepatic glucose production, decreased glucose uptake by muscle and adipose tissue, and increased adipose lipolysis with production of free fatty acids (FFA).
1. Insulin resistance and obesity: Although obesity is the most common cause of insulin resistance and increases the risk of T2D, most people with obesity and insulin resistance do not develop diabetes. In the absence of a defect in β-cell function, obese individuals can compensate for insulin resistance with elevated levels of insulin. For example, Figure 25.7A shows that insulin secretion is two to three times higher in obese subjects than it is in lean individuals. This higher insulin concentration compensates for the diminished effect of the hormone (as a result of insulin resistance) and produces blood glucose levels similar to those observed in lean individuals (Fig. 25.7B).
2.
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Insulin resistance and type 2 diabetes: Insulin resistance alone will not lead to T2D. Rather, T2D develops in insulin-resistant individuals who also show impaired β-cell function. Insulin resistance and subsequent risk for the development of T2D is commonly observed in individuals who are obese, physically inactive, or elderly and in the 3%–5% of pregnant women who develop gestational diabetes. These patients are unable to sufficiently compensate for insulin resistance with increased insulin release. Figure 25.8 shows the time course for the development of hyperglycemia and the loss of β-cell function.
3.
Causes of insulin resistance: Insulin resistance increases with weight gain and decreases with weight loss, and excess adipose tissue (particularly in the abdomen) is key in the development of insulin resistance. Adipose is not simply an energy storage tissue, but also a secretory tissue. With obesity, there are changes in adipose secretions that result in insulin resistance (Fig. 25.9). These include secretion of proinflammatory cytokines such as interleukin 6 and tumor necrosis factor-α by activated macrophages (inflammation is associated with insulin resistance); increased synthesis of leptin, a protein with proinflammatory effects (see p. 353 for additional effects of leptin); and decreased secretion of adiponectin (see p. 350), a protein with anti-inflammatory effects. The net result is chronic, low-grade inflammation. One effect of insulin resistance is increased lipolysis and production of FFA (see Fig. 25.9). FFA availability decreases use of glucose, contributing to hyperglycemia, and increases ectopic deposition of TAG in liver (hepatic steatosis). [Note: Steatosis results in nonalcoholic fatty liver disease (NAFLD). If accompanied by inflammation, a more serious condition, nonalcoholic steatohepatitis (NASH), can develop.] FFA also have a proinflammatory effect. In the long term, FFA impair insulin signaling. [Note: Adiponectin increases FA β-oxidation (see p. 349). Consequently, a decrease in this adipocyte protein contributes to FFA availability.]
B. Dysfunctional β cells
In T2D, the pancreas initially retains β-cell capacity, resulting in insulin levels that vary from above normal to below normal. However, with time, the β cell becomes increasingly dysfunctional and fails to secrete enough insulin to correct the prevailing hyperglycemia. For example, insulin levels are high in typical, obese, T2D patients but not as high as in similarly obese individuals who do not have diabetes. Thus, the natural progression of the disease results in a declining ability to control hyperglycemia with endogenous secretion of insulin (Fig. 25.10). Deterioration of β-cell function may be accelerated by the toxic effects of sustained hyperglycemia and elevated FFA and a proinflammatory environment.
C. Metabolic changes
The abnormalities of glucose and TAG metabolism in T2D are the result of insulin resistance expressed primarily in liver, skeletal muscle, and white adipose tissue (Fig. 25.11).
1.
Hyperglycemia: Hyperglycemia is caused by increased hepatic production of glucose, combined with diminished use of glucose by muscle and adipose tissues. Ketonemia is usually minimal or absent in patients with T2D because the presence of insulin, even in the presence of insulin resistance, restrains hepatic ketogenesis.
2.
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Dyslipidemia: In the liver, FFA are converted to TAG, which are packaged and secreted in VLDL. Dietary TAG–rich chylomicrons are synthesized and secreted by the intestinal mucosal cells following a meal. Because lipoprotein TAG degradation catalyzed by lipoprotein lipase in adipose tissue is low in diabetes, the plasma chylomicron and VLDL levels are elevated, resulting in hypertriacylglycerolemia (see Fig. 25.10). Low levels of high-density lipoproteins are also associated with T2D, likely as a result of increased degradation.
D. Treatment
The goal in treating T2D is to maintain blood glucose concentrations within normal limits and to prevent the development of long-term complications. Weight reduction, exercise, and medical nutrition therapy (dietary modifications) often correct the hyperglycemia of newly diagnosed T2D. Oral hypoglycemic agents, such as metformin (decreases hepatic gluconeogenesis), sulfonylureas (increase insulin secretion; see p. 310), thiazolidinediones (decrease FFA levels and increase peripheral insulin sensitivity), α-glucosidase inhibitors (decrease absorption of dietary carbohydrate), and SGLT inhibitors (decrease renal reabsorption of glucose), or subq insulin therapy may be required to achieve satisfactory plasma glucose levels. [Note: Bariatric surgery in morbidly obese individuals with T2D has been shown to result in disease remission in most patients. Remission may not be permanent.]
IV. CHRONIC EFFECTS AND PREVENTION
As noted previously, available therapies moderate the hyperglycemia of diabetes but fail to completely normalize metabolism. The long-standing elevation of blood glucose is associated with the chronic vascular complications of diabetes including cardiovascular disease (CVD) and stroke (macrovascular complications) as well as retinopathy, nephropathy, and neuropathy (microvascular). Intensive insulin treatment (see p. 340) delays the onset and slows the progression of some long-term complications. For example, the incidence of retinopathy decreases as control of blood glucose improves and HbA1c levels decrease (Fig. 25.12). [Note: Data concerning the effect of tight control on CVD in T2D are less clear.] The benefits of tight control of blood glucose outweigh the increased risk of severe hypoglycemia in most patients. How hyperglycemia causes the chronic complications of diabetes is unclear. In cells in which glucose uptake is not dependent on insulin, elevated blood glucose leads to increased intracellular glucose and its metabolites. For example, increased intracellular sorbitol contributes to cataract formation (see p. 140) in diabetes. Additionally, hyperglycemia promotes glycation of cellular proteins in a reaction analogous to the formation of HbA1c. These glycated proteins undergo additional reactions and become advanced glycation end products (AGE) that mediate some of the early microvascular changes of diabetes and can reduce wound healing. Some AGE bind to a membrane receptor (RAGE), causing the release of proinflammatory molecules. There is currently no preventative treatment for T1D. The risk for T2D can be significantly decreased by a combined regimen of medical nutrition therapy, weight loss, exercise, and aggressive control of hypertension and dyslipidemias. For example, Figure 25.13 shows the incidence of disease in normal and overweight individuals with varying degrees of exercise.
V. CHAPTER SUMMARY
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Diabetes mellitus is a heterogeneous group of syndromes characterized by an elevation of fasting blood glucose that is caused by a relative or absolute deficiency of insulin (Fig. 25.14). Diabetes is the leading cause of adult blindness and amputation and a major cause of renal failure, nerve damage, heart attacks, and stroke. Diabetes can be classified into two groups, type 1 (T1D) and type 2 (T2D). T1D constitutes ~10% of >29 million cases of diabetes in the United States. The disease is characterized by an absolute deficiency of insulin caused by an autoimmune attack on the pancreatic β cells. This destruction requires an environmental stimulus (such as a viral infection) and a genetic determinant that causes the β cell to be mistakenly identified as “nonself.” The metabolic abnormalities of T1D include hyperglycemia, diabetic ketoacidosis (DKA), and hypertriacylglycerolemia that result from a deficiency of insulin. Those with T1D must rely on exogenous insulin delivered subcutaneously to control hyperglycemia and ketoacidosis. T2D has a strong genetic component. It results from a combination of insulin resistance and dysfunctional β cells. Insulin resistance is the decreased ability of target tissues, such as liver, white adipose, and skeletal muscle, to respond properly to normal (or elevated) circulating concentrations of insulin. Obesity is the most common cause of insulin resistance. However, most people with obesity and insulin resistance do not develop diabetes. In the absence of a defect in β-cell function, obese individuals without diabetes can compensate for insulin resistance with elevated levels of insulin. Insulin resistance alone will not lead to T2D. Rather, T2D develops in insulin-resistant individuals who also show impaired β-cell function. The acute metabolic alterations observed in T2D are milder than those described for the insulin-dependent form of the disease, in part because insulin secretion in T2D, although inadequate, does restrain ketogenesis and blunts the development of DKA. Available treatments for diabetes moderate the hyperglycemia but fail to completely normalize metabolism. The long-standing elevation of blood glucose is associated with the chronic complications of diabetes including cardiovascular disease and stroke (macrovascular) as well as retinopathy, nephropathy, and neuropathy (microvascular).
Choose the ONE best answer.
5.1. Three patients being evaluated for gestational diabetes are given an oral glucose tolerance test. Based on the data shown below, which patient is prediabetic?
A. Patient #1 B. Patient #2 C. Patient #3
D. None
Correct answer = B. Patient #2 has a normal fasting blood glucose (FBG) but an impaired glucose tolerance (GT) as reflected in her blood glucose level at 2 hours and, so, is described as prediabetic. Patient #1 has a normal FBG and GT, whereas patient #3 has diabetes.
5.2. Relative or absolute lack of insulin in humans would result in which one of the following reactions in the liver?
A. Decreased activity of hormone-sensitive lipase
B. Decreased gluconeogenesis from lactate
C. Decreased glycogenolysis
D. Increased formation of 3-hydroxybutyrate
E. Increased glycogenesis
Correct answer = D. Low insulin levels favor the liver producing ketone bodies, using acetyl coenzyme A generated by β-oxidation of the fatty acids provided by hormone-sensitive lipase (HSL) in adipose tissue (not liver). Low insulin also causes activation of HSL, decreased glycogen synthesis, and increased gluconeogenesis and glycogenolysis.
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5.3. Which one of the following is characteristic of untreated diabetes regardless of the type?
A. Hyperglycemia
B. Ketoacidosis
C. Low levels of hemoglobin A1c
D. Normal levels of C-peptide
E. Obesity
F. Simple inheritance pattern
Correct answer = A. Elevated blood glucose occurs in type 1 diabetes (T1D) as a result of a lack of insulin. In type 2 diabetes (T2D), hyperglycemia is due to a defect in β-cell function and insulin resistance. The hyperglycemia results in elevated hemoglobin A1c levels. Ketoacidosis is rare in T2D, whereas obesity is rare in T1D. C (connecting)-peptide is a measure of insulin synthesis. It would be virtually absent in T1D and initially increased then decreased in T2D. Both forms of the disease show complex genetics.
5.4. An obese individual with type 2 diabetes typically:
A. benefits from receiving insulin about 6 hours after a meal.
B. has a lower plasma level of glucagon than does a normal individual.
C. has a lower plasma level of insulin than does a normal individual early in the disease process.
D. shows improvement in glucose tolerance if body weight is reduced.
E. shows sudden onset of symptoms.
Correct answer = D. Many individuals with type 2 diabetes are obese, and almost all show some improvement in blood glucose with weight reduction. Symptoms usually develop gradually. These patients have elevated insulin levels and usually do not require insulin (certainly not 6 hours after a meal) until late in the disease. Glucagon levels are typically normal.
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I. OVERVIEW
Obesity is a disorder of body weight regulatory systems characterized by an accumulation of excess body fat. In primitive societies, in which daily life required a high level of physical activity and food was only available intermittently, a genetic tendency favoring storage of excess calories as fat may have had a survival value. Today, however, the sedentary lifestyle and abundance and wide variety of palatable, inexpensive foods in industrialized societies has undoubtedly contributed to an obesity epidemic. As adiposity has increased, so has the risk of developing associated diseases, such as type 2 diabetes (T2D), cardiovascular disease (CVD), hypertension, cancer, and arthritis. Particularly alarming is the explosion of obesity in children and adolescents, which has shown a threefold increase in prevalence over the last four decades. [Note: Approximately 17% of those age 2–19 years are obese.] In the United States, the lifetime risk of becoming overweight or obese is ~50% and 25%, respectively. Obesity has increased globally, and, by some estimates, there are more obese than undernourished individuals worldwide.
II. ASSESSMENT
Because the amount of body fat is difficult to measure directly, it is usually determined from an indirect measure, the body mass index (BMI), which has been shown to correlate with the amount of body fat in most individuals. [Note: Exceptions are athletes who have large amounts of lean muscle mass.] Measuring the waist size with a tape measure is also used to screen for obesity, because this measurement reflects the amount of fat in the central abdominal area of the body. The presence of excess central fat is associated with an increased risk for morbidity and mortality, independent of the BMI. [Note: A waist size ≥40 in (men) and ≥35 in (women) is considered a risk factor.]
A. Body mass index
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The BMI (defined as weight in kg/[height in m]2) provides a measure of relative weight, adjusted for height. This allows comparisons within and between populations. The healthy range for the BMI is between 18.5 and 24.9. Individuals with a BMI between 25 and 29.9 are considered overweight, those with a BMI ≥30 are defined as obese, and a BMI >40 is considered severely (morbidly) obese (Fig. 26.1). These cutoffs are based on studies examining the relationship of BMI to premature death and are similar in men and women. Nearly two thirds of U.S. adults are overweight, and more than one third of those are obese. Children with a BMI-for-age above the 95th percentile are considered obese.
B. Anatomic differences in fat deposition
The anatomic distribution of body fat has a major influence on associated health risks. A waist/hip ratio (WHR) >0.8 for women and >1.0 for men is defined as android, apple-shaped, or upper-body obesity and is associated with more fat deposition in the trunk (Fig. 26.2A). In contrast, a lower WHR reflects a preponderance of fat distributed in the hips and thighs and is called gynoid, pear-shaped, or lower-body obesity. It is defined as a WHR of <0.8 for women and <1.0 for men. The pear shape, more commonly found in women, presents a much lower risk of metabolic disease, and some studies indicate that it may actually be protective. Thus, the clinician can use simple indices of body shape to identify those who may be at higher risk for metabolic diseases associated with obesity.
About 80%–90% of human body fat is stored in subcutaneous (subq) depots in the abdominal (upper body) and the gluteal-femoral (lower body) regions. The remaining 10%–20% is in visceral depots located deep within the abdominal cavity (Fig. 26.2B). Excess fat in visceral and abdominal subq stores increases health risks associated with obesity.
C. Biochemical differences in regional fat depots
The regional types of fat described above are biochemically different. Subq adipocytes from the lower body, particularly in women, are larger, very efficient at fat (triacylglycerol [TAG]) deposition, and tend to mobilize fatty acids (FA) more slowly than subq adipocytes from the upper body. Visceral adipocytes are the most metabolically active. In obese individuals, both abdominal subcutaneous and visceral depots have high rates of lipolysis and contribute to increased availability of free fatty acids (FFA). These metabolic differences may contribute to the higher health risk found in individuals with upper body (abdominal) obesity. [Note: FFA impair insulin signaling and are proinflammatory (see p. 343).] 1.
Endocrine function: White adipose tissue, once thought to be a passive reservoir of TAG, is now known to play an active role in body weight regulatory systems. For example, the adipocyte is an endocrine cell that secretes a number of protein regulators (adipokines), such as the hormones leptin and adiponectin. Leptin regulates appetite as well as metabolism (see p. 352). Adiponectin reduces FFA levels in the blood (by increasing FA oxidation in muscles) and has been associated with improved lipid profiles, increased insulin sensitivity resulting in better glycemic control, and reduced inflammation in patients with diabetes. [Note: Adiponectin levels decrease as body weight increases, whereas leptin levels increase.] 2.
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Importance of portal circulation: With obesity, there is increased release of FFA and secretion of proinflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α), from adipose tissue. [Note: Cytokines are small proteins that regulate the immune system.] One hypothesis for why abdominal adipose depots have such a large influence on metabolic dysfunction in obesity is that the FFA and cytokines released from these depots enter the portal vein and, therefore, have direct access to the liver. In the liver, they may lead to insulin resistance (see p. 343) and increased hepatic synthesis of TAG, which are released as components of very-low-density lipoprotein particles and contribute to the hypertriacylglycerolemia associated with obesity. By contrast, FFA from lower body subq adipose depots enter the general circulation, where they can be oxidized in muscle and, therefore, reach the liver in lower concentration.
D. Adipocyte size and number
As TAG are stored, adipocytes can expand to an average of two to three times their normal volume (Fig. 26.3). However, the ability of fat cells to expand is limited. With prolonged overnutrition, preadipocytes within adipose tissue are stimulated to proliferate and differentiate into mature fat cells, increasing the number of adipocytes. Thus, most obesity is due to a combination of increased fat cell size (hypertrophy) and number (hyperplasia). Obese individuals can have up to five times the normal number of adipocytes. [Note: Like other tissues, the adipose tissue undergoes continuous remodeling. Contrary to early dogma, we now know that adipocytes can die. The estimated average lifespan of an adipocyte is 10 years.] If excess calories cannot be accommodated within adipose tissue, the excess FA “spill over” into other tissues, such as muscle and the liver. The amount of this ectopic fat is strongly associated with insulin resistance. With weight loss in an obese individual, the size of the fat cells is reduced, but the number is not usually affected. Thus, a normal amount of body fat is achieved by decreasing the size of the fat cell below normal. However, small fat cells are very efficient at reaccumulating fat, and this may drive appetite and weight regain.
III. BODY WEIGHT REGULATION
The body weight of most individuals tends to be relatively stable over time. This observation prompted the hypothesis that each individual has a biologically predetermined “set point” for body weight. The body attempts to add to adipose stores when the body weight falls below the set point and to lose adipose from stores when the body weight rises above the set point. Thus, the body defends the set point. For example, with weight loss, appetite increases and energy expenditure falls, whereas with overfeeding, appetite falls and energy expenditure may slightly increase (Fig. 26.4). However, a strict set point model explains neither why some individuals fail to revert to their starting weight after a period of overeating nor the current epidemic of obesity.
A. Genetic contributions
It is now evident that genetic mechanisms play a major role in determining body weight.
1.
Biologic origin: The importance of genetics as a determinant of obesity is indicated by the observation that children who are adopted usually show a body weight that correlates with their biologic rather than adoptive parents. Furthermore, identical twins have very similar BMI (Fig. 26.5), whether reared together or apart, and their BMI are more similar than those of nonidentical, dizygotic twins.
2.
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Mutations: Rare, single gene mutations can cause human obesity. For example, mutations in the gene for leptin (causing decreased production) or its receptor (decreased function) result in hyperphagia (increased appetite for and consumption of food) and severe obesity (Fig. 26.6), underscoring the importance of the leptin system in regulating human body weight (see IV below). [Note: Most obese humans have elevated leptin levels but are resistant to the appetite-regulating effects of this hormone.]
B. Environmental and behavioral contributions
The epidemic of obesity occurring over the last several decades cannot be simply explained by changes in genetic factors, which are stable on this short time scale. Clearly, environmental factors, such as the ready availability of palatable, energy-dense foods, play a role. Furthermore, sedentary lifestyles decrease physical activity and enhance the tendency to gain weight. Eating behaviors, such as portion size, variety of foods consumed, an individual’s food preferences, and the number of people present during eating, also influence food consumption. However, it is important to note that many individuals in this same environment do not become obese. The susceptibility to obesity appears to be explained, at least in part, by an interaction of an individual’s genes and his or her environment and can be influenced by additional factors such as maternal under-or overnutrition that may “set” the body regulatory systems to defend a higher or lower level of body fat. Thus, epigenetic changes (see p.
476) likely influence the risk for obesity.
IV. MOLECULAR INFLUENCES
The cause of obesity can be summarized in a deceptively simple application of the first law of thermodynamics: Obesity results when energy (caloric) intake exceeds energy expenditure. However, the mechanism underlying this imbalance involves a complex interaction of biochemical, neurologic, environmental, and psychologic factors. The basic neural and humoral pathways that regulate appetite, energy expenditure, and body weight involve systems that regulate short-term food intake (meal to meal), and signals for the long-term (day to day, week to week, year to year) regulation of body weight (Fig. 26.7).
and overnourished (B) states. CCK = cholecystokinin; PYY = peptide YY.
A.
Long-term signals reflect the status of fat (TAG) stores.
1.
Leptin: Leptin is an adipocyte peptide hormone that is made and secreted in proportion to the size of fat stores. It acts on the brain to regulate food intake and energy expenditure. When we consume more calories than we need, body fat increases, and leptin production by adipocytes increases. The body adapts by increasing energy use (increasing activity) and decreasing appetite (an anorexigenic effect). When body fat decreases, the opposite effects occur. Unfortunately, most obese individuals are leptin resistant, and the leptin system may be better at preventing weight loss than preventing weight gain. [Note: Leptin’s effects are mediated through binding to receptors in the arcuate nucleus of the hypothalamus.] 2.
Insulin: Obese individuals are also hyperinsulinemic. Like leptin, insulin acts on hypothalamic neurons to dampen appetite. (See Chapter 23 for the effects of insulin on metabolism.) [Note: Obesity is associated with insulin resistance (see p. 342).]
B. Short-term signals
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Short-term signals from the gastrointestinal (GI) tract control hunger and satiety, which affect the size and number of meals over a time course of minutes to hours. In the absence of food intake (between meals), the stomach produces ghrelin, an orexigenic (appetite-stimulating) hormone that drives hunger. As food is consumed, GI hormones, including cholecystokinin and peptide YY, among others, induce satiety (an anorexigenic effect), thereby terminating eating, through actions on gastric emptying and neural signals to the hypothalamus. Within the hypothalamus, neuropeptides (such as orexigenic neuropeptide Y [NPY] and anorexigenic α-melanocyte–stimulating hormone [α-MSH]) and neurotransmitters (such as anorexigenic serotonin and dopamine) are important in regulating hunger and satiety. Long-term and short-term signals interact, insofar as leptin increases secretion of α-MSH and decreases secretion of NPY. Thus, there are many complex regulatory loops that control the size and number of meals in relationship to the status of body fat stores. [Note: α-MSH, a cleavage product of proopiomelanocortin, binds to the melanocortin-4 receptor (MC4R). Loss-of-function mutations to MC4R are associated with early-onset obesity.]
V. METABOLIC EFFECTS
The primary metabolic effects of obesity include dyslipidemias, glucose intolerance, and insulin resistance expressed primarily in the liver, skeletal muscle, and adipose tissue. These metabolic abnormalities reflect molecular signals originating from the increased mass of adipocytes (see Fig. 25.9, p. 343, and Fig. 26.7). [Note: About 30% of obese individuals do not show these metabolic abnormalities.]
A. Metabolic syndrome
Abdominal obesity is associated with a cluster of metabolic abnormalities (hyperglycemia, insulin resistance, hyperinsulinemia, dyslipidemia [low levels of high-density lipoprotein (HDL) and elevated TAG], and hypertension) that is referred to as the metabolic syndrome (Fig. 26.8). It is a risk factor for CVD and T2D. The low-grade, chronic, systemic inflammation seen with obesity contributes to the pathogenesis of insulin resistance and T2D and likely plays a role in metabolic syndrome. In obesity, adipocytes release proinflammatory mediators such as IL-6 and TNF-α. Additionally, levels of adiponectin, which normally dampens inflammation and sensitizes tissues to insulin, are low.
B. Nonalcoholic liver disease
Obesity is associated with ectopic deposition of TAG in the liver (hepatic steatosis) and results in increased risk for nonalcoholic fatty liver disease ([NAFLD], see p. 343).
VI. OBESITY AND HEALTH
Obesity is correlated with an increased risk of death (Fig. 26.9) and is a risk factor for a number of chronic conditions, including T2D, dyslipidemias, hypertension, CVD, some cancers, gallstones, arthritis, gout, pelvic floor disorders (for example, urinary incontinence), NAFLD, and sleep apnea. The relationship between obesity and associated morbidities is stronger among individuals age <55 years. After age 74 years, there is no longer an association between increased BMI and mortality. [Note: Obesity also has social consequences (for example, stigmatization and discrimination).] Weight loss in obese individuals leads to decreased blood pressure, plasma TAG, and blood glucose levels. HDL increase.
VII. WEIGHT REDUCTION
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Weight reduction can help reduce the complications of obesity. To achieve weight reduction, the obese patient must decrease energy intake or increase energy expenditure, although decreasing energy intake is thought to contribute more to inducing weight loss. Typically, a plan for weight reduction combines dietary change; increased physical activity; and behavioral modification, which can include nutrition education and meal planning, recording food intake through food diaries, modifying factors that lead to overeating, and relearning cues to satiety. Medications or surgery may be recommended. Once weight loss is achieved, weight maintenance is a separate process that requires vigilance because the majority of patients regain weight after they stop their weight-loss efforts.
B. Caloric restriction
Dieting is the most commonly practiced approach to weight control. Because 1 lb of adipose tissue corresponds to ~3,500 kcal, the effect that caloric restriction will have on the amount of adipose tissue can be estimated. Weight loss on calorie-restricted diets is determined primarily by caloric intake and not nutrient composition. [Note: However, compositional aspects can affect glycemic control and the blood lipid profile.] Caloric restriction is ineffective over the long term for many individuals. Over 90% of people who attempt to lose weight regain the lost weight when dietary intervention is suspended. Nonetheless, although few individuals will reach their ideal weight with treatment, weight losses of 10% of body weight over a 6-month period often reduce blood pressure and lipid levels and enhance control of T2D.
A. Physical activity
An increase in physical activity can create an energy deficit. Although adding exercise to a hypocaloric regimen may not produce a greater weight loss initially, exercise is a key component of programs directed at maintaining weight loss. In addition, physical activity increases cardiopulmonary fitness and reduces the risk of CVD, independent of weight loss. Persons who combine caloric restriction and exercise with behavioral treatment may expect to lose ~5%–10% of initial body weight over a period of 4–6 months. Studies show that individuals who maintain their exercise program regain less weight after their initial weight loss.
C. Pharmacologic treatment
The U.S. Food and Drug Administration has approved several weight-loss medications for use in adults. They include orlistat (decreases absorption of dietary fat), lorcaserin and phentermine in combination with topiramate (promote satiety through serotonin signaling), liraglutide (decreases appetite by activating the glucagon-like peptide 1 receptor), and buproprion in combination with naltrexone (increase metabolism by increasing norepinephrine). Their effects on weight reduction tend to be modest. [Note: Pharmacologic activation of brown adipocytes (see p. 79) is being explored.]
D. Surgical treatment
Gastric bypass and restriction surgeries are effective in causing weight loss in severely obese individuals. Through mechanisms that remain poorly understood, these operations greatly improve glycemic control in morbidly obese diabetic individuals. [Note: Implantation of a device that electrically stimulates the vagus nerve to decrease food intake has been approved.]
VIII. CHAPTER SUMMARY
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Obesity, the accumulation of excess body fat, results when energy (caloric) intake exceeds energy expenditure (Fig. 26.10). Obesity is increasing in industrialized countries because of a reduction in daily energy expenditure and an increase in energy intake resulting from the increasing availability of palatable, inexpensive foods. The body mass index (BMI) is easy to determine and highly correlated to body fat. Nearly 69% of U.S. adults are overweight (BMI ≥25), and >33% of this group are obese (BMI ≥30). The anatomic distribution of body fat has a major influence on associated health risks. Excess fat located in the abdomen (upper body, apple shape), as reflected in waist size, is associated with greater risk for hypertension, insulin resistance, diabetes, dyslipidemia, and coronary heart disease as compared to fat located in the hips and thighs (lower body, pear shape). A person’s weight is determined by genetic and environmental factors. Appetite is influenced by afferent, or incoming, signals (that is, neural signals, circulating hormones such as leptin, and metabolites) that are integrated by the hypothalamus. These diverse signals prompt release of hypothalamic peptides (such as neuropeptide Y and α-melanocyte– stimulating hormone) and activate outgoing, efferent neural signals. Obesity is correlated with an increased risk of death and is also a risk factor for a number of chronic conditions. Weight reduction is achieved best with negative energy balance, that is, by decreasing caloric intake and increasing physical activity. Virtually all diets that limit particular groups of foods or macronutrients lead to short-term weight loss. Long-term maintenance of weight loss is difficult to achieve. Modest reduction in food intake occurs with pharmacologic treatment. Surgical procedures, such as gastric bypass, designed to limit food intake are an option for the severely obese patient who has not responded to other treatments.
Choose the ONE best answer. For Questions 26.1 and 26.2, use the following scenario.
A 40-year-old woman, 5 ft, 1 in (155 cm) tall and weighing 188 lb (85.5 kg), seeks your advice on how to lose weight. Her waist measured 41 in and her hips 39 in. The remainder of the physical examination and the blood laboratory data were all within the normal range. Her only child (who is age 14 years), her sister, and both of her parents are overweight. The patient recalls being overweight throughout her childhood and adolescence. Over the past 15 years, she had been on seven different diets for periods of 2 weeks to 3 months, losing from 5 to 25 lb each time. On discontinuation of the diets, she regained weight, returning to 185–190 lb.
6.1. Calculate and interpret the body mass index for the patient.
Body mass index (BMI) = weight in kg/(height in m)2 = 85.5/1.552 = 35.6. Because her BMI is >30, the patient is classified as obese.
6.2. Which one of the following statements best describes the patient?
A. She has approximately the same number of adipocytes as an individual of normal weight, but each adipocyte is larger.
B. She shows an apple pattern of fat distribution.
C. She would be expected to show higher-than-normal levels of adiponectin.
D. She would be expected to show lower-than-normal levels of circulating leptin.
E. She would be expected to show lower-than-normal levels of circulating triacylglycerols.
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Correct answer = B. Her waist/hip ratio (WHR) is 1.05 (41/39). Apple shape is defined as a WHR of >0.8 for women and >1.0 for men. Therefore, she has an apple pattern of fat distribution, more commonly seen in males. Compared with other women of the same body weight who have a gynoid (pear-shaped) fat pattern, her android fat pattern places her at greater risk for diabetes, hypertension, dyslipidemia, and coronary heart disease. Individuals with marked obesity and a history dating to early childhood have a fat depot made up of too many adipocytes, each fully loaded with triacylglycerol (TAG). Plasma leptin levels are proportional to fat mass, suggesting that resistance to leptin, rather than its deficiency, occurs in human obesity. Adiponectin levels decrease with increasing fat mass. The elevated circulating free fatty acids characteristic of obesity are carried to the liver and converted to TAG. The TAG are released as components of very-low-density lipoproteins, resulting in elevated plasma TAG levels, or are stored in the liver, resulting in hepatic steatosis.
Nutrition: Overview and Macronutrients
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I. OVERVIEW
Nutrients are the constituents of food necessary to sustain the normal functions of the body. All energy (calories) is provided by three classes of nutrients: fats, carbohydrates, and protein (Fig. 27.1). Because the intake of these energy-rich molecules is larger (g amounts) than that of the other dietary nutrients, they are called macronutrients. This chapter focuses on the kinds and amounts of macronutrients that are needed to maintain optimal health and prevent chronic disease. Those nutrients needed in lesser amounts (mg or µg), vitamins and minerals, are called micronutrients and are considered in Chapters 28 and 29.
II. DIETARY REFERENCE INTAKES
Committees of U.S. and Canadian experts organized by the Food and Nutrition Board of the Institute of Medicine of the National Academy of Sciences have compiled Dietary Reference Intakes (DRI), which are estimates of the amounts of nutrients required to prevent deficiencies and maintain optimal health and growth. The DRI expands on the Recommended Dietary Allowances (RDA), which have been published with periodic revisions since 1941. Unlike the RDA, the DRI establishes upper limits on the consumption of some nutrients and incorporates the role of nutrients in lifelong health, going beyond deficiency diseases. Both the DRI and the RDA refer to long-term average daily nutrient intakes, because it is not necessary to consume the full RDA every day.
A. Definition
The DRI consists of four dietary reference standards for the intake of nutrients designated for specific life stage (age) groups, physiologic states, and gender (Fig. 27.2).
1.
Estimated average requirement: The average daily nutrient intake level estimated to meet the requirement of one half of the healthy individuals in a particular life stage and gender group is the Estimated Average Requirement (EAR). It is useful in estimating the actual requirements in groups and individuals.
2.
Recommended dietary allowance: The RDA is the average daily nutrient intake level that is sufficient to meet the requirements of nearly all (97%– 98%) individuals in a particular life stage and gender group. The RDA is not the minimal requirement for healthy individuals, but it is intentionally set to provide a margin of safety for most individuals. The EAR serves as the foundation for setting the RDA. If the standard deviation (SD) of the EAR is available and the requirement for the nutrient is normally distributed, the RDA is set at 2 SD above the EAR (that is, RDA = EAR + 2 SDEAR).
3.
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Adequate intake: An Adequate Intake (AI) is set instead of an RDA if sufficient scientific evidence is not available to calculate an EAR or RDA. The AI is based on estimates of nutrient intake by a group (or groups) of apparently healthy people. For example, the AI for young infants, for whom human milk is the recommended sole source of food for the first 6 months, is based on the estimated daily mean nutrient intake supplied by human milk for healthy, full-term infants who are exclusively breast-fed.
4.
Tolerable upper intake level: The highest average daily nutrient intake level that is likely to pose no risk of adverse health effects to almost all individuals in the general population is the Tolerable Upper Intake Level (UL, or TUL). As intake increases above the UL, the potential risk of adverse effects may increase. The UL is useful because of the increased availability of fortified foods and the increased use of dietary supplements. For some nutrients, there may be insufficient data on which to develop a UL.
B. Using the dietary reference intakes
Most nutrients have a set of DRI (Fig. 27.3). Usually a nutrient has an EAR and a corresponding RDA. Most are set by age and gender and may be influenced by special factors, such as pregnancy and lactation in women (see p. 372). When the data are not sufficient to estimate an EAR (or an RDA), an AI is designated. Intakes below the EAR need to be improved because the probability of adequacy is ≤50% (Fig. 27.4). Intakes between the EAR and RDA likely need to be improved because the probability of adequacy is <98%, and intakes at or above the RDA can be considered adequate. Intakes above the AI can be considered adequate. Intakes between the UL and the RDA can be considered to have no risk for adverse effects. [Note: Because the DRI is designed to meet the nutritional needs of the healthy, it does not include any special needs of the sick.]
III. ENERGY REQUIREMENT IN HUMANS
The Estimated Energy Requirement (EER) is the average dietary energy intake predicted to maintain an energy balance (that is, the calories consumed are equal to the energy expended) in a healthy adult of a defined age, gender, and height whose weight and level of physical activity are consistent with good health. Differences in the genetics, body composition, metabolism, and behavior of individuals make it difficult to accurately predict a person’s caloric requirements. However, some simple approximations can provide useful estimates. For example, sedentary adults require ~30 kcal/kg/day to maintain body weight, moderately active adults require 35 kcal/kg/day, and very active adults require 40 kcal/kg/day.
A. Energy content of food
The energy content of food is calculated from the heat released by the total combustion of food in a calorimeter. It is expressed in kilocalories (kcal, or Cal). The standard conversion factors for determining the metabolic caloric value of fat, protein, and carbohydrate are shown in Figure 27.5. Note that the energy content of fat is more than twice that of carbohydrate or protein, whereas the energy content of ethanol is intermediate between those of fat and carbohydrate. [Note: The joule (J) is a unit of energy widely used in countries other than the United States. One cal = 4.2 J; 1 kcal (1 Cal, 1 food calorie) = 4.2 kJ. For uniformity, many scientists are promoting the use of joules rather than calories in the United States. However, kcal still predominates and is used throughout this text.]
Figure27.5Averageenergyavailablefromthemacronutrientsandalcohol.
B. Use of food energy in the body
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The energy generated by metabolism of the macronutrients is used for three energy-requiring processes that occur in the body: resting metabolic rate (RMR), physical activity, and the thermic effect of food. The number of kcal expended by these processes in a 24-hour period is the total energy expenditure (TEE).
1. Resting metabolic rate: RMR is the energy expended by an individual in a resting, postabsorptive state. It represents the energy required to carry out the normal body functions, such as respiration, blood flow, and ion transport. RMR can be determined by measuring oxygen (O2) consumed or carbon dioxide (CO2) produced (indirect calorimetry). [Note: The ratio of CO2 to O2 is the respiratory quotient (RQ). It reflects the substrate being oxidized for energy (Fig. 27.6).] RMR also can be estimated using equations that include sex and age (RMR reflects lean muscle mass, which is highest in men and the young) as well as height and weight. A commonly used rough estimate is 1 kcal/kg/hour for men and 0.9 kcal/kg/hour for women. [Note: A basal metabolic rate (BMR) can be determined if more stringent environmental conditions are used, but it is not routinely done. RMR is ~10% higher than the BMR.] In an adult, the 24-hour RMR, known as the resting energy expenditure (REE), is ~1,800 kcal for men (70 kg) and 1,300 kcal for women (50 kg). From 60%–75% of the TEE in sedentary individuals is attributable to the REE (Fig. 27.7). [Note: Hospitalized individuals are commonly hypercatabolic, and the RMR is multiplied by an injury factor that ranges from 1.0 (mild infection) to 2.0 (severe burns) in calculating their TEE.] 2.
Physical activity: Muscular activity provides the greatest variation in the TEE. The amount of energy consumed depends on the duration and intensity of the exercise. This energy cost is expressed as a multiple of the RMR (range is 1.1 to >8.0) that is referred to as the physical activity ratio (PAR) or the metabolic equivalent of the task (MET). In general, a lightly active person requires ~30%–50% more calories than the RMR (see Fig. 27.7), whereas a highly active individual may require ≥100% calories above the RMR.
3.
Thermic effect of food: The production of heat by the body increases as much as 30% above the resting level during the digestion and absorption of food. This is called the thermic effect of food, or diet-induced thermogenesis. The thermic response to food intake may amount to 5%– 10% of the TEE.
IV. ACCEPTABLE MACRONUTRIENT DISTRIBUTION RANGES
Acceptable Macronutrient Distribution Ranges (AMDR) are defined as a range of intakes for a particular macronutrient that is associated with reduced risk of chronic disease while providing adequate amounts of essential nutrients. The AMDR for adults is 45%–65% of their total calories from carbohydrates, 20%– 35% from fat, and 10%–35% from protein (Fig. 27.8). The biologic properties of dietary fat, carbohydrate, and protein are described below.
V. DIETARY FATS
The incidence of a number of chronic diseases is significantly influenced by the kinds and amounts of nutrients consumed (Fig. 27.9). Dietary fats most strongly influence the incidence of coronary heart disease (CHD), but evidence linking dietary fat and the risk for cancer or obesity is much weaker.
Earlier recommendations emphasized decreasing the total amount of dietary fat. Unfortunately, this resulted in increased consumption of refined grains and added sugars. Data now show that the type of fat is a more important risk factor than the total amount of fat.
A. Plasma lipids and coronary heart disease
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Plasma cholesterol may arise from the diet or from endogenous biosynthesis. In either case, cholesterol is transported between the tissues in combination with protein and phospholipids as lipoproteins.
1. Low-density and high-density lipoproteins: The level of plasma cholesterol is not precisely regulated but, rather, varies in response to diet. Elevated levels of total cholesterol (hypercholesterolemia) result in an increased risk for CHD (Fig. 27.10). A much stronger correlation exists between CHD and the level of cholesterol in low-density lipoproteins ([LDL-C] see p. 234). As LDL-C increases, CHD increases. In contrast, elevated levels of high-density lipoprotein cholesterol (HDL
C) have been associated with a decreased risk for heart disease (see p. 235). [Note: Elevated plasma triacylglycerol (TAG) is associated with CHD, but a causative relationship has yet to be demonstrated.] Abnormal levels of plasma lipids (dyslipidemias) act in combination with smoking, obesity, sedentary lifestyle, insulin resistance, and other risk factors to increase the risk of CHD.
2. Benefits of lowering plasma cholesterol: Dietary or drug treatment of hypercholesterolemia has been shown to be effective in decreasing LDLC, increasing HDL-C, and reducing the risk for cardiovascular events. The diet-induced changes in plasma cholesterol concentrations are modest, typically 10%–20%, whereas treatment with statin drugs decreases plasma cholesterol by 30%–60% (see p. 224). [Note: Dietary and drug treatment can also lower TAG.]
B. Dietary fats and plasma lipids
TAG are quantitatively the most important class of dietary fats. The influence of TAG on blood lipids is determined by the chemical nature of their constituent fatty acids. The absence or presence and number of double bonds (saturated versus mono-and polyunsaturated), the location of the double bonds (ω-6 versus ω-3), and the cis versus trans configuration of the unsaturated fatty acids are the most important structural features that influence blood lipids.
1. Saturated fats: TAG composed primarily of fatty acids whose hydrocarbon chains do not contain any double bonds are referred to as saturated fats. Consumption of saturated fats is positively associated with high levels of total plasma cholesterol and LDL-C and an increased risk of CHD. The main sources of saturated fatty acids are dairy and meat products and some vegetable oils, such as coconut and palm oils (a major source of fat in Latin America and Asia, although not in the United States). Many experts strongly advise limiting intake of saturated fats to <10% of total caloric intake and replacing them with unsaturated fats (and whole grains).
Saturated fatty acids with carbon chain lengths of 14 (myristic) and 16 (palmitic) are most potent in increasing the plasma cholesterol level. Stearic acid (18 carbons, found in many foods including chocolate) has little effect on blood cholesterol.
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2. Monounsaturated fats: TAG containing primarily fatty acids with one double bond are referred to as monounsaturated fats. Monounsaturated fatty acids (MUFA) are generally obtained from plant-based oils. When substituted for saturated fatty acids in the diet, MUFA lower both total plasma cholesterol and LDL-C and maintain or increase HDL-C. This ability of MUFA to favorably modify lipoprotein levels may explain, in part, the observation that Mediterranean cultures, with diets rich in olive oil (high in monounsaturated oleic acid), show a low incidence of CHD. [Note: Although there is no AMDR for MUFA, a common recommendation is 10%–20% of caloric intake.] a. The Mediterranean diet: The Mediterranean diet is an example of a diet rich in MUFA (from olive oil) and polyunsaturated fatty acids or PUFA (from fish oils, plant oils, and some nuts) but low in saturated fat. For example, Figure 27.11 shows the composition of the Mediterranean diet in comparison with both a Western diet similar to that consumed in the United States and a typical low-fat diet. The Mediterranean diet contains seasonally fresh food, with an abundance of plant material, low amounts of red meat, and olive oil as the principal source of fat. The Mediterranean diet is associated with decreased plasma total cholesterol and LDL-C, decreased TAG, and increased HDL-C when compared with a typical Western diet higher in saturated fats.
3. Polyunsaturated fats: TAG containing primarily fatty acids with more than one double bond are referred to as polyunsaturated fats. The effects of PUFA on cardiovascular disease are influenced by the location of the double bonds within the molecule.
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a. ω-6 Fatty acids: These are long-chain PUFA, with the first double bond beginning at the sixth bond position when starting from the methyl (ω) end of the fatty acid molecule. [Note: They are also called n-6 fatty acids (see p. 182).] Consumption of fats containing ω-6 PUFA, principally linoleic acid (18:2 [9,12]), obtained from vegetable oils, lowers plasma cholesterol when substituted for saturated fats. Plasma LDL-C is lowered, but HDL-C, which protects against CHD, is also lowered, partially offsetting the benefits of lowering LDL-C. Nuts, avocados, olives, soybeans, and various oils, including sunflower and corn oil, are common sources of these fatty acids. The AMDR for linoleic acid is 5%–10%. [Note: The lower recommendation for intake of PUFA relative to MUFA is because of concern that free radical– mediated oxidation (peroxidation) of PUFA may lead to deleterious products.] b. ω-3 Fatty acids: These are long-chain PUFA, with the first double bond beginning at the third bond position from the methyl (ω) end. Dietary ω-3 PUFA suppress cardiac arrhythmias, reduce plasma TAG, decrease the tendency for thrombosis, lower blood pressure, and substantially reduce risk of cardiovascular mortality (Fig. 27.12), but they have little effect on LDL-C or HDL-C levels. Evidence suggests that they have anti-inflammatory effects. The ω-3 PUFA, principally α-linolenic acid, 18:3(9,12,15), are found in plant oils, such as flaxseed and canola, and some nuts, such as walnuts. The AMDR for αlinolenic acid is 0.6%–1.2%. Fish oil contains the long-chain ω-3 docosahexaenoic acid (DHA, 22:6) and eicosapentaenoic acid (EPA, 20:5). Two fatty fish (for example, salmon) meals per week are recommended. For patients with documented CHD, 1 g/day of fish oils is recommended, while 2–4 g/day is prescribed to lower TAG. [Note: DHA is included in infant formulas to promote brain development.] Linoleic and α-linolenic acids are essential fatty acids (EFA) required for membrane fluidity and synthesis of eicosanoids (see p. 213). EFA deficiency, caused primarily by fat malabsorption, is characterized by scaly dermatitis as a result of the depletion of skin ceramides with long-chain fatty acids (see p. 206).
= eicosapentaenoic acid (20:5); DHA = docosahexaenoic acid (22:6).
4. Trans fatty acids: Trans fatty acids (Fig. 27.13) are chemically classified as unsaturated fatty acids but behave more like saturated fatty acids in the body because they elevate LDL-C and lower HDL-C, thereby increasing the risk of CHD. Trans fatty acids do not occur naturally in plants but occur in small amounts in animals. However, trans fatty acids are formed during the hydrogenation of vegetable oils (for example, in the manufacture of margarine and partially hydrogenated vegetable oil). Trans fatty acids are a major component of many commercial baked goods, such as cookies, and most deep-fried foods. Many manufacturers have reformulated their products to be free of trans fats. In 2006, the U.S. Food and Drug Administration required that Nutrition Facts labels (see p.
370) portray the trans fat content of packaged food. By 2018, virtually no industrial trans fatty acids will be permitted in food.
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5. Dietary cholesterol: Cholesterol is found only in animal products. The effect of dietary cholesterol on plasma cholesterol (Fig. 27.14) is less important than the amount and types of fatty acids consumed. Many experts recommend ≤300 mg/day. However, having an upper limit has become controversial.
concentrations to an increase in dietary cholesterol intake.
C. Other dietary factors affecting coronary heart disease
Moderate consumption of alcohol (up to 1 drink/day for women and up to 2 drinks/day for men) decreases the risk of CHD, because there is a positive correlation between moderate alcohol (ethanol) consumption and the plasma concentration of HDL-C. However, because of the potential dangers of alcohol abuse, health professionals are reluctant to recommend increased alcohol consumption to their patients. Red wine may provide cardioprotective benefits in addition to those resulting from its alcohol content (for example, red wine contains phenolic compounds that inhibit lipoprotein oxidation; see p. 235). [Note: These antioxidants are also present in raisins and grape juice.] Figure 27.15 summarizes the effects of dietary fats. [Note: Recent studies (including meta-analyses) have raised questions concerning the current guidelines for dietary fat in the prevention of CHD.]
VI. DIETARY CARBOHYDRATES
The primary role of dietary carbohydrates is to provide energy. Although self-reported caloric intake in the United States peaked in 2003 and is now declining, the incidence of obesity has dramatically increased (see p. 349). During this same period, carbohydrate consumption has significantly increased (as fat consumption decreased), leading some observers to link obesity with carbohydrate consumption. However, obesity has also been related to increasingly inactive lifestyles and to calorie-dense foods served in expanded portion size. Carbohydrates are not inherently fattening.
A. Classification
Dietary carbohydrates are classified as simple sugars (monosaccharides and disaccharides), complex sugars (polysaccharides), and fiber.
1. Monosaccharides: Glucose and fructose are the principal monosaccharides found in food. Glucose is abundant in fruits, sweet corn, corn syrup, and honey. Free fructose is found together with free glucose in honey and fruits (for example, apples).
a. High-fructose corn syrup: High-fructose corn syrups (HFCS) are corn syrups that have undergone enzymatic processing to convert their glucose into fructose and have then been mixed with pure corn syrup (100% glucose) to produce a desired sweetness. In the United States, HFCS 55 (containing 55% fructose and 42% glucose) is commonly used as a substitute for sucrose in beverages, including soft drinks, with HFCS 42 used in processed foods. The composition and metabolism of HFCS and sucrose are similar, the major difference being that HFCS is ingested as a mixture of monosaccharides (Fig. 27.16). Most studies have shown no significant difference between sucrose and HFCS meals in either postprandial glucose or insulin responses. [Note: The rise in the use of HFCS parallels the rise in obesity, but a causal relationship has not been demonstrated.] (B) leads to absorption of glucose plus fructose.
2.
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Disaccharides: The most abundant disaccharides are sucrose (glucose + fructose), lactose (glucose + galactose), and maltose (glucose + glucose). Sucrose is ordinary table sugar and is abundant in molasses and maple syrup. Lactose is the principal sugar found in milk. Maltose is a product of enzymic digestion of polysaccharides. It is also found in significant quantities in beer and malt liquors. The term “sugar” refers to monosaccharides and disaccharides. “Added sugars” are those sugars and syrups (such as HFCS) added to foods during processing or preparation.
3.
Polysaccharides: Complex carbohydrates are polysaccharides (most often polymers of glucose) that do not have a sweet taste. Starch is an example of a complex carbohydrate that is found in abundance in plants. Common sources include wheat and other grains, potatoes, dried peas and beans (legumes), and vegetables.
4.
Fiber: Dietary fiber is defined as the nondigestible, nonstarch carbohydrates and lignin (a noncarbohydrate polymer of aromatic alcohols) present intact in plants. Soluble fiber is the edible part of plants that is resistant to digestion and absorption in the human small intestine but is completely or partially fermented by bacteria to short-chain fatty acids in the large intestine. Insoluble fiber passes through the digestive track largely unchanged. Dietary fiber provides little energy but has several beneficial effects. First, it adds bulk to the diet (Fig. 27.17). Fiber can absorb 10–15 times its own weight in water, drawing fluid into the lumen of the intestine and increasing bowel motility and promoting bowel movements (laxation). Soluble fiber delays gastric emptying and can result in a sensation of fullness (satiety). This delayed emptying also results in reduced spikes in blood glucose following a meal. Second, consumption of soluble fiber has been shown to lower LDL-C levels by increasing fecal bile acid excretion and interfering with bile acid reabsorption (see p. 225). For example, diets rich (25–50 g/day) in the soluble fiber oat bran are associated with a modest, but significant, reduction in risk for CHD by lowering total cholesterol and LDL-C levels. Also, fiber-rich diets decrease the risk for constipation, hemorrhoids, and diverticulosis. The AI for dietary fiber is 25 g/day for women and 38 g/day for men. However, most American diets are far lower in fiber at ~15 g/day. [Note: “Functional fiber” is the term used for isolated fiber that has proven health benefits such as commercially available fiber supplements.]
B. Dietary carbohydrate and blood glucose
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Some carbohydrate-containing foods produce a rapid rise followed by a steep fall in blood glucose concentration, whereas others result in a gradual rise followed by a slow decline (Fig. 27.18). Thus, they differ in their glycemic response (GR). [Note: Fiber blunts the GR.] The glycemic index (GI) ranks carbohydrate-rich foods on a scale of 0–100 based on the GR they cause relative to the GR caused by the same amount (50 g) of carbohydrate eaten in the form of white bread or glucose. A low GI is <55, whereas a high GI is ≥70. Evidence suggests that a low-GI diet improves glycemic control in diabetic individuals. Food with a low GI tends to create a sense of satiety over a longer period of time and may be helpful in limiting caloric intake. [Note: How much a typical serving size of a food raises blood glucose is referred to as the glycemic load (GL). A food (for example, carrots) can have a high GI and a low GL.]
C. Carbohydrate requirements
Carbohydrates are not essential nutrients, because the carbon skeletons of most amino acids can be converted into glucose (see p. 261). However, the absence of dietary carbohydrate leads to ketogenesis (see p. 195) and degradation of body protein whose constituent amino acids provide carbon skeletons for gluconeogenesis (see p. 118). The RDA for carbohydrate is set at 130 g/day for adults and children, based on the amount of glucose used by carbohydrate-dependent tissues, such as the brain and erythrocytes. However, this level of intake is usually exceeded. Adults should consume 45%–65% of their total calories from carbohydrates. It is now recommended that added sugars represent no more than 10% of total energy intake because of concerns that they may displace nutrient-rich foods from the diet. [Note: Added sugars are associated with increased body weight and type 2 diabetes.]
D. Simple sugars and disease
There is no direct evidence that the consumption of simple sugars naturally present in food is harmful. Contrary to folklore, diets high in sucrose do not lead to diabetes or hypoglycemia. Also contrary to popular belief, carbohydrates are not inherently fattening. They yield 4 kcal/g (the same as protein and less than one half that of fat; see Fig. 27.5) and result in fat synthesis only when consumed in excess of the body’s energy needs. However, there is an association between sucrose consumption and dental caries, particularly in the absence of fluoride treatment (see p. 405).
VII. DIETARY PROTEIN
The AMDR for protein is 10%–35%. Dietary protein provides the essential amino acids (see Fig. 20.2, p. 262). Nine of the 20 amino acids needed for the synthesis of body proteins are essential (that is, they cannot be synthesized in humans).
A. Protein quality
The quality of a dietary protein is a measure of its ability to provide the essential amino acids (EAA) required for tissue maintenance. Most government agencies have adopted the Protein Digestibility–Corrected Amino Acid Score (PDCAAS) as the standard by which to evaluate protein quality. PDCAAS is based on the profile of EAA after correcting for the digestibility of the protein. The highest possible score under these guidelines is 1.00. This amino acid score provides a method to balance intakes of poorer-quality proteins with high-quality dietary proteins.
1.
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Proteins from animal sources: Proteins from animal sources (meat, poultry, milk, and fish) have a high quality because they contain all the EAA in proportions similar to those required for synthesis of human tissue proteins (Fig. 27.19), and they are more readily digested. [Note: Gelatin prepared from animal collagen is an exception. It has a low biologic value as a result of deficiencies in several EAA.] 2.
Proteins from plant sources: Plant proteins have a lower quality than do animal proteins. However, proteins from different plant sources may be combined in such a way that the result is equivalent in nutritional value to animal protein. For example, wheat (lysine deficient but methionine rich) may be combined with kidney beans (methionine poor but lysine rich) to produce a higher biologic value than either of the component proteins (Fig. 27.20). [Note: Animal proteins can also complement the biologic value of plant proteins.]
B. Nitrogen balance
Nitrogen balance occurs when the amount of nitrogen consumed equals that of the nitrogen excreted in the urine (primarily as urinary urea nitrogen, or UUN), sweat, and feces. Most healthy adults are normally in nitrogen balance. [Note: There is, on average, 1 g nitrogen in 6.25 g protein.] 1.
Positive nitrogen balance: This occurs when nitrogen intake exceeds nitrogen excretion. It is observed during situations in which tissue growth occurs, for example, in childhood, pregnancy, or during recovery from an emaciating illness.
2.
Negative nitrogen balance: This occurs when nitrogen loss is greater than nitrogen intake. It is associated with inadequate dietary protein; lack of an essential amino acid; or during physiologic stresses, such as trauma, burns, illness, or surgery.
Nitrogen (N) balance (g Nin – g Nout) in a 24-hour period can be determined by the formula, N balance = protein intake in g/6.25 – (UUN + 4 g), where 4 g accounts for urinary loss in forms other than UUN plus loss in skin and feces.
C. Protein requirements
The amount of dietary protein required in the diet varies with its biologic value. The greater the proportion of animal protein in the diet, the less protein is required. The RDA for protein is computed for proteins of mixed biologic value at 0.8 g/kg of body weight for adults, or ~56 g of protein for a 70-kg individual. People who exercise strenuously on a regular basis may benefit from extra protein to maintain muscle mass, and a daily intake of ~1 g/kg has been recommended for athletes. Women who are pregnant or lactating require up to 30 g/day in addition to their basal requirements. To support growth, infants should consume 2 g/kg/day. [Note: Disease states influence protein needs. Protein restriction may be needed in kidney disease, whereas burns require increased protein intake.] 1.
Consumption of excess protein: There is no physiologic advantage to the consumption of more protein than the RDA. Protein consumed in excess of the body’s needs is deaminated, and the resulting carbon skeletons are metabolized to provide energy or acetyl coenzyme A for fatty acid synthesis. When excess protein is eliminated from the body as urinary nitrogen, it is often accompanied by increased urinary calcium, thereby increasing the risk of nephrolithiasis (kidney stones) and osteoporosis.
2.
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The protein-sparing effect of carbohydrates: The dietary protein requirement is influenced by the carbohydrate content of the diet. When the intake of carbohydrates is low, amino acids are deaminated to provide carbon skeletons for the synthesis of glucose that is needed as a fuel by the central nervous system. If carbohydrate intake is <130 g/day, substantial amounts of protein are metabolized to provide precursors for gluconeogenesis. Therefore, carbohydrate is considered to be “protein-sparing,” because it allows amino acids to be used for repair and maintenance of tissue protein rather than for gluconeogenesis.
D. Protein-energy (calorie) malnutrition
In developed countries, protein-energy malnutrition (PEM), also known as protein-energy undernutrition (PEU), is most commonly seen in patients with medical conditions that decrease appetite or alter how nutrients are digested or absorbed or in hospitalized patients with major trauma or infections. [Note: Such highly catabolic patients frequently require intravenous (IV, or parenteral) or tube-based (enteral) administration of nutrients.] PEM may also be seen in children or the elderly who are malnourished. In developing countries, an inadequate intake of protein and/or calories is the primary cause of PEM. Affected individuals show a variety of symptoms, including a depressed immune system with a reduced ability to resist infection. Death from secondary infection is common. PEM is a spectrum of degrees of malnutrition, and two extreme forms are kwashiorkor and marasmus (Fig. 27.21). [Note: Marasmic kwashiorkor has features of both forms.] 1.
Kwashiorkor: Kwashiorkor occurs when protein deprivation is relatively greater than the reduction in total calories. Protein deprivation is associated with severely decreased synthesis of visceral protein. Kwashiorkor is commonly seen in developing countries in children after weaning at about age 1 year, when their diet consists predominantly of carbohydrates. Typical symptoms include stunted growth, skin lesions, depigmented hair, anorexia, fatty liver, bilateral pitting edema, and decreased serum albumin concentration. Edema results from the lack of adequate blood proteins, primarily albumin, to maintain the distribution of water between blood and tissues. It may mask muscle and fat loss. Therefore, chronic malnutrition is reflected in the level of serum albumin. [Note: Because caloric intake from carbohydrates may be adequate, insulin levels suppress lipolysis and proteolysis. Kwashiorkor is, therefore, nonadapted malnutrition.] 2.
Marasmus: Marasmus occurs when calorie deprivation is relatively greater than the reduction in protein. It usually occurs in developing countries in children younger than age 1 year when breast milk is supplemented or replaced with watery gruels of native cereals that are usually deficient in both protein and calories. Typical symptoms include arrested growth, extreme muscle wasting and depletion of subcutaneous fat (emaciation), weakness, and anemia (Fig. 27.22). Individuals with marasmus do not show the edema observed in kwashiorkor. [Note: Refeeding severely malnourished individuals can result in hypophosphatemia (see p. 400), because any available phosphate is used to phosphorylate carbohydrate intermediates. Milk is frequently given because it is rich in phosphate.]
Cachexia, a wasting disorder characterized by loss of appetite and muscle atrophy (with or without increased lipolysis) that cannot be reversed by conventional nutritional support, is seen with a number of chronic diseases, such as cancer and chronic pulmonary and renal disease. It is associated with decreased treatment tolerance and response and decreased survival time.
VIII. NUTRITION TOOLS
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A set of tools has been developed that gives consumers information about what (and how much) they should eat as well as the nutritional content of the foods they do eat. Additional tools allow medical professionals to assess whether or not the nutritional needs of an individual are being met.
A. MyPlate
MyPlate was designed by the U.S. Department of Agriculture (USDA) to graphically illustrate its recommendations as to what food groups and how much of each should be consumed daily. In MyPlate, the relative amounts of each of five food groups (vegetables, grains, protein, fruit, and dairy) are represented by the relative size of their section on the plate (Fig. 27.23). The number of servings depends on variables that include age and sex. [Note: MyPlate replaced MyPyramid in 2011.]
B. Nutrition facts label
Most types of packaged goods are required to have a Nutrition Facts label, or “food label” (Fig. 27.24), that includes the size of a single serving, the Cal it provides, and the number of servings per container. In addition, a percent daily value (%DV) is shown for most nutrients listed. [Note: The %DV is based on a 2,000-Cal diet for healthy adults.] 1.
Percent daily value: The %DV compares the amount of a given nutrient in a single serving of a product to the recommended daily intake for that nutrient. For example, the %DV for the micronutrients listed, as well as for total carbohydrates and fiber, are based on their recommended minimum daily intake. Thus, if the label lists 20% for calcium, one serving provides 20% of the minimum recommended amount of calcium needed each day. In contrast, the %DV for saturated fat, cholesterol, and sodium are based on their recommended maximum daily intake, and the %DV reflects what percentage of this maximum a serving provides. There is no %DV for protein because the recommended intake depends on body weight (see p. 367). [Note: “Sugars” represents mono-and disaccharides. The remainder of the carbohydrate (total carbohydrate – [fiber + sugars]) is the oligo-and polysaccharides.] 2.
Proposed revisions: In 2014, the USDA proposed the following changes to the Nutrition Facts label for implementation by 2018: Added sugars, vitamin D, and potassium are to be included; vitamins A and C, total fat, and Cal from fat are to be removed; and serving size is to be adjusted to reflect the amounts people are now consuming. Additionally, design changes to highlight key parts of the label were proposed (Fig. 27.25). [Note: The proposed addition/removal of certain micronutrients is based on newer data on the risk for underingestion.]
C. Nutrition assessment
Nutrition assessment evaluates nutritional status based on clinical information. It includes (but is not limited to) dietary history, anthropometric measures, and laboratory data. [Note: Assessment findings may result in medical nutrition therapy, which is the treatment of medical conditions through changes in diet (for example, replacement of long-chain TAG with medium-chain TAG in malabsorption disorders) and/or the method of intake (for example, enteral [tube] or parenteral [IV] feeding).] 1.
Dietary history: This is a record of food intake over a period of time. For a food diary, the specific types and exact amounts of food eaten are recorded in “real time” (as soon as possible after eating) for a period of 3–7 days. Retrospective approaches include a food frequency questionnaire (for example, what fruits were eaten and how often they were eaten in a typical day, week, or month) and a 24-hour recall of the specific foods and the amounts eaten in the last 24 hours.
2.
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Anthropometric measures: These are physical measures of the body. They include (but are not limited to) weight, height, body mass index (an indicator of obesity, see p. 349), skin-fold thickness (an indicator of subcutaneous fat), and waist circumference (an indicator of abdominal fat, see p. 349). [Note: Ideal body weight can be calculated using the Hamwi method: 106 lb (for males) or 100 lb (for females) for the first 5 ft of height + 5 lb for every inch over 5 ft, with an adjustment of −10% for a small frame and + 10% for a large one.] 3.
Laboratory data: These are obtained by tests performed on body fluids, tissues, and waste. They can include plasma LDL-C (for cardiovascular risk), fecal fat (for malabsorption), red cell indices (for vitamin deficiencies), and N balance and serum proteins (such as albumin and transthyretin [prealbumin]) for protein–energy status. [Note: These proteins are made in the liver and transport molecules such as fatty acids and thyroxine (see p. 406) through blood. Low albumin levels correlate with increased morbidity and mortality in hospitalized patients. The short half-life (2–3 days) of transthyretin as compared to that of albumin (20 days) has led to its use in monitoring the progress of hospitalized patients.]
Nutritional insufficiency can be the result of inadequate nutrient intake (caused, for example, by an inability to eat, loss of appetite, or decreased availability), inadequate absorption, decreased utilization, increased excretion, or increased requirements.
IX. NUTRITION AND THE LIFE STAGES
Macronutrient energy sources, micronutrients, EFA, and EAA are required at every life stage. Additionally, each stage has specific nutrition needs.
A. Infancy, childhood, and adolescence
The rapid growth and development in infancy (birth to age 1 year) and childhood (age 1 year to adolescence) necessitate higher energy and protein needs relative to body size than are required in subsequent life stages. In adolescence, the marked increases in height and weight that occur increase nutritional needs. Growth charts (Fig. 27.26) are used to compare an individual’s stature (height) and/or weight to the expected values for others of the same age (≤20 years) and sex. They are based on data from large numbers of normal individuals over time. [Note: Deviations from the expected growth curve, as reflected in the crossing of two or more percentile lines, raise concern.] 1. Infants: Ideal infant nutrition is based on human breast milk because it provides calories and most micronutrients in amounts appropriate for the human infant. Carbohydrates, protein, and fat are present in a 7:3:1 ratio. [Note: In addition to the disaccharide lactose, human milk contains nearly 200 unique oligosaccharides. About 90% of the microbiota (the population of microbes) in the breast-fed infant’s intestine is represented by one type, Bifidobacterium infantis, which expresses all the enzymes needed to degrade these complex sugars. The sugars, in turn, act as prebiotics that support the growth of B. infantis, a probiotic (helpful bacteria).] Breast milk is low in vitamin D, however, and exclusively breast-fed babies require vitamin D supplementation. [Note: Human milk provides antibodies and other proteins that reduce the risk of infection.]
The microbiota in and on the human body plus their genomes are referred to as the microbiome. It is acquired at birth from the environment and changes with the life stages. The gut microbiome influences host nutrition by facilitating processing of food consumed and is itself influenced by that food. Its relationship with undernutrition, obesity, and diabetes is under investigation.
2.
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Children: As with infants, children have increased need for calories and nutrients. The primary concerns in this stage, however, are deficiencies of iron and calcium.
3.
Adolescents: In the teen years, the increases in height and weight increase the need for calories, protein, calcium, iron, and phosphorus. Eating patterns in this stage can result in overconsumption of fat, sodium, and sugar and underconsumption of vitamin A, thiamine, and folic acid. [Note: Eating disorders and obesity are concerns in this age group.]
B. Adulthood
Overnutrition is a concern in young adults, whereas malnutrition is a concern in older adults.
1.
Young adults: Nutrition in young adults focuses on the maintenance of good health and the prevention of disease. The goal is a diet rich in plant-based foods (with a focus on fiber and whole grains), limited intake of saturated fat and trans fatty acids, and balanced intake of ω-3 and ω-6 PUFA.
2.
Pregnant or lactating women: The requirements for calories, protein, and virtually all micronutrients increase in pregnancy and lactation. Supplementation with folic acid (to prevent neural tube defects [see p. 379]), vitamin D, calcium, iron, iodine, and DHA is typically recommended.
3.
Older adults: Aging increases the risk of malnutrition. Decreased appetite resulting from a reduced sense of taste (dysgeusia) and smell (hyposmia) decreases nutrient intake. [Note: Physical limitations, including problems with dentition, and psychosocial factors, such as isolation, may also play a role in reduced intake.] Inadequate intake of protein, calcium, and vitamins D and B12 is common. B12 deficiency can result from decreased absorption caused by achlorhydria (reduced stomach acid, see p. 381). In aging, lean muscle mass decreases and fat increases, resulting in decreased RMR. [Note: Drug–nutrient interactions can occur at any life stage but are more common as the number of medications increases as in aging.]
Monoamine oxidase inhibitors (MAOI), used to treat depression (see p.
287) and early Parkinson disease, can interact with tyramine-containing foods. Tyramine is a monoamine derived from the decarboxylation of tyrosine during the curing, aging, or fermentation of food (Fig. 27.27). It causes the release of norepinephrine, increasing blood pressure and heart rate. Patients who take an MAOI and consume such foods are at risk for a hypertensive crisis.
X. CHAPTER SUMMARY
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The Dietary Reference Intakes (DRI) provide estimates of the amounts of nutrients required to prevent deficiencies and maintain optimal health and growth. They consist of the Estimated Average Requirement (EAR), the average daily nutrient intake level estimated to meet the requirement of 50% of the healthy individuals in a particular life stage (age) and gender group; the Recommended Dietary Allowance (RDA), the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97%–98%) individuals in a life stage and gender group; the Adequate Intake (AI), which is set instead of an RDA if sufficient scientific evidence is not available to calculate the RDA; and the Tolerable Upper Intake Level (UL), the highest average daily nutrient intake level that is likely to pose no risk of adverse health effects to almost all individuals in the general population. The energy generated by the metabolism of the macronutrients (9 kcal/g of fat and 4 kcal/g of protein or carbohydrate) is used for three energy-requiring processes that occur in the body: resting metabolic rate, physical activity, and the thermic effect of food. Acceptable Macronutrient Distribution Ranges (AMDR) are defined as the ranges of intake for a particular macronutrient that are associated with reduced risk of chronic disease while providing adequate amounts of essential nutrients. Adults should consume 45%–65% of their total calories from carbohydrates, 20%–35% from fat, and 10%–35% from protein (Fig. 27.28). Elevated levels of cholesterol in low-density lipoproteins (LDL-C) result in increased risk for coronary heart disease (CHD). In contrast, elevated levels of cholesterol in high-density lipoproteins (HDL-C) have been associated with a decreased risk for CHD. Dietary or drug treatment of hypercholesterolemia is effective in decreasing LDL-C, increasing HDLC, and reducing the risk for CHD. Consumption of saturated fats is strongly associated with high levels of total plasma and LDL-C. When substituted for saturated fatty acids in the diet, monounsaturated fats lower both total plasma cholesterol and LDL-C but maintain or increase HDL-C. Consumption of fats containing ω-6 polyunsaturated fatty acids lowers plasma LDL-C, but HDL-C, which protects against CHD, is also lowered. Dietary ω-3 polyunsaturated fats suppress cardiac arrhythmias and reduce plasma triacylglycerols, decrease the tendency for thrombosis, and substantially reduce the risk of cardiovascular mortality. Carbohydrates provide energy and fiber to the diet. When they are consumed as part of a diet in which caloric intake is equal to energy expenditure, they do not promote obesity. Dietary protein provides essential amino acids. Protein quality is a measure of its ability to provide the essential amino acids required for tissue maintenance. Proteins from animal sources, in general, have a higher-quality protein than that derived from plants. However, proteins from different plant sources may be combined in such a way that the result is equivalent in nutritional value to animal protein. Positive nitrogen (N) balance occurs when N intake exceeds N excretion. It is observed in situations in which tissue growth occurs, for example, in childhood, pregnancy, or during recovery from an emaciating illness. Negative N balance occurs when N losses are greater than N intake. It is associated with inadequate dietary protein; lack of an essential amino acid; or during physiologic stresses such as trauma, burns, illness, or surgery. Kwashiorkor occurs when protein deprivation is relatively greater than the reduction in total calories. It is characterized by edema. Marasmus occurs when calorie deprivation is relatively greater than the reduction in protein. No edema is seen. Both are extreme forms of protein-energy malnutrition (PEM). Nutrition Facts labels give consumers information about the nutritional content of packaged foods. Medical assessment of nutritional status includes dietary history, anthropometric measures, and laboratory data. Each life stage has specific nutrition needs. Growth charts are used to monitor the growth pattern of an individual from birth through adolescence. Drug–nutrient interactions are of concern, especially in older adults.
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Choose the ONE best answer.
7.1. For the child shown at right, which of the statements would support a diagnosis of kwashiorkor? The child:
A. appears plump due to increased deposition of fat in adipose tissue.
B. displays abdominal and peripheral edema.
C. has a serum albumin level above normal.
D. has markedly decreased weight for height.
The correct answer = B. Kwashiorkor is caused by inadequate protein intake in the presence of fair to good energy (calorie) intake. Typical findings in a patient with kwashiorkor include abdominal and peripheral edema (note the swollen belly and legs) caused largely by a decreased serum albumin concentration. Body fat stores are depleted, but weight for height can be normal because of edema. Treatment includes a diet adequate in calories and protein.
7.2. Which one of the following statements concerning dietary fat is correct?
A. Coconut oil is rich in monounsaturated fats, and olive oil is rich in saturated fats.
B. Fatty acids containing trans double bonds, unlike the naturally occurring cis isomers, raise high-density lipoprotein cholesterol levels.
C. The polyunsaturated fatty acids linoleic and linolenic acids are required components.
D. Triacylglycerols obtained from plants generally contain less unsaturated fatty acids than do those from animals.
Correct answer = C. Humans are unable to make linoleic and linolenic fatty acids. Consequently, these fatty acids are essential in the diet. Coconut oil is rich in saturated fats, and olive oil is rich in monounsaturated fats. Trans fatty acids raise plasma levels of low-density lipoprotein cholesterol, not high-density lipoprotein cholesterol. Triacylglycerols obtained from plants generally contain more unsaturated fatty acids than do those from animals.
7.3. Given the information that a 70-kg man is consuming a daily average of 275 g of carbohydrate, 75 g of protein, and 65 g of fat, which one of the following conclusions can reasonably be drawn?
A. About 20% of calories are derived from fats.
B. The diet contains a sufficient amount of fiber.
C. The individual is in nitrogen balance.
D. The proportions of carbohydrate, protein, and fat in the diet conform to current recommendations.
E. The total energy intake per day is about 3,000 kcal.
Correct answer = D. The total energy intake is (275 g carbohydrate × 4 kcal/g) + (75 g protein × 4 kcal/g) + (65 g fat × 9 kcal/g) = 1,100 + 300 + 585 = 1,985 total kcal/day. The percentage of calories derived from carbohydrate is 1,100/1,985 = 55, from protein is 300/1,985 = 15, and from fat is 585/1,985 = 30. These are very close to current recommendations. The amount of fiber or nitrogen balance cannot be deduced from the data presented. If the protein is of low biologic value, a negative nitrogen balance is possible.
7.6. In chronic bronchitis, excessive mucus production causes airway obstruction that results in hypoxemia (low blood oxygen level), impaired expiration, and hypercapnia (carbon dioxide retention). Why might a high-fat, low-carbohydrate diet be recommended for a patient with chronic obstructive pulmonary disease caused by chronic bronchitis?
A. Fat contains more oxygen atoms relative to carbon or hydrogen atoms than do carbohydrates.
B. Fat is calorically less dense than carbohydrates.
C. Fat metabolism generates less carbon dioxide.
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D. The respiratory quotient (RQ) for fat is higher than the RQ for carbohydrates.
Correct answer = C. A treatment goal for the chronic obstructive pulmonary disease (COPD) caused by acute bronchitis is to insure appropriate nutrition without increasing the respiratory quotient (RQ), which is the ratio of carbon dioxide (CO2) produced to oxygen consumed, thereby minimizing the production of CO2. Less CO2 is produced from the metabolism of fat (RQ = 0.7) than from the catabolism of carbohydrate (RQ =1.0). Fat contains fewer oxygen atoms. Fat is calorically denser than is carbohydrate. [Note: RQ is determined by indirect calorimetry.] 7.7. A 32-year-old man who was rescued from a house fire was admitted to the hospital with burns over 45% of his body (severe burns). The man weighs 154 lb (70 kg) and is 72 in (183 cm) tall. Which one of the following is the best rapid estimate of the immediate daily caloric needs of this patient?
A. 1,345 kcal B. 1,680 kcal C. 2,690 kcal D. 3,360 kcal
Correct answer = D. A commonly used rough estimate of the total energy expenditure (TEE) for men is 1 kcal/1 kg body weight/24 hours. [Note: It is 0.8 kcal for women.] For this patient, that value is 1,680 kcal (1 kcal/kg/hour × 24 hours × 70 kg). In addition, an injury factor of 2 for severe burns must be included in the calculation: 1,680 kcal × 2 = 3,360 kcal.
7.8. Which one of the following is the best advice to give a patient who asks about the notation “%DV” (percent daily value) on the Nutrition Facts label?
A. Achieve 100% daily value for each nutrient each day.
B. Select foods that have the highest percent daily value for all nutrients.
C. Select foods with a low percent daily value for the micronutrients.
D. Select foods with a low percent daily value for saturated fat.
Correct answer = D. The percent daily value (%DV) compares the amount of a given nutrient in a single serving of a product to the recommended daily intake for that nutrient. The %DV for the micronutrients listed on the label, as well as for total carbohydrates and fiber, are based on their recommended minimum daily intake, whereas the %DV for saturated fat, cholesterol, and sodium are based on their recommended maximum daily intake.
For Questions 27.7 and 27.8, use the following case.
A sedentary 50-year-old man weighing 176 lb (80 kg) requests a physical. He denies any health problems. Routine blood analysis is unremarkable except for plasma total cholesterol of 295 mg/dl. (Reference value is <200 mg.) The man refuses drug therapy for his hypercholesterolemia. Analysis of a 1-day dietary recall showed the following: 7.4. Decreasing which one of the following dietary components would have the greatest effect in lowering the patient’s plasma cholesterol?
A. Carbohydrates
B. Cholesterol
C. Fiber
D. Monounsaturated fat
E. Polyunsaturated fat
F. Saturated fat
Correct answer = F. The intake of saturated fat most strongly influences plasma cholesterol in this diet. The patient is consuming a high-calorie, high-fat diet with 42% of the fat as saturated fat. The most important dietary recommendations are to lower total caloric intake, substitute monounsaturated and polyunsaturated fats for saturated fats, and increase dietary fiber. A decrease in dietary cholesterol would be helpful but is not a primary objective.
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7.5. What information would be necessary to estimate the patient’s total energy expenditure?
The daily basal energy expenditure (estimated resting metabolic rate/hour × 24 hours) and a physical activity ratio (PAR) based on the type and duration of physical activities are needed variables. An additional 10% would be added to account for the thermic effect of food. Note that if the patient were hospitalized, an injury factor (IF) would be included in the calculation, and the PAR would be modified. Tables of PAR and IF are available.
Micronutrients: Vitamins 28
For additional ancillary materials related to this chapter, please visit thePoint.
I. OVERVIEW
Vitamins are chemically unrelated organic compounds that cannot be synthesized in adequate quantities by humans and, therefore, must be supplied by the diet. Nine vitamins (folic acid, cobalamin, ascorbic acid, pyridoxine, thiamine, niacin, riboflavin, biotin, and pantothenic acid) are classified as water soluble. Because they are readily excreted in the urine, toxicity is rare. However, deficiencies can occur quickly. Four vitamins (A, D, K, and E) are termed fat soluble (Fig. 28.1). They are released, absorbed, and transported (in chylomicrons, see p. 227) with dietary fat. They are not readily excreted, and significant quantities are stored in the liver and adipose tissue. In fact, consumption of vitamins A and D in excess of the Dietary Reference Intakes (see Chapter 27) can lead to accumulation of toxic quantities of these compounds. Vitamins are required to perform specific cellular functions. For example, many of the water-soluble vitamins are precursors of coenzymes for the enzymes of intermediary metabolism. In contrast to the water-soluble vitamins, only one fat-soluble vitamin (vitamin K) has a coenzyme function.
II. FOLIC ACID (VITAMIN B9)
Folic acid (or, folate), which plays a key role in one-carbon metabolism, is essential for the biosynthesis of several compounds. Folic acid deficiency is probably the most common vitamin deficiency in the United States, particularly among pregnant women and individuals with alcoholism. [Note: Leafy, dark-green vegetables are a good source of folic acid.]
A. Function
Tetrahydrofolate (THF), the reduced, coenzyme form of folate, receives one-carbon fragments from donors such as serine, glycine, and histidine and transfers them to intermediates in the synthesis of amino acids, purine nucleotides, and thymidine monophosphate (TMP), a pyrimidine nucleotide incorporated into DNA (Fig. 28.2).
B. Nutritional anemias
Anemia is a condition in which the blood has a lower than normal concentration of hemoglobin, which results in a reduced ability to transport oxygen (O2). Nutritional anemias (that is, those caused by inadequate intake of one or more essential nutrients) can be classified according to the size of the red blood cells (RBC), or mean corpuscular volume (MCV), observed in the blood (Fig. 28.3). Microcytic anemia (MCV below normal), caused by lack of iron, is the most common form of nutritional anemia. The second major category of nutritional anemia, macrocytic (MCV above normal), results from a deficiency in folic acid or vitamin B12. [Note: These macrocytic anemias are commonly called megaloblastic because a deficiency of either vitamin (or both) causes accumulation of large, immature RBC precursors, known as megaloblasts, in the bone marrow and the blood (Fig. 28.4). Hypersegmented neutrophils are also seen.] 1.
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Folate and anemia: Inadequate serum levels of folate can be caused by increased demand (for example, pregnancy and lactation; see p. 372), poor absorption caused by pathology of the small intestine, alcoholism, or treatment with drugs (for example, methotrexate) that are dihydrofolate reductase inhibitors (see Fig. 28.2). A folate-free diet can cause a deficiency within a few weeks. A primary result of folic acid deficiency is megaloblastic anemia (see Fig. 28.4), caused by diminished synthesis of purine nucleotides and TMP, which leads to an inability of cells (including RBC precursors) to make DNA and, therefore, an inability to divide.
2.
Folate and neural tube defects: Spina bifida and anencephaly, the most common neural tube defects (NTD), affect ~3,000 pregnancies in the United States annually. Folic acid supplementation before conception and during the first trimester has been shown to significantly reduce NTD. Therefore, all women of childbearing age are advised to consume 0.4 mg/day (400 µg/day) of folic acid to reduce the risk of having a pregnancy affected by NTD and ten times that amount if a previous pregnancy was affected. Adequate folate nutrition must occur at the time of conception because critical folate-dependent development occurs in the first weeks of fetal life, at a time when many women are not yet aware of their pregnancy. In 1998, the U.S. Food and Drug Administration authorized the addition of folic acid to wheat flour and enriched grain products, resulting in a dietary supplementation of ~0.1 mg/day. This supplementation allows ~50% of all reproductive-aged women to receive 0.4 mg of folate from all sources.
III. COBALAMIN (VITAMIN B12)
Vitamin B12 is required in humans for two essential enzymatic reactions: the remethylation of homocysteine (Hcy) to methionine and the isomerization of methylmalonyl coenzyme A (CoA), which is produced during the degradation of some amino acids (isoleucine, valine, threonine, and methionine) and fatty acids (FA) with odd numbers of carbon atoms (Fig. 28.5). When cobalamin is deficient, unusual (branched) FA accumulate and become incorporated into cell membranes, including those of the central nervous system (CNS). This may account for some of the neurologic manifestations of vitamin B12 deficiency. [Note: Folic acid (as N5-methyl THF) is also required in the remethylation of
Hcy. Therefore, deficiency of B12 or folate results in elevated Hcy levels.]
A. Structure and coenzyme forms
Cobalamin contains a corrin ring system that resembles the porphyrin ring of heme (see p. 279), but differs in that two of the pyrrole rings are linked directly rather than through a methene bridge. Cobalt (see p. 407) is held in the center of the corrin ring by four coordination bonds with the nitrogens of the pyrrole groups. The remaining coordination bonds of the cobalt are with the nitrogen of 5,6-dimethylbenzimidazole and with cyanide in commercial preparations of the vitamin in the form of cyanocobalamin (Fig. 28.6). The physiologic coenzyme forms of cobalamin are 5′deoxyadenosylcobalamin and methylcobalamin, in which cyanide is replaced with 5′-deoxyadenosine or a methyl group, respectively (see Fig. 28.6).
B. Distribution
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Vitamin B12 is synthesized only by microorganisms, and it is not present in plants. Animals obtain the vitamin preformed from their intestinal microbiota (see p. 371) or by eating foods derived from other animals. Cobalamin is present in appreciable amounts in liver, red meat, fish, eggs, dairy products, and fortified cereals.
C. Folate trap hypothesis
The effects of cobalamin deficiency are most pronounced in rapidly dividing cells, such as the erythropoietic tissue of bone marrow and the mucosal cells of the intestine. Such tissues need both the N5,N10-methylene and N10-formyl forms of THF for the synthesis of nucleotides required for DNA replication (see pp. 292 and 303). However, in vitamin B12 deficiency, the utilization of the N5-methyl form of THF in the B12 dependent methylation of Hcy to methionine is impaired. Because the methylated form cannot be converted directly to other forms of THF, folate is trapped in the N5-methyl form, which accumulates. The levels of the other forms decrease. Thus, cobalamin deficiency leads to a deficiency of the THF forms needed in purine and TMP synthesis, resulting in the symptoms of megaloblastic anemia.
D. Clinical indications for cobalamin
In contrast to other water-soluble vitamins, significant amounts (2–5 mg) of vitamin B12 are stored in the body. As a result, it may take several years for the clinical symptoms of B12 deficiency to develop as a result of decreased intake of the vitamin. [Note: Deficiency happens much more quickly (in months) if absorption is impaired (see below). The Schilling test evaluates B12 absorption.] B12 deficiency can be determined by the level of methylmalonic acid in blood, which is elevated in individuals with low intake or decreased absorption of the vitamin.
1. Pernicious anemia: Vitamin B12 deficiency is most commonly seen in patients who fail to absorb the vitamin from the intestine (Fig. 28.7). B12 is released from food in the acidic environment of the stomach. [Note:
Malabsorption of cobalamin in the elderly is most often due to reduced secretion of gastric acid (achlorhydria).] Free B12 then binds a glycoprotein (R-protein or haptocorrin), and the complex moves into the intestine. B12 is released from the R-protein by pancreatic enzymes and binds another glycoprotein, intrinsic factor (IF). The cobalamin–IF complex travels through the intestine and binds to a receptor (cubilin) on the surface of mucosal cells in the ileum. The cobalamin is transported into the mucosal cell and, subsequently, into the general circulation, where it is carried by its binding protein (transcobalamin). B12 is taken up and stored in the liver, primarily. It is released into bile and efficiently reabsorbed in the ileum. Severe malabsorption of vitamin B12 leads to pernicious anemia. This disease is most commonly a result of an autoimmune destruction of the gastric parietal cells that are responsible for the synthesis of IF (lack of IF prevents B12 absorption). [Note: Patients who have had a partial or total gastrectomy become IF deficient and, therefore, B12 deficient.] Individuals with cobalamin deficiency are usually anemic (folate recycling is impaired), and they show neuropsychiatric symptoms as the disease develops. The CNS effects are irreversible. Pernicious anemia requires lifelong treatment with either high-dose oral B12 or intramuscular injection of cyanocobalamin. [Note: Supplementation works even in the absence of IF because ~1% of B12 uptake is by IF-independent diffusion.]
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Folic acid supplementation can partially reverse the hematologic abnormalities of B12 deficiency and, therefore, can mask a cobalamin deficiency. Thus, to prevent the later CNS effects of B12 deficiency, therapy for megaloblastic anemia is initiated with both vitamin B12 and folic acid until the cause of the anemia can be determined.
IV. ASCORBIC ACID (VITAMIN C)
The active form of vitamin C is ascorbic acid (Fig. 28.8). Its main function is as a reducing agent. Vitamin C is a coenzyme in hydroxylation reactions (for example, hydroxylation of prolyl and lysyl residues in collagen; see p. 47), where its role is to keep the iron (Fe) of hydroxylases in the reduced, ferrous (Fe+2) form. Thus, vitamin C is required for the maintenance of normal connective tissue as well as for wound healing. Vitamin C also facilitates the absorption of dietary nonheme iron from the intestine by reduction of the ferric form (Fe+3) to Fe+2 (see p. 403).
A. Deficiency
Ascorbic acid deficiency results in scurvy, a disease characterized by sore and spongy gums, loose teeth, fragile blood vessels, hemorrhage, swollen joints, bone changes, and fatigue (Fig. 28.9). Many of the deficiency symptoms can be explained by the decreased hydroxylation of collagen, resulting in defective connective tissue. A microcytic anemia caused by decreased absorption of iron may also be seen.
B. Chronic disease prevention
Vitamin C is one of a group of nutrients that includes vitamin E (see p. 395) and β-carotene (see p. 386), which are known as antioxidants. [Note: Vitamin C regenerates the functional, reduced form of vitamin E.] Consumption of diets rich in these compounds is associated with a decreased incidence of some chronic diseases, such as cardiovascular disease (CVD) and certain cancers. However, clinical trials involving supplementation with the isolated antioxidants have failed to demonstrate any convincing preventive effects.
V. PYRIDOXINE (VITAMIN B6)
Vitamin B6 is a collective term for pyridoxine, pyridoxal, and pyridoxamine, all derivatives of pyridine. They differ only in the nature of the functional group attached to the ring (Fig. 28.10). Pyridoxine occurs primarily in plants, whereas pyridoxal and pyridoxamine are found in foods obtained from animals. All three compounds can serve as precursors of the biologically active coenzyme, pyridoxal phosphate (PLP). PLP functions as a coenzyme for a large number of enzymes, particularly those that catalyze reactions involving amino acids, for example, in the transsulfuration of Hcy to cysteine (see p. 264). [Note: PLP is also required by glycogen phosphorylase (see p. 128).]
A. Clinical indications for pyridoxine
Isoniazid, a drug commonly used to treat tuberculosis, can induce a vitamin B6 deficiency by forming an inactive derivative with PLP. Thus, dietary supplementation with B6 is an adjunct to isoniazid treatment. Otherwise, dietary deficiencies in pyridoxine are rare but have been observed in newborn infants fed formulas low in B6, in women taking oral contraceptives, and in those with alcoholism.
B. Toxicity
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Vitamin B6 is the only water-soluble vitamin with significant toxicity. Neurologic symptoms (sensory neuropathy) occur at intakes above 500 mg/day, an amount nearly 400 times the recommended dietary allowance (RDA) and over 5 times the tolerable upper limit (UL). (See Chapter 27 for a discussion of RDA and UL.) Substantial improvement, but not complete recovery, occurs when the vitamin is discontinued.
VI. THIAMINE (VITAMIN B1)
Thiamine pyrophosphate (TPP) is the biologically active form of the vitamin, formed by the transfer of a pyrophosphate group from ATP to thiamine (Fig.
28.11). TPP serves as a coenzyme in the formation or degradation of α-ketols by transketolase (Fig. 28.12A) and in the oxidative decarboxylation of α-keto acids (Fig. 28.12B).
A. Clinical indications for thiamine
The oxidative decarboxylation of pyruvate and α-ketoglutarate, which plays a key role in energy metabolism of most cells, is particularly important in tissues of the CNS. In thiamine deficiency, the activity of these two dehydrogenase-catalyzed reactions is decreased, resulting in decreased production of ATP and, therefore, impaired cellular function. TPP is also required by branched-chain α-keto acid dehydrogenase of muscle (see p. 266). [Note: It is the decarboxylase of each of these α-keto acid dehydrogenase multienzyme complexes that requires TPP.] Thiamine deficiency is diagnosed by an increase in erythrocyte transketolase activity observed with addition of TPP.
1.
Beriberi: This severe thiamine-deficiency syndrome is found in areas where polished rice is the major component of the diet. Adult beriberi is classified as dry (characterized by peripheral neuropathy, especially in the legs) or wet (characterized by edema because of dilated cardiomyopathy).
2.
Wernicke-Korsakoff syndrome: In the United States, thiamine deficiency, which is seen primarily in association with chronic alcoholism, is due to dietary insufficiency or impaired intestinal absorption of the vitamin. Some individuals with alcoholism develop Wernicke-Korsakoff syndrome, a thiamine-deficiency state characterized by mental confusion, gait ataxia, nystagmus (a to-and-fro motion of the eyeballs), and ophthalmoplegia (weakness of eye muscles) with Wernicke encephalopathy as well as memory problems and hallucinations with Korsakoff dementia. The syndrome is treatable with thiamine supplementation, but recovery of memory is typically incomplete.
VII. NIACIN (VITAMIN B3)
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Niacin, or nicotinic acid, is a substituted pyridine derivative. The biologically active coenzyme forms are nicotinamide adenine dinucleotide (NAD+) and its phosphorylated derivative, nicotinamide adenine dinucleotide phosphate (NADP+), as shown in Figure 28.13. Nicotinamide, a derivative of nicotinic acid that contains an amide instead of a carboxyl group, also occurs in the diet. Nicotinamide is readily deaminated in the body and, therefore, is nutritionally equivalent to nicotinic acid. NAD+ and NADP+ serve as coenzymes in oxidation–reduction reactions in which the coenzyme undergoes reduction of the pyridine ring by accepting two electrons from a hydride ion, as shown in Figure 28.14. The reduced forms of NAD+ and NADP+ are NADH and NADPH, respectively. [Note: A metabolite of tryptophan, quinolinate, can be converted to NAD(P). In comparison, 60 mg of tryptophan = 1 mg of niacin.] to NADH. [Note: The hydride ion consists of a hydrogen (H) atom plus an electron.] = phosphate.
A. Distribution
Niacin is found in unrefined and enriched grains and cereal, milk, and lean meats (especially liver).
B. Clinical indications for niacin 1.
Deficiency: A deficiency of niacin causes pellagra, a disease involving the skin, gastrointestinal tract, and CNS. The symptoms of pellagra progress through the three Ds: dermatitis (photosensitive), diarrhea, and dementia. If untreated, death (a fourth D) occurs. Hartnup disorder, characterized by defective absorption of tryptophan, can result in pellagra-like symptoms. [Note: Corn is low in both niacin and tryptophan. Corn-based diets can cause pellagra.] 2.
Hyperlipidemia treatment: Niacin at doses of 1.5 g/day, or 100 times the RDA, strongly inhibits lipolysis in adipose tissue, the primary producer of circulating free fatty acids (FFA). The liver normally uses these circulating FFA as a major precursor for triacylglycerol (TAG) synthesis. Thus, niacin causes a decrease in liver TAG synthesis, which is required for very-low-density lipoprotein ([VLDL] see p. 230) production. Low-density lipoprotein (LDL, the cholesterol-rich lipoprotein) is derived from VLDL in the plasma. Thus, both plasma TAG (in VLDL) and cholesterol (in LDL) are lowered. Therefore, niacin is particularly useful in the treatment of type IIb hyperlipoproteinemia, in which both VLDL and LDL are elevated. The high doses of niacin required can cause acute, prostaglandin-mediated flushing. Aspirin can reduce this side effect by inhibiting prostaglandin synthesis (see p. 214). Itching may also occur. [Note: Niacin raises high-density lipoprotein and lowers Lp(a) levels (see p. 237).]
VIII. RIBOFLAVIN (VITAMIN B2)
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The two biologically active forms of B2 are flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), formed by the transfer of an adenosine monophosphate moiety from ATP to FMN (Fig. 28.15). FMN and FAD are each capable of reversibly accepting two hydrogen atoms, forming FMNH2 or FADH2, respectively. FMN and FAD are bound tightly, sometimes covalently, to flavoenzymes (for example, NADH dehydrogenase [FMN] and succinate dehydrogenase [FAD]) that catalyze the oxidation or reduction of a substrate. Riboflavin deficiency is not associated with a major human disease, although it frequently accompanies other vitamin deficiencies. Deficiency symptoms include dermatitis, cheilosis (fissuring at the corners of the mouth), and glossitis (the tongue appearing smooth and dark). [Note: Because riboflavin is light sensitive, phototherapy for hyperbilirubinemia (see p. 285) may require supplementation with the vitamin.]
IX. BIOTIN (VITAMIN B7)
Biotin is a coenzyme in carboxylation reactions, in which it serves as a carrier of activated carbon dioxide (CO2) (see Fig. 10.3, p. 119, for the mechanism of biotin-dependent carboxylations). Biotin is covalently bound to the ε-amino group of lysine residues in biotin-dependent enzymes (Fig. 28.16). Biotin deficiency does not occur naturally because the vitamin is widely distributed in food. Also, a large percentage of the biotin requirement in humans is supplied by intestinal bacteria. However, the addition of raw egg white to the diet as a source of protein can induce symptoms of biotin deficiency, namely, dermatitis, hair loss, loss of appetite, and nausea. Raw egg white contains the glycoprotein avidin, which tightly binds biotin and prevents its absorption from the intestine. With a normal diet, however, it has been estimated that 20 eggs/day would be required to induce a deficiency syndrome. [Note: Inclusion of raw eggs in the diet is not recommended because of the possibility of salmonellosis caused by infection with Salmonella enterica.]
Multiple carboxylase deficiency results from decreased ability to add biotin to carboxylases during their synthesis or to remove it during their degradation. Treatment is biotin supplementation.
X. PANTOTHENIC ACID (VITAMIN B5)
Pantothenic acid is a component of CoA, which functions in the transfer of acyl groups (Fig. 28.17). CoA contains a thiol group that carries acyl compounds as activated thiol esters. Examples of such structures are succinyl CoA, fatty acyl CoA, and acetyl CoA. Pantothenic acid is also a component of the acyl carrier protein domain of fatty acid synthase (see p. 184). Eggs, liver, and yeast are the most important sources of pantothenic acid, although the vitamin is widely distributed. Pantothenic acid deficiency is not well characterized in humans, and no RDA has been established.
XI. VITAMIN A
Vitamin A is a fat-soluble vitamin that comes primarily from animal sources as retinol (preformed vitamin A), a retinoid. The retinoids, a family of structurally related molecules, are essential for vision, reproduction, growth, and maintenance of epithelial tissues. They also play a role in immune function. Retinoic acid, derived from oxidation of retinol, mediates most of the actions of the retinoids, except for vision, which depends on retinal, the aldehyde derivative of retinol.
A. Structure
The retinoids include the natural forms of vitamin A, retinol and its metabolites (Fig. 28.18), and synthetic forms (drugs).
1.
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Retinol: A primary alcohol containing a β-ionone ring with an unsaturated side chain, retinol is found in animal tissues as a retinyl ester with long-chain FA. It is the storage form of vitamin A.
2.
Retinal: This is the aldehyde derived from the oxidation of retinol. Retinal and retinol can readily be interconverted.
3.
Retinoic acid: This is the acid derived from the oxidation of retinal. Retinoic acid cannot be reduced in the body and, therefore, cannot give rise to either retinal or retinol.
4.
β-Carotene: Plant foods contain β-carotene (provitamin A), which can be oxidatively and symmetrically cleaved in the intestine to yield two molecules of retinal. In humans, the conversion is inefficient, and the vitamin A activity of β-carotene is only about 1/12 that of retinol.
B. Absorption and transport to the liver
Retinyl esters from the diet are hydrolyzed in the intestinal mucosa, releasing retinol and FFA (Fig. 28.19). Retinol derived from esters and from the reduction of retinal from β-carotene cleavage is reesterified to long-chain FA within the enterocytes and secreted as a component of chylomicrons into the lymphatic system. Retinyl esters contained in chylomicron remnants are taken up by, and stored in, the liver. [Note: All fat-soluble vitamins are carried in chylomicrons.]
C. Release from the liver
When needed, retinol is released from the liver and transported through the blood to extrahepatic tissues by retinol-binding protein complexed with transthyretin (see Fig. 28.19). The ternary complex binds to a transport protein on the surface of the cells of peripheral tissues, permitting retinol to enter. An intracellular retinol-binding protein carries retinol to sites in the nucleus where the vitamin regulates transcription in a manner analogous to that of steroid hormones.
D. Retinoic acid mechanism of action
Retinol is oxidized to retinoic acid. Retinoic acid binds with high affinity to specific receptor proteins (retinoic acid receptors [RAR]) present in the nucleus of target tissues such as epithelial cells (Fig. 28.20). The activated retinoic acid–RAR complex binds to response elements on DNA and recruits activators or repressors to regulate retinoid-specific RNA synthesis, resulting in control of the production of specific proteins that mediate several physiologic functions. For example, retinoids control the expression of the gene for keratin in most epithelial tissues of the body. [Note: The RAR proteins are part of the superfamily of transcriptional regulators that includes the nuclear receptors for steroid and thyroid hormones and vitamin D, all of which function in a similar way (see p. 240).]
E. Functions 1.
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Visual cycle: Vitamin A is a component of the visual pigments of rod and cone cells. Rhodopsin, the visual pigment of the rod cells in the retina, consists of 11-cis retinal bound to the protein opsin (see Fig. 28.19). When rhodopsin, a G protein–coupled receptor, is exposed to light, a series of photochemical isomerizations occurs, which results in the bleaching of rhodopsin and release of all-trans retinal and opsin. This process activates the G protein transducin, triggering a nerve impulse that is transmitted by the optic nerve to the brain. Regeneration of rhodopsin requires isomerization of all-trans retinal back to 11-cis retinal. All-trans retinal is reduced to all-trans retinol, esterified, and isomerized to 11-cis retinol that is oxidized to 11-cis retinal. The latter combines with opsin to form rhodopsin, thus completing the cycle. Similar reactions are responsible for color vision in the cone cells.
2.
Epithelial cell maintenance: Vitamin A is essential for normal differentiation of epithelial tissues and mucus secretion and, thus, supports the body’s barrier-based defense against pathogens.
3.
Reproduction: Retinol and retinal are essential for normal reproduction, supporting spermatogenesis in the male and preventing fetal resorption in the female. Retinoic acid is inactive in maintaining reproduction and in the visual cycle but promotes growth and differentiation of epithelial cells.
F. Distribution
Liver, kidney, cream, butter, and egg yolk are good sources of preformed vitamin A. Yellow, orange, and dark-green vegetables and fruits are good sources of the carotenes (provitamin A).
G. Requirement
The RDA for adults is 900 retinol activity equivalents (RAE) for males and 700 RAE for females. In comparison, 1 RAE = 1 µg of retinol, 12 µg of β carotene, or 24 µg of other carotenoids.
H. Clinical indications for vitamin A
Although chemically related, retinoic acid and retinol have distinctly different therapeutic applications. Retinol and its carotenoid precursor are used as dietary supplements, whereas various forms of retinoic acid are useful in dermatology (Fig. 28.21).
1.
Deficiency: Vitamin A, administered as retinol or retinyl esters, is used to treat patients who are deficient in the vitamin. Night blindness (nyctalopia) is one of the earliest signs of vitamin A deficiency. The visual threshold is increased, making it difficult to see in dim light. Prolonged deficiency leads to an irreversible loss in the number of visual cells. Severe deficiency leads to xerophthalmia, a pathologic dryness of the conjunctiva and cornea, caused, in part, by increased keratin synthesis. If untreated, xerophthalmia results in corneal ulceration and, ultimately, in blindness because of the formation of opaque scar tissue. The condition is most commonly seen in children in developing tropical countries. Over 500,000 children worldwide are blinded each year by xerophthalmia caused by insufficient vitamin A in the diet.
2.
Skin conditions: Dermatologic problems such as acne are effectively treated with retinoic acid or its derivatives (see Fig. 28.21). Mild cases of acne and skin aging are treated with tretinoin (all-trans retinoic acid). Tretinoin is too toxic for systemic (oral) administration in treating skin conditions and is confined to topical application. [Note: Oral tretinoin is used in treating acute promyelocytic leukemia.] In patients with severe cystic acne unresponsive to conventional therapies, isotretinoin (13-cis retinoic acid) is administered orally. An oral synthetic retinoid is used to treat psoriasis.
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I. Retinoid toxicity 1.
Vitamin A: Excessive intake of vitamin A (but not carotene) produces a toxic syndrome called hypervitaminosis A. Amounts exceeding 7.5 mg/day of retinol should be avoided. Early signs of chronic hypervitaminosis A are reflected in the skin, which becomes dry and pruritic (because of decreased keratin synthesis); in the liver, which becomes enlarged and can become cirrhotic; and in the CNS, where a rise in intracranial pressure may mimic the symptoms of a brain tumor. Pregnant women, in particular, should not ingest excessive quantities of vitamin A because of its potential for teratogenesis (causing congenital malformations in the developing fetus). UL is 3,000 µg preformed vitamin A/day. [Note: Vitamin A promotes bone growth. In excess, however, it is associated with decreased bone mineral density and increased risk of fractures.] 2.
Isotretinoin: The drug, an isomer of retinoic acid, is teratogenic and absolutely contraindicated in women with childbearing potential unless they have severe, disfiguring cystic acne that is unresponsive to standard therapies. Pregnancy must be excluded before treatment begins, and birth control must be used. Prolonged treatment with isotretinoin can result in an increase in TAG and cholesterol, providing some concern for an increased risk of CVD.
XII. VITAMIN D
The D vitamins are a group of sterols that have a hormone-like function. The active molecule, 1,25-dihydroxycholecalciferol ([1,25-diOH-D3], or calcitriol), binds to intracellular receptor proteins. The 1,25-diOH-D3–receptor complex interacts with response elements in the nuclear DNA of target cells in a manner similar to that of vitamin A (see Fig. 28.20) and either selectively stimulates or represses gene transcription. The most prominent actions of calcitriol are to regulate the serum levels of calcium and phosphorus.
A. Distribution 1.
Endogenous vitamin precursor: 7-Dehydrocholesterol, an intermediate in cholesterol synthesis, is converted to cholecalciferol in the dermis and epidermis of humans exposed to sunlight and transported to liver bound to vitamin D–binding protein.
2.
Diet: Ergocalciferol (vitamin D2), found in plants, and cholecalciferol (vitamin D3), found in animal tissues, are sources of preformed vitamin D activity (Fig. 28.22). Vitamin D2 and vitamin D3 differ chemically only in the presence of an additional double-bond and methyl group in the plant sterol. Dietary vitamin D is packaged into chylomicrons. [Note: Preformed vitamin D is a dietary requirement only in individuals with limited exposure to sunlight.]
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B. Metabolism 1. 1,25-Dihydroxycholecalciferol formation: Vitamins D2 and D3 are not biologically active but are converted in vivo to calcitriol, the active form of the D vitamin, by two sequential hydroxylation reactions (Fig. 28.23). The first hydroxylation occurs at the 25 position and is catalyzed by a specific 25-hydroxylase in the liver. The product of the reaction, 25hydroxycholecalciferol ([25-OH-D3], calcidiol), is the predominant form of vitamin D in the serum and the major storage form. 25-OH-D3 is further hydroxylated at the 1 position by 25-hydroxycholecalciferol 1 hydroxylase found primarily in the kidney, resulting in the formation of 1,25-diOH-D3 (calcitriol). [Note: Both hydroxylases are cytochrome P450 proteins (see p. 149).] 2. Hydroxylation regulation: Calcitriol is the most potent vitamin D metabolite. Its formation is tightly regulated by the level of serum phosphate (PO43−) and calcium ions (Ca2+) as shown in Figure 28.24. 25
Hydroxycholecalciferol 1-hydroxylase activity is increased directly by low serum PO43− or indirectly by low serum Ca2+ , which triggers the secretion of parathyroid hormone (PTH) from the chief cells of the parathyroid gland. PTH upregulates the 1-hydroxylase. Thus, hypocalcemia caused by insufficient dietary Ca2+ results in elevated levels of serum 1,25-diOH-D3. [Note: 1,25-diOH-D3 inhibits expression of PTH, forming a negative feedback loop. It also inhibits activity of the 1-hydroxylase.]
C. Function
The overall function of calcitriol is to maintain adequate serum levels of Ca2+ . It performs this function by 1) increasing uptake of Ca2+ by the intestine, 2) minimizing loss of Ca2+ by the kidney by increasing reabsorption, and 3) stimulating resorption (demineralization) of bone when blood Ca2+ is low (see Fig. 28.23).
1.
Effect on the intestine: Calcitriol stimulates intestinal absorption of Ca2+ by first entering the intestinal cell and binding to a cytosolic receptor. The 1,25-diOH-D3–receptor complex then moves to the nucleus where it selectively interacts with response elements on the DNA. As a result, Ca2+ uptake is enhanced by increased expression of the calcium-binding protein calbindin. Thus, the mechanism of action of 1,25-diOH-D3 is typical of steroid hormones (see p. 240).
2.
Effect on bone: Bone is composed of collagen and crystals of Ca5(PO4)3OH (hydroxylapatite). When blood Ca2+ is low, 1,25-diOH-D3 stimulates bone resorption by a process that is enhanced by PTH. The result is an increase in serum Ca2+ . Therefore, bone is an important reservoir of Ca2+ that can be mobilized to maintain serum levels. [Note: PTH and calcitriol also work together to prevent renal loss of Ca2+.]
D. Distribution and requirement
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Vitamin D occurs naturally in fatty fish, liver, and egg yolk. Milk, unless it is artificially fortified, is not a good source. The RDA for individuals ages 1–70 years is 15 µg/day and 20 µg/day if over age 70 years. Experts disagree, however, on the optimal level of vitamin D needed to maintain health. [Note: 1 µg vitamin D = 40 international units (IU).] Because breast milk is a poor source of vitamin D, supplementation is recommended for breastfed babies.
E. Clinical indications for vitamin D 1. Nutritional rickets: Vitamin D deficiency causes a net demineralization of bone, resulting in rickets in children and osteomalacia in adults (Fig. 28.25). Rickets is characterized by the continued formation of the collagen matrix of bone, but incomplete mineralization results in soft, pliable bones. In osteomalacia, demineralization of preexisting bones increases their susceptibility to fracture. Insufficient exposure to daylight and/or deficiencies in vitamin D consumption occur predominantly in infants and the elderly. Vitamin D deficiency is more common in the northern latitudes, because less vitamin D synthesis occurs in the skin as a result of reduced exposure to ultraviolet light. [Note: Loss-of-function mutations in the vitamin D receptor result in hereditary vitamin D– deficient rickets.] 2.
Renal osteodystrophy: Chronic kidney disease causes decreased ability to form active vitamin D as well as increased retention of PO43− , resulting in hyperphosphatemia and hypocalcemia. The low blood Ca2+ causes a rise in PTH and associated bone demineralization with release of Ca2+ and PO43− . Supplementation with vitamin D is an effective therapy.
However, supplementation must be accompanied by PO43− reduction therapy to prevent further bone loss and precipitation of calcium phosphate crystals.
3.
Hypoparathyroidism: Lack of PTH causes hypocalcemia and hyperphosphatemia. [Note: PTH increases phosphate excretion.] Patients may be treated with vitamin D and calcium supplementation.
F. Toxicity
Like all fat-soluble vitamins, vitamin D can be stored in the body and is only slowly metabolized. High doses (100,000 IU for weeks or months) can cause loss of appetite, nausea, thirst, and weakness. Enhanced Ca2+ absorption and bone resorption results in hypercalcemia, which can lead to deposition of calcium salts in soft tissue (metastatic calcification). The UL is 100 µg/day (4,000 IU/day) for individuals ages 9 years or older, with a lower level for those under age 9 years. [Note: Toxicity is only seen with use of supplements. Excess vitamin D produced in the skin is converted to inactive forms.]
XIII. VITAMIN K
The principal role of vitamin K is in the posttranslational modification of a number of proteins (most of which are involved with blood clotting), in which it serves as a coenzyme in the carboxylation of certain glutamic acid residues in these proteins. Vitamin K exists in several active forms, for example, in plants as phylloquinone (or vitamin K1), and in intestinal bacteria as menaquinone (or vitamin K2). A synthetic form of vitamin K, menadione, is able to be converted to K2.
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A. Function 1. γ-Carboxyglutamate formation: Vitamin K is required in the hepatic synthesis of the blood clotting proteins, prothrombin (factor [F]II) and FVII, FIX, and FX. (See online Chapter 35.) Formation of the functional clotting factors requires the vitamin K–dependent carboxylation of several glutamic acid residues to γ-carboxyglutamate (Gla) residues (Fig. 28.26). The carboxylation reaction requires γ-glutamyl carboxylase, O2,
CO2, and the hydroquinone form of vitamin K (which gets oxidized to the epoxide form). The formation of Gla residues is sensitive to inhibition by warfarin, a synthetic analog of vitamin K that inhibits vitamin K epoxide reductase (VKOR), the enzyme required to regenerate the functional hydroquinone form of vitamin K.
2.
Prothrombin interaction with membranes: The Gla residues are good chelators of positively charged calcium ions, because of their two adjacent, negatively charged carboxylate groups. With prothrombin, for example, the prothrombin–calcium complex is able to bind to negatively charged membrane phospholipids on the surface of damaged endothelium and platelets. Attachment to membrane increases the rate at which the proteolytic conversion of prothrombin to thrombin can occur (Fig. 28.27).
3.
γ-Carboxyglutamate residues in other proteins: Gla residues are also present in proteins other than those involved in forming a blood clot. For example, osteocalcin and matrix Gla protein of bone and proteins C and S (involved in limiting the formation of blood clots) also undergo γcarboxylation.
Figure28.27RoleofvitaminKinbloodcoagulation.CO2=carbondioxide.
B. Distribution and requirement
Vitamin K is found in cabbage, kale, spinach, egg yolk, and liver. The adequate intake for vitamin K is 120 µg/day for adult males and 90 µg for adult females. There is also synthesis of the vitamin by the gut microbiota.
C. Clinical indications for vitamin K 1.
Deficiency: A true vitamin K deficiency is unusual because adequate amounts are generally obtained from the diet and produced by intestinal bacteria. If the bacterial population in the gut is decreased (for example, by antibiotics), the amount of endogenously formed vitamin is decreased, and this can lead to hypoprothrombinemia in the marginally malnourished individual (for example, a debilitated geriatric patient). This condition may require supplementation with vitamin K to correct the bleeding tendency. In addition, certain cephalosporin antibiotics (for example, cefamandole) cause hypoprothrombinemia, apparently by a warfarin-like mechanism that inhibits VKOR. Consequently, their use in treatment is usually supplemented with vitamin K. Deficiency can also affect bone health.
2.
Deficiency in the newborn: Because newborns have sterile intestines, they initially lack the bacteria that synthesize vitamin K. Because human milk provides only about one fifth of the daily requirement for vitamin K, it is recommended that all newborns receive a single intramuscular dose of vitamin K as prophylaxis against hemorrhagic disease of the newborn.
D. Toxicity
Prolonged administration of large doses of menadione can produce hemolytic anemia and jaundice in the infant, because of toxic effects on the RBC membrane. Therefore, it is no longer used to treat vitamin K deficiency. No UL for the natural form has been set.
XIV. VITAMIN E
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The E vitamins consist of eight naturally occurring tocopherols, of which αtocopherol is the most active (Fig. 28.28). Vitamin E functions as an antioxidant in prevention of nonenzymic oxidations (for example, oxidation of LDL (see p.
232) and peroxidation of polyunsaturated FA by O2 and free radicals). [Note: Vitamin C regenerates active vitamin E.]
A. Distribution and requirements
Vegetable oils are rich sources of vitamin E, whereas liver and eggs contain moderate amounts. The RDA for α-tocopherol is 15 mg/day for adults. The vitamin E requirement increases as the intake of polyunsaturated FA increases to limit FA peroxidation.
B. Deficiency
Newborns have low reserves of vitamin E, but breast milk (and formulas) contain the vitamin. Very-low-birth-weight infants may be given supplements to prevent the hemolysis and retinopathy associated with vitamin E deficiency. When observed in adults, deficiency is usually associated with defective lipid absorption or transport. [Note: Abetalipoproteinemia, caused by a defect in the formation of chylomicrons (and VLDL), results in vitamin E deficiency (see p. 231).]
C. Clinical indications for vitamin E
Vitamin E is not recommended for the prevention of chronic disease, such as CVD or cancer. Clinical trials using vitamin E supplementation have been uniformly disappointing. For example, subjects in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study trial who received high doses of vitamin E not only lacked cardiovascular benefit but also had an increased incidence of stroke. [Note: Vitamins E and C are used to slow the progression of age-related macular degeneration.]
D. Toxicity
Vitamin E is the least toxic of the fat-soluble vitamins, and no toxicity has been observed at doses of 300 mg/day (UL = 1,000 mg/day).
Populations consuming diets high in fruits and vegetables show decreased incidence of some chronic diseases. However, clinical trials have failed to show a definitive benefit from supplements of folic acid; vitamins A, C, or E; or antioxidant combinations for the prevention of cancer or CVD.
XV. CHAPTER SUMMARY
The vitamins are summarized in Figure 28.29 on pp. 396–397.
Choose the ONE best answer.
For Questions 28.1–28.5, match the vitamin deficiency to the clinical consequence.
8.1. Bleeding 8.2. Diarrhea and dermatitis 8.3. Neural tube defects 8.4. Night blindness (nyctalopia) 8.5. Sore, spongy gums and loose teeth
Correct answers = H, B, A, C, E. Vitamin K is required for formation of the γcarboxyglutamate residues in several proteins required for blood clotting. Consequently, a deficiency of vitamin K results in a tendency to bleed. Niacin deficiency is characterized by the three Ds: diarrhea, dermatitis, and dementia (and death, a fourth D, if untreated). Folic acid deficiency can result in neural tube defects in the developing fetus. Night blindness is one of the first signs of vitamin A deficiency. Rod cells in the retina detect white and black images and work best in low light, for example, at night. Rhodopsin, the visual pigment of the rod cells, consists of 11-cis retinal bound to the protein opsin. Vitamin C is required for the hydroxylation of proline and lysine during collagen synthesis.
Severe vitamin C deficiency (scurvy) results in defective connective tissue, characterized by sore and spongy gums, loose teeth, capillary fragility, anemia, and fatigue.
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8.6. A 52-year-old woman presents with fatigue of several months’ duration. Blood studies reveal a macrocytic anemia, reduced levels of hemoglobin, elevated levels of homocysteine, and normal levels of methylmalonic acid. Which of the following is most likely deficient in this woman?
A. Folic acid
B. Folic acid and vitamin B12
C. Iron
D. Vitamin C
Correct answer = A. Macrocytic anemia is seen with deficiencies of folic acid, vitamin B12, or both. Vitamin B12 is utilized in only two reactions in the body: the remethylation of homocysteine (Hcy) to methionine, which also requires folic acid (as tetrahydrofolate [THF]), and the isomerization of methylmalonyl coenzyme A to succinyl coenzyme A, which does not require THF. The elevated Hcy and normal methylmalonic acid levels in the patient’s blood reflect a deficiency of folic acid as the cause of the macrocytic anemia. Iron deficiency causes microcytic anemia, as can vitamin C deficiency.
8.7. A 10-month-old African American girl, whose family recently located from Maine to Virginia, is being evaluated for the bowed appearance of her legs. The parents report that the baby is still being breastfed and takes no supplements. Radiologic studies confirm the suspicion of rickets caused by vitamin D deficiency. Which one of the following statements concerning vitamin D is correct?
A. A deficiency results in an increased secretion of calbindin.
B. Chronic kidney disease results in overproduction of 1,25dihydroxycholecalciferol (calcitriol).
C. 25-Hydroxycholecalciferol (calcidiol) is the active form of the vitamin.
D. It is required in the diet of individuals with limited exposure to sunlight.
E. Its actions are mediated through binding to G protein–coupled receptors.
F. It opposes the effect of parathyroid hormone.
Correct answer = D. Vitamin D is required in the diet of individuals with limited exposure to sunlight, such as those living at northern latitudes like Maine and those with dark skin. Note that breast milk is low in vitamin D, and the lack of supplementation increases the risk of a deficiency. Vitamin D deficiency results in decreased synthesis of calbindin. Chronic kidney disease decreases production of calcitriol (1,25-dihydroxycholecalciferol), the active form of the vitamin. Vitamin D binds to nuclear receptors and alters gene transcription. Its effects are synergistic with parathyroid hormone.
8.8. Why might a deficiency of vitamin B6 result in a fasting hypoglycemia? Deficiency of what other vitamin could also result in hypoglycemia?
Vitamin B6 is required for glycogen degradation by glycogen phosphorylase. A deficiency would result in fasting hypoglycemia. Additionally, a deficiency of biotin (required by pyruvate carboxylase of gluconeogenesis) would also result in fasting hypoglycemia.
Micronutrients: Minerals 29
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I. OVERVIEW
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Minerals are inorganic substances (elements) required in small amounts by the body. They function in a number of processes including formation of bones and teeth, fluid balance, nerve conduction, muscle contraction, signaling, and catalysis. [Note: Several minerals are essential enzyme cofactors.] Like the organic vitamins (see Chapter 28), minerals are micronutrients required in mg or µg amounts. Those required by adults in the largest amounts (>100 mg/day) are referred to as the macrominerals. Minerals required in amounts between 1 and 100 mg/day are the microminerals (trace minerals). Ultratrace minerals are required in amounts <1 mg/day (Fig. 29.1). [Note: The classification of specific minerals into these categories can vary among sources.] Mineral concentrations in the body are influenced by their rates of absorption and excretion.
II. MACROMINERALS
The macrominerals include calcium (Ca2+), phosphorus ([P] as inorganic phosphate [Pi, or PO43−]), magnesium (Mg2+), sodium (Na+), chloride (Cl−), and potassium (K+). [Note: The free ionic forms are electrolytes.]
A. Calcium and phosphorus
These macrominerals are considered together because they are components of hydroxylapatite (Ca5[PO4]3OH), which makes up bones and teeth.
1. Calcium: Ca2+ is the most abundant mineral in the body, with ~98% being found in bones. The remainder is involved in a number of processes such as signaling, muscle contraction, and blood clotting. Ca2+ binds to a variety of proteins including calmodulin (see p. 133), phospholipase A2 (see p. 213), and protein kinase C (see p. 205) and alters their activity. [Note: Calbindin is a vitamin D–induced intracellular Ca2+-binding protein involved in Ca2+ absorption in the intestine (see p. 392).] Dairy products, many green vegetables (for example, broccoli, but not spinach), and fortified orange juice are good dietary sources. Although dietary deficiency syndromes are unknown, average Ca2+ intake in the United States is insufficient for optimal bone health. Toxicity is seen only with supplements (tolerable upper limit [UL] = 2,500 mg/day for adults). Hypercalcemia (elevated serum Ca2+) can result from overproduction of parathyroid hormone (PTH). This may cause constipation and kidney stones. Hypocalcemia (low serum Ca2+) can result from a deficiency of PTH or vitamin D. It can lead to bone demineralization (resorption). [Note: The hormonal regulation of serum Ca2+ levels was presented in the vitamin D section of Chapter 28 and is reviewed in 3. below.]
Bone mass increases from infancy through the early reproductive years and then shows an age-related loss in both men and women that increases the risk for fracture. This loss is greatest in postmenopausal Caucasian women. Some studies have shown that supplementation with Ca2+ and vitamin D decreases this risk.
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2. Phosphorus: Free phosphate (Pi) is the most abundant intracellular anion. However, 85% of the body’s phosphorus is in the form of inorganic hydroxylapatite, with most of the remainder in intracellular organic compounds such as phospholipids, nucleic acids, ATP, and creatine phosphate. Phosphate is supplied as ATP for kinases and as Pi for phosphorylases (for example, glycogen phosphorylase, see p. 128). [Note: Its addition (by kinases) or removal (by phosphatases) is an important means of covalent regulation of enzymes (see Chapter 24).] Phosphorus is widely distributed in food (milk is a good source), and dietary deficiency is rare. Hypophosphatemia can be caused by refeeding carbohydrates to malnourished patients (refeeding syndrome, see p. 369), overuse of aluminum-containing antacids (aluminum chelates Pi), and increased urinary loss in response to increased production of PTH (see below). Muscle weakness is a common symptom. Hyperphosphatemia is caused primarily by decreased PTH levels. The excess Pi can combine with Ca2+ and form crystals that deposit in soft tissue (metastatic calcification). [Note: The Ca2+/Pi ratio is important for bone formation (the ratio is ~2/1 in bone), and some experts are concerned that replacement of Ca2+-rich milk by Ca2+-poor, Pi-rich soft drinks can affect bone health.] 3. Hormonal regulation: Serum levels of Ca2+ and Pi are primarily controlled by calcitriol (1,25-dihydroxycholecalciferol, the active form of vitamin D) and PTH, both of which respond to a decrease in serum Ca2+ . Calcitriol, produced by the kidneys, increases serum Ca2+ and Pi by increasing bone resorption and intestinal absorption and renal reabsorption of Ca2+ and Pi (Fig. 29.2). PTH (from the parathyroid glands) increases serum Ca2+ by increasing bone resorption, increasing renal reabsorption of Ca2+ , and activating the renal 1-hydroxylase that produces calcitriol from calcidiol (see p. 390) (Fig. 29.3). In contrast to calcitriol, PTH decreases Pi reabsorption in the kidneys, lowering serum Pi. [Note: High serum Pi increases PTH and decreases calcitriol.] A third hormone, calcitonin (from the C cells of the thyroid gland), responds to elevated serum Ca2+ levels by promoting bone mineralization and increasing renal excretion of Ca2+ (and Pi).
B. Magnesium
About 60% of the body’s Mg2+ is in bone, but it accounts for just 1% of the bone mass. The mineral is required by a variety of enzymatic reactions, including phosphorylation by kinases (Mg2+ binds the ATP cosubstrate) and phosphodiester bond formation by DNA and RNA polymerases. Mg2+ is widely distributed in foods, but the average intake in the United States is below the recommended level. Hypomagnesemia can result from decreased absorption or increased excretion of Mg2+ . Symptoms include hyperexcitability of skeletal muscles and nerves and cardiac arrhythmias. With hypermagnesemia, hypotension is seen. [Note: Magnesium sulfate is used in the treatment of preeclampsia, a hypertensive disorder of pregnancy.]
C. Sodium, chloride, and potassium
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These macrominerals are considered together because they play important roles in several physiologic processes. For example, they maintain water balance, osmotic equilibrium, acid–base balance (pH), and the electrical gradients across cell membranes (membrane potential) that are essential for the functioning of neurons and myocytes. [Note: These processes are discussed in Lippincott’s Illustrated Reviews: Physiology.] 1. Sodium and chloride: Na+ and Cl− are primarily extracellular electrolytes. They are readily absorbed from foods containing salt (NaCl), much of which comes from processed foods. [Note: Na+ is required for the intestinal absorption (and renal reabsorption) of glucose and galactose (see p. 87) and free amino acids (see p. 249) by Na+-linked transporters. Cl− is used to form hydrochloric acid required for digestion (see p. 248).] In the United States, the average daily consumption of NaCl is 1.5–3 times the adequate intake (AI) of 3.8 mg/day (UL = 5.8 g/day). Dietary deficiency is rare.
a.
Hypertension: Na+ intake is related to blood pressure (BP). Ingestion of Na+ stimulates thirst centers in the brain and secretion of antidiuretic hormone from the pituitary, leading to water retention. This results in an increase in plasma volume and, consequently, an increase in BP. Chronic hypertension can damage the heart, kidneys, and blood vessels. Modest reductions in Na+ intake have been shown to result in modest reductions in BP. [Note: Some populations (for example, African Americans) are “salt sensitive” and have larger responses to Na+.] b.
Hyper-and hyponatremia: Hypernatremia, typically caused by excess water loss, and hyponatremia, typically caused by decreased ability to excrete water, can result in severe brain damage. [Note: Chronic hyponatremia increases Ca2+ excretion and can result in osteoporosis (low bone mass).] 2. Potassium: In contrast to Na+, K+ is primarily an intracellular electrolyte. [Note: The concentration differential of Na+ and K+ across the cell membrane is maintained by the Na+/K+ ATPase (Fig. 29.4).] In contrast to Na+ and Cl−, K+ (like Mg2+) is underingested in Western diets because its primary sources, fruits and vegetables, are underingested. [Note: Increasing dietary K+ decreases BP by increasing Na+ excretion.] There is a narrow range for normal serum K+ levels, and even modest changes (up or down, resulting in hyper-or hypokalemia) can result in cardiac arrhythmias and skeletal muscle weakness. [Note: Hypokalemia can result from the inappropriate use of laxatives to lose weight.] No UL for K+ has been established.
III. MICROMINERALS (TRACE MINERALS)
The trace minerals include copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn). They are required by adults in amounts between 1 and 100 mg/day.
A. Copper
Cu is a key component of several enzymes that play critical functions in the body (Fig. 29.5). These include ferroxidases such as the ceruloplasmin and hephaestin involved in the oxidation of ferrous iron (Fe2+) to the ferric form (Fe3+) that is required for its intracellular storage or transport through blood (see B.1. below). Meat, shellfish, nuts, and whole grains are good dietary sources of Cu. Dietary deficiency is uncommon. If a deficiency does develop, anemia may be seen because of the effect on Fe metabolism. Toxicity from dietary sources is rare (UL = 10 mg/day). Menkes syndrome and Wilson disease are genetic causes of Cu deficiency and Cu overload, respectively.
1.
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Menkes syndrome: In Menkes syndrome (“kinky hair” disease), a rare X-linked (1:140,000 males) disorder, efflux of dietary Cu out of intestinal enterocytes into the circulation by a Cu-transporting ATPase (ATP7A) is impaired. This results in systemic Cu deficiency. Consequently, urinary and serum free (unbound) Cu are low, as is the concentration of ceruloplasmin, which carries over 90% of the Cu in the circulation (Fig. 29.6). Progressive neurologic degeneration and connective tissue disorders are seen, as are changes to hair. Parenteral administration of Cu has been used as a treatment with varying success. [Note: The mildest form of Menkes syndrome is called occipital horn syndrome.] 2.
Wilson disease: In Wilson disease, an autosomal-recessive (AR) disorder affecting 1:35,000 live births, efflux of excess Cu from the liver by ATP7B is impaired. Cu accumulates in the liver; leaks into the blood; and is deposited in the brain, eyes, kidneys, and skin. In contrast to Menkes syndrome, urinary and serum free Cu are high (see Fig. 29.6). Hepatic dysfunction and neurologic and psychiatric symptoms are seen. Kayser-Fleischer rings (corneal deposits of Cu) may be present (Fig. 29.7). Life-long use of Cu-chelating agents, such as penicillamine, is the treatment.
The bioavailability (percent of the amount ingested that is able to be absorbed) of a mineral can be influenced by other minerals. For example, excess Zn decreases the absorption of Cu, and Cu is needed for the absorption of Fe.
B. Iron
The adult body typically contains 3–4 g of Fe. It is a component of many proteins, both catalytic (for example, hydroxylases such as prolyl hydroxylase, see p. 47) and noncatalytic. Iron can be linked to sulfur (S) as seen in the Fe–S proteins of the electron transport chain (see p. 75), or it can be part of the heme prosthetic group (see p. 25) in proteins such as hemoglobin (~70% of all Fe), myoglobin, and the cytochromes. [Note: Free ionic Fe is toxic because it can cause production of the hydroxyl radical, a reactive oxygen species (ROS).] Dietary Fe is available as Fe2+ in heme (animal sources) and Fe3+ in nonheme sources (plants). Heme iron is less abundant, but it is better absorbed. Meat, poultry, some shellfish, ready-toeat cereals, lentils, and molasses are good dietary sources of Fe. About 10% of ingested Fe is absorbed. This amount, ~1−2 mg/day, is sufficient to replace Fe lost from the body primarily by the sloughing of cells.
1.
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Absorption, storage, and transport: Intestinal uptake of heme is by a heme carrier protein (Fig. 29.8). Within the enterocytes, heme oxygenase releases Fe2+ from heme (see p. 282). Nonheme Fe is taken up via the apical membrane protein divalent metal ion transporter-1 (DMT-1). [Note: Vitamin C enhances absorption of nonheme Fe because it is the coenzyme for duodenal cytochrome b (Dcytb), a ferrireductase that reduces Fe3+ to Fe2+.] Absorbed Fe2+ from heme and nonheme sources has two possible fates: It can be 1) oxidized to Fe3+ and stored by the intracellular protein ferritin (up to 4,500 Fe3+/ferritin) or 2) transported out of the enterocyte by the basolateral membrane protein ferroportin, oxidized by the Cu-containing membrane protein hephaestin, and taken up by the plasma transport protein transferrin (2 Fe3+/transferrin), as shown in Figure 29.8. [Note: Cells other than enterocytes use the Cu-containing plasma protein ceruloplasmin in place of hephaestin.] In normal individuals, transferrin (Tf) is about one third saturated with Fe3+ . Ferroportin, the only known exporter of Fe from cells to the blood in humans, is regulated by the hepatic peptide hepcidin that induces internalization and lysosomal degradation of ferroportin. Therefore, hepcidin is the central molecule in Fe homeostasis. [Note: Transcription of hepcidin is suppressed when Fe is deficient.] 2.
Recycling: Macrophages phagocytose old and/or damaged red blood cells (RBC), freeing heme Fe that is sent out of the cells via ferroportin, oxidized by ceruloplasmin, and transported by Tf as described above. This recycled Fe meets ~90% of our daily need, which is predominantly for erythropoiesis.
3.
Uptake: Tf-bound Fe3+ from enterocytes and macrophages binds to receptors (TfR) on erythroblasts and other Fe-requiring cells and is taken up by receptor-mediated endocytosis. The Fe3+ is released from Tf for use (or stored on ferritin), and the TfR (and Tf) is recycled in a process similar to the receptor-mediated endocytosis seen with low-density lipoprotein particles (see p. 231). [Note: Regulation of the translation of the messenger RNA for ferritin and the TfR by iron regulatory proteins and iron-responsive elements is discussed on p. 474.] 4.
Deficiency: Fe deficiency can result in a microcytic, hypochromic anemia (Fig. 29.9), the most common anemia in the United States, as a result of decreased hemoglobin synthesis and, consequently, decreased
RBC size. Treatment is the administration of Fe.
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5. Excess: Fe overload can occur with accidental ingestion. [Note: Acute Fe poisoning is the most common cause of poisoning deaths of children age <6 years (UL = 40 mg/day for children, 45 mg/day for adults).] Treatment is use of an Fe chelator. Overload can also occur with genetic defects. An example is hereditary hemochromatosis (HH), an AR disorder of Fe overload found primarily in those of Northern European ancestry. It is most commonly caused by mutations to the HFE (high iron) gene. Hyperpigmentation with hyperglycemia (“bronze diabetes”) and damage to the liver (a major storage site for Fe), pancreas, and heart may be seen. In HH, serum Fe and Tf saturation are elevated. Treatment is phlebotomy or use of Fe chelators. [Note: Fe overload is seen with mutations to proteins of Fe metabolism that result in inappropriately low levels of hepcidin. It can result in hemosiderosis (the deposition of hemosiderin, an intracellular, insoluble storage form of Fe).]
C. Manganese
Mn is important for the function of several enzymes (Fig. 29.10). Whole grains, legumes (for example, beans and peas), nuts, and tea (especially green tea) are good sources of the mineral. Consequently, Mn deficiency in humans is rare. Toxicity from foods and/or supplements is also rare (UL = 11 mg/day for adults).
D. Zinc
Zn plays important structural and catalytic functions in the body. Zinc fingers are supersecondary structures (motifs, see p. 18) in proteins (for example, transcription factors) that bind to DNA and regulate gene expression (Fig. 29.11). Hundreds of enzymes require Zn for activity. Examples include alcohol dehydrogenase, which oxidizes ethanol to acetaldehyde (see p. 317); carbonic anhydrase, which is important in the bicarbonate buffer system (see p. 30); porphobilinogen synthase of heme synthesis, which is inhibited by lead (lead replaces the zinc; see p. 279); and the nonmitochondrial isoform of superoxide dismutase (SOD), which also requires Cu (see Fig. 29.5). Dietary sources of Zn include meat, fish, eggs, and dairy products. Phytates (phosphate storage molecules in some plant products) irreversibly bind Zn in the intestine, decreasing its absorption, and can result in a deficiency. [Note: Phytates may also bind Ca2+ and nonheme Fe.] Several drugs (for example, penicillamine) chelate metals, and their use may cause Zn deficiency. [Note: Severe deficiency is seen with a defect in the intestinal transporter for Zn that results in the malabsorption disorder acrodermatitis enteropathica. Symptoms include rashes, slowed growth and development, diarrhea, and immune deficiencies. Vision problems may also occur because Zn is needed in the metabolism of vitamin A.]
Eukaryotic cells infected with bacteria can restrict availability of the essential micronutrients Fe, Mn, and Zn to the pathogens. This decreases the intracellular survival of the pathogen and is known as “nutritional immunity.”
E. Other microminerals
Chromium (Cr) and fluorine (F) also play roles in the body. Cr potentiates the action of insulin by an unknown mechanism. It is found in fruits, vegetables, dairy products, and meat. F (as fluoride [F−]) is added to water in many parts of the world to reduce the incidence of dental caries (Fig. 29.12). F− replaces the hydroxyl group of hydroxylapatite, forming fluoroapatite that is more resistant to the enamel-dissolving acid produced by mouth bacteria.
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IV. ULTRATRACE MINERALS
The ultratrace minerals include iodine (I), selenium (Se), and molybdenum (Mo). They are required by adults in amounts <1 mg/day.
A. Iodine
I is utilized in the synthesis of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) that are required for development, growth, and metabolism. Circulating iodide (I−) is taken up (“trapped”) and concentrated in the epithelial follicular cells of the thyroid gland. It then is sent into the colloid of the follicular lumen where it is oxidized to iodine (I2) by thyroperoxidase (TPO), as shown in Figure 29.13. TPO then uses I2 to iodinate selected tyrosine residues in thyroglobulin (Tg), forming monoiodinated tyrosine (MIT) and diiodinated tyrosine (DIT), as shown in Figure 29.14. [Note: Tg is synthesized and secreted into colloid by follicular cells.] The coupling of two DIT on Tg gives T4, whereas coupling one MIT and one DIT gives T3. The iodinated Tg is endocytosed and stored in follicular cells until needed, at which time it is proteolytically digested to release T3 and T4, which are secreted into the circulation (see Fig. 29.13).
Under normal conditions, ~90% of secreted thyroid hormone is T4 that is carried by transthyretin. In target tissues (for example, the liver and developing brain), T4 is converted to T3 (the more active form) by Se-containing deiodinases. T3 binds to a nuclear receptor that binds DNA at thyroid response elements and functions as a transcription factor. [Note:
Thyroid hormone production is controlled by thyrotropin (thyroid-stimulating hormone ([TSH]) from the anterior pituitary. TSH secretion is itself controlled by thyrotropin-releasing hormone (TRH) from the hypothalamus.] 1.
Hypothyroidism: Underingestion of I can result in goiter (enlargement of the thyroid in response to excessive stimulation by TSH), as shown in Figure 29.15. More severe deficiency results in hypothyroidism that is characterized by fatigue, weight gain, decreased thermogenesis, and decreased metabolic rate (see p. 359). If hormone deficiency occurs during fetal and infant development (congenital hypothyroidism), irreversible intellectual disability (formerly called “cretinism”), hearing loss, spasticity, and short stature can result. In the United States, dairy products, seafood, and meat are the primary sources of I. The use of iodized salt has greatly reduced dietary I deficiency. [Note: Autoimmune destruction of TPO is a cause of Hashimoto thyroiditis (a primary hypothyroidism).] 2.
Hyperthyroidism: This condition is the result of overproduction of thyroid hormone. Although it can be caused by overingestion of I-containing supplements (UL = 1.1 g/day for adults), the most common cause of hyperthyroidism is Graves disease, in which an antibody that mimics the effect of TSH is produced, resulting in dysregulated production of thyroid hormone. This can cause nervousness, weight loss, increased perspiration and heart rate, protruding eyes (exophthalmos, Fig. 29.16), and goiter.
B. Selenium
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Se is present in ~25 human proteins (selenoproteins) as a constituent of the amino acid selenocysteine, which is derived from serine (see p. 268). Selenoproteins include glutathione peroxidase that oxidizes glutathione in the reduction of hydrogen peroxide, a ROS, to water (see p. 148); thioredoxin reductase that reduces thioredoxin, a coenzyme of ribonucleotide reductase (see p. 297); and deiodinases that remove I from thyroid hormones. Meat, dairy products, and grains are important dietary sources. Keshan disease, first identified in China, is a cardiomyopathy caused by eating foods produced from Se-deficient soil. Toxicity (selenosis) caused by overingestion of supplements causes brittle nails and hair. Cutaneous and neurologic effects may also be seen (UL = 400 µg in adults).
C. Molybdenum
Mo functions as a cofactor for a small number of mammalian oxidases (Fig. 29.17). Legumes are important dietary sources. No dietary deficiency syndromes are known. Mo has low toxicity in humans (UL = 2 mg/day in adults).
Cobalt (Co), an ultratrace mineral, is a component of vitamin B12 (cobalamin, see p. 379), which is required as methylcobalamin in the remethylation of homocysteine to methionine (see p. 264) or adenosylcobalamin in the isomerization of methylmalonyl coenzyme A (CoA) to succinyl CoA (see p. 194). No Recommended Dietary Allowance or Daily Reference Intake (see p. 358) has been established for Co.
V. CHAPTER SUMMARY
The minerals are summarized in Figure 29.18 on p. 408.
For Questions 29.1–29.7, match the mineral to the most appropriate description.
A. Calcium
B. Chloride
C. Copper
D. Iodine
E. Iron
F. Magnesium
G. Manganese
H. Molybdenum
I. Phosphorus
J. Potassium
K. Selenium
L. Sodium
M. Zinc 9.1. Elevated levels of which mineral may result in hypertension in certain populations?
9.2. Which mineral is the major extracellular anion?
9.3. A decrease of which mineral is seen in refeeding syndrome and with overuse of aluminum-containing antacids?
9.4. Which mineral is a constituent of some amino acids found in proteins involved in antioxidant defense, thyroid hormone metabolism, and redox reactions?
9.5. Which mineral is required for the formation of a supersecondary protein structure that allows binding to DNA? (Its deficiency can result in a dermatitis.) 9.6. Deficiency of which mineral can cause bone pain, tetany (intermittent muscle spasms), paresthesia (a “pins and needles” sensation), and an increased tendency to bleed?
9.7. Deficiency of which mineral can result in goiter and a decreased metabolic rate?
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Correct answers = L, B, I, K, M, A, D. Hypernatremia (elevation of serum sodium) can lead to water retention that can cause hypertension in salt-sensitive populations (for example, African Americans). Chloride is the major extracellular anion. [Note: Sodium is the major extracellular cation, potassium is the major intracellular cation, and phosphate is the major intracellular anion. The concentration differential across the membrane is maintained by active transport.] Carbohydrate metabolism involves the generation of phosphorylated intermediates. Refeeding severely malnourished individuals traps phosphate and results in hypophosphatemia. Muscle weakness is a common symptom. Selenocysteine, an amino acid formed from serine and selenium, is found in proteins (selenoproteins) such as glutathione peroxidase, deiodinases, and thioredoxin reductase. Zinc fingers are a type of structural motif found in proteins (for example, transcription factors) that bind to DNA. Severe deficiency of zinc as a result of mutations to its intestinal transporter can result in acrodermatitis enteropathica, which is characterized by dermatitis, diarrhea, and alopecia. Calcium is required for bone mineralization, muscle contraction, nerve conduction, and blood clotting. Its deficiency will affect all of these processes. Thyroid hormones are iodinated tyrosines released by proteolytic digestion of thyroglobulin. Underingestion of iodine causes enlargement of the thyroid in an attempt to increase hormone synthesis. [Note: Goiter can also result if too much hormone is made, as in Graves disease, or if too little is made, as in Hashimoto disease. Both are autoimmune diseases.] Thyroid hormone increases the resting metabolic rate.
9.8. DiGeorge syndrome is a congenital condition that results in structural anomalies and failure of the thymus and parathyroid glands to develop. Clinical manifestations include recurrent infections as a consequence of a deficiency in T cells. Which one of the following is an expected clinical consequence of the deficiency in parathyroid hormone?
A. Increased bone resorption
B. Increased calcium reabsorption in the kidney
C. Increased serum calcitriol
D. Increased serum phosphate Correct answer = D. Parathyroid hormone (PTH) increases bone resorption (demineralization) resulting in the release of calcium and phosphate. It also increases the renal reabsorption of calcium, because PTH activates the renal hydroxylase that converts calcidiol to calcitriol. PTH also increases the renal excretion of phosphate. With the hypoparathyroidism of DiGeorge syndrome, all of these activities of PTH are impaired. Consequently, hypocalcemia and hyperphosphatemia are seen.
For questions 29.9 and 29.10, match the signs and symptoms to the pathology.
A. Graves disease
B. Hereditary hemochromatosis
C. Hypercalcemia
D. Hyperphosphatemia
E. Keshan disease
F. Menkes syndrome
G. Selenosis
H. Wilson disease 9.9. A 28-year-old male is seen for complaints of recent, severe, upper-rightquadrant pain. He also reports some difficulty with fine motor tasks. No jaundice is observed on physical examination. Laboratory tests were remarkable for elevated liver function tests (serum aspartate and alanine aminotransferases) and elevated urinary calcium and phosphate. Ophthalmology consult revealed Kayser-Fleischer rings in the cornea. The patient was started on penicillamine and zinc.
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Correct answer = H. The patient has Wilson disease, an autosomal-recessive disorder that decreases copper efflux from the liver because of mutations to the hepatic copper transport protein ATP7B. Some copper leaks into the blood and is deposited in the brain, eyes, kidney, and skin. This results in liver and kidney damage, neurologic effects, and corneal changes caused by the excess copper. Administration of the metal chelator penicillamine is the treatment. [Note: Because zinc is also chelated, supplementation with zinc is common.] Graves disease results in hyperthyroidism. Hereditary hemochromatosis is a disorder of iron overload. Keshan disease is the result of selenium deficiency, whereas selenosis is caused by selenium excess. Menkes syndrome is the result of a systemic deficiency in copper as a result of mutations to ATP7A, an intestinal copper transport protein.
9.10. A 52-year-old female is seen because of unplanned changes in the pigmentation of her skin that give her a tanned appearance. Physical examination shows hyperpigmentation, hepatomegaly, and mild scleral icterus. Laboratory tests are remarkable for elevated serum transaminases (liver function tests) and fasting blood glucose. Results of other tests are pending.
Correct answer = B. The patient has hereditary hemochromatosis, a disease of iron overload that results from inappropriately low levels of hepcidin caused primarily by mutations to the HFE (high iron) gene. Hepcidin regulates ferroportin, the only known iron export protein in humans, by increasing its degradation. The increase in iron with hepcidin deficiency causes hyperpigmentation and hyperglycemia (“bronze diabetes”). Phlebotomy or use of iron chelators is the treatment. [Note: Pending lab tests would show an increase in serum iron and transferrin saturation.]
UNIT VII Storage and Expression of Genetic Information
DNA Structure, Replication, and Repair
For additional ancillary materials related to this chapter, please visit thePoint.
I. OVERVIEW
Nucleic acids are required for the storage and expression of genetic information. There are two chemically distinct types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid ([RNA] see Chapter 31). DNA, the repository of genetic information (or, genome), is present not only in chromosomes in the nucleus of eukaryotic organisms, but also in mitochondria and the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have a single chromosome but may also contain nonchromosomal DNA in the form of plasmids. The genetic information found in DNA is copied and transmitted to daughter cells through DNA replication. The DNA contained in a fertilized egg encodes the information that directs the development of an organism. This development may involve the production of billions of cells. Each cell is specialized, expressing only those functions that are required for it to perform its role in maintaining the organism. Therefore, DNA must be able not only to replicate precisely each time a cell divides, but also to have the information that it contains be selectively expressed. Transcription (RNA synthesis) is the first stage in the expression of genetic information (see Chapter 31). Next, the code contained in the nucleotide sequence of messenger RNA molecules is translated (protein synthesis; see Chapter 32), thus completing gene expression. The regulation of gene expression is discussed in Chapter 33.
The flow of information from DNA to RNA to protein is termed the “central dogma” of molecular biology (Fig. 30.1) and is descriptive of all organisms, with the exception of some viruses that have RNA as the repository of their genetic information.
II. DNA STRUCTURE
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DNA is a polymer of deoxyribonucleoside monophosphates (dNMP) covalently linked by 3′→5′-phosphodiester bonds. With the exception of a few viruses that contain single-stranded DNA (ssDNA), DNA exists as a double-stranded molecule (dsDNA), in which the two strands wind around each other, forming a double helix. [Note: The sequence of the linked dNMP is primary structure, whereas the double helix is secondary structure.] In eukaryotic cells, DNA is found associated with various types of proteins (known collectively as nucleoprotein) present in the nucleus, whereas the protein–DNA complex is present in a non–membrane-bound region known as the nucleoid in prokaryotes.
A. 3′→5′-Phosphodiester bonds
Phosphodiester bonds join the 3′-hydroxyl group of the deoxypentose of one nucleotide to the 5′-hydroxyl group of the deoxypentose of an adjacent nucleotide through a phosphoryl group (Fig. 30.2). The resulting long, unbranched chain has polarity, with both a 5′-end (the end with the free phosphate) and a 3′-end (the end with the free hydroxyl) that are not attached to other nucleotides. By convention, the bases located along the resulting deoxyribose-phosphate backbone are always written in sequence from the 5′-end of the chain to the 3′-end. For example, the sequence of bases in the DNA shown in Figure 30.2D (5′-TACG-3′) is read “thymine, adenine, cytosine, guanine.” Phosphodiester linkages between nucleotides can be hydrolyzed enzymatically by a family of nucleases, deoxyribonucleases for DNA and ribonucleases for RNA, or cleaved hydrolytically by chemicals. [Note: Only RNA is cleaved by alkali.]
B. Double helix
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In the double helix, the two chains are coiled around a common axis called the helical axis. The chains are paired in an antiparallel manner (that is, the 5′-end of one strand is paired with the 3′-end of the other strand), as shown in Figure 30.3. In the DNA helix, the hydrophilic deoxyribose-phosphate backbone of each chain is on the outside of the molecule, whereas the hydrophobic bases are stacked inside. The overall structure resembles a twisted ladder. The spatial relationship between the two strands in the helix creates a major (wide) groove and a minor (narrow) groove. These grooves provide access for the binding of regulatory proteins to their specific recognition sequences along the DNA chain. [Note: Certain anticancer drugs, such as dactinomycin (actinomycin D), exert their cytotoxic effect by intercalating into the narrow groove of the DNA double helix, thereby interfering with DNA (and RNA) synthesis.] 1. Base-pairing: The bases of one strand of DNA are paired with the bases of the second strand, so that an adenine (A) is always paired with a thymine (T), and a cytosine (C) is always paired with a guanine (G). [Note: The base pairs are perpendicular to the helical axis (see Fig. 30.3).] Therefore, one polynucleotide chain of the DNA double helix is always the complement of the other. Given the sequence of bases on one chain, the sequence of bases on the complementary chain can be determined (Fig. 30.4). [Note: The specific base-pairing in DNA leads to the Chargaff rule, which states that in any sample of dsDNA, the amount of A equals the amount of T, the amount of G equals the amount of C, and the total amount of purines (A + G) equals the total amount of pyrimidines (T + C).] The base pairs are held together by hydrogen bonds: two between A and T and three between G and C (Fig. 30.5). These hydrogen bonds, plus the hydrophobic interactions between the stacked bases, stabilize the structure of the double helix.
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2. DNA strand separation: The two strands of the double helix separate when hydrogen bonds between the paired bases are disrupted. Disruption can occur in the laboratory if the pH of the DNA solution is altered so that the nucleotide bases ionize, or if the solution is heated. [Note: Covalent phosphodiester bonds are not broken by such treatment.] When DNA is heated, the temperature at which one half of the helical structure is lost is defined as the melting temperature (Tm). The loss of helical structure in DNA, called denaturation, can be monitored by measuring its absorbance at 260 nm. [Note: ssDNA has a higher relative absorbance at this wavelength than does dsDNA.] Because there are three hydrogen bonds between G and C but only two between A and T, DNA that contains high concentrations of A and T denatures at a lower temperature than does G-and C-rich DNA (Fig. 30.6). Under appropriate conditions, complementary DNA strands can reform the double helix by the process called renaturation (or, reannealing). [Note: Separation of the two strands over short regions occurs during both DNA and RNA synthesis.] 3. Structural forms: There are three major structural forms of DNA: the B form (described by Watson and Crick in 1953), the A form, and the Z form. The B form is a right-handed helix with 10 base pairs (bp) per 360° turn (or twist) of the helix, and with the planes of the bases perpendicular to the helical axis. Chromosomal DNA is thought to consist primarily of B-DNA (Fig. 30.7 shows a space-filling model of B-DNA). The A form is produced by moderately dehydrating the B form. It is also a right-handed helix, but there are 11 bp per turn, and the planes of the base pairs are tilted 20° away from the perpendicular to the helical axis. The conformation found in DNA–RNA hybrids (see p. 418) or RNA–RNA double-stranded regions is probably very close to the A form. Z-DNA is a left-handed helix that contains 12 bp per turn (see Fig. 30.7). [Note: The deoxyribose-phosphate backbone zigzags, hence, the name Z-DNA.] Stretches of Z-DNA can occur naturally in regions of DNA that have a sequence of alternating purines and pyrimidines (for example, poly GC). Transitions between the B and Z helical forms of DNA may play a role in regulating gene expression.
C. Linear and circular DNA molecules
Each chromosome in the nucleus of a eukaryote consists of one long, linear molecule of dsDNA, which is bound by a complex mixture of proteins (histone and nonhistone, see p. 425) to form chromatin. Eukaryotes have closed, circular, dsDNA molecules in their mitochondria, as do plant chloroplasts. A prokaryotic organism typically contains a single, circular, dsDNA molecule. [Note: Circular DNA is “supercoiled,” that is, the double helix crosses over on itself one or more times. Supercoiling can result in overwinding (positive supercoiling) or underwinding (negative supercoiling) of DNA. Supercoiling, a type of tertiary structure, compacts DNA.] Each prokaryotic chromosome is associated with nonhistone proteins that help compact the DNA to form a nucleoid. In addition, most species of bacteria also contain small, circular, extrachromosomal DNA molecules called plasmids. Plasmid DNA carries genetic information and undergoes replication that may or may not be synchronized to chromosomal division. [Note: The use of plasmids as vectors in recombinant DNA technology is described in Chapter 34.]
Plasmids may carry genes that convey antibiotic resistance to the host bacterium and may facilitate the transfer of genetic information from one bacterium to another.
III. STEPS IN PROKARYOTIC DNA REPLICATION
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When the two strands of dsDNA are separated, each can serve as a template for the replication (synthesis) of a new complementary strand. This produces two daughter molecules, each of which contains two DNA strands (one old, one new) in an antiparallel orientation (see Fig. 30.3). This process is called semiconservative replication because, although the parental duplex is separated into two halves (and, therefore, is not conserved as an entity), each of the parental strands remains intact in one of the two new duplexes (Fig. 30.8). The enzymes involved in DNA replication are template-directed, magnesium (Mg2+)requiring polymerases that can synthesize the complementary sequence of each strand with extraordinary fidelity. The reactions described in this section were first known from studies of the bacterium Escherichia coli (E. coli), and the description given below refers to the process in prokaryotes. DNA synthesis in higher organisms is more complex but involves the same types of mechanisms. In either case, initiation of DNA replication commits the cell to continue the process until the entire genome has been replicated.
A. Complementary strand separation
In order for the two complementary strands of the parental dsDNA to be replicated, they must first separate (or “melt”) over a small region, because the polymerases use only ssDNA as a template. In prokaryotic organisms, DNA replication begins at a single, unique nucleotide sequence, a site called the origin of replication, or ori (oriC in E. coli), as shown in Figure 30.9A. [Note: This sequence is referred to as a consensus sequence, because the order of nucleotides is essentially the same at each site.] The ori includes short, AT-rich segments that facilitate melting. In eukaryotes, replication begins at multiple sites along the DNA helix (Fig. 30.9B). Having multiple origins of replication provides a mechanism for rapidly replicating the great length of eukaryotic DNA molecules.
B. Replication fork formation
As the two strands unwind and separate, synthesis occurs at two replication forks that move away from the origin in opposite directions (bidirectionally), generating a replication bubble (see Fig. 30.9). [Note: The term “replication fork” derives from the Y-shaped structure in which the tines of the fork represent the separated strands (Fig. 30.10).] 1. Required proteins: Initiation of DNA replication requires the recognition of the origin (start site) by a group of proteins that form the prepriming complex. These proteins are responsible for melting at the ori, maintaining the separation of the parental strands, and unwinding the double helix ahead of the advancing replication fork. In E. coli, these proteins include the following.
a.
DnaA protein: DnaA protein initiates replication by binding to specific nucleotide sequences (DnaA boxes) within oriC. Binding causes an AT-rich region (the DNA unwinding element) in the origin to melt. Melting (strand separation) results in a short, localized region of ssDNA.
b.
DNA helicases: These enzymes bind to ssDNA near the replication fork and then move into the neighboring double-stranded region, forcing the strands apart (in effect, unwinding the double helix). Helicases require energy provided by ATP hydrolysis (see Fig. 30.10). Unwinding at the replication fork causes supercoiling in other regions of the DNA molecule. [Note: DnaB is the principal helicase of replication in E. coli. Binding of this hexameric protein to DNA requires DnaC.] c.
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Single-stranded DNA–binding protein: This protein binds to the ssDNA generated by helicases (see Fig. 30.10). Binding is cooperative (that is, the binding of one molecule of single-stranded binding [SSB] protein makes it easier for additional molecules of SSB protein to bind tightly to the DNA strand). The SSB proteins are not enzymes, but rather serve to shift the equilibrium between dsDNA and ssDNA in the direction of the single-stranded forms. These proteins not only keep the two strands of DNA separated in the area of the replication origin, thus providing the single-stranded template required by polymerases, but also protect the DNA from nucleases that degrade ssDNA.
2. Solving the problem of supercoils: As the two strands of the double helix are separated, a problem is encountered, namely, the appearance of positive supercoils in the region of DNA ahead of the replication fork as a result of overwinding (Fig. 30.11) and negative supercoils in the region behind the fork. The accumulating positive supercoils interfere with further unwinding of the double helix. [Note: Supercoiling can be demonstrated by tightly grasping one end of a helical telephone cord while twisting the other end. If the cord is twisted in the direction of tightening the coils, the cord will wrap around itself in space to form positive supercoils. If the cord is twisted in the direction of loosening the coils, the cord will wrap around itself in the opposite direction to form negative supercoils.] To solve this problem, there is a group of enzymes called DNA topoisomerases, which are responsible for removing supercoils in the helix by transiently cleaving one or both of the DNA strands.
a.
Type I DNA topoisomerases: These enzymes reversibly cleave one strand of the double helix. They have both strand-cutting and strand-resealing activities. They do not require ATP, but rather appear to store the energy from the phosphodiester bond they cleave, reusing the energy to reseal the strand (Fig. 30.12). Each time a transient nick is created in one DNA strand, the intact DNA strand is passed through the break before it is resealed, thus relieving (relaxing) accumulated supercoils. Type I topoisomerases relax negative supercoils (that is, those that contain fewer turns of the helix than does relaxed DNA) in
E. coli and both negative and positive supercoils (that is, those that contain fewer or more turns of the helix than does relaxed DNA) in many prokaryotic cells (but not E. coli) and in eukaryotic cells.
b.
Type II DNA topoisomerases: These enzymes bind tightly to the DNA double helix and make transient breaks in both strands. The enzyme then causes a second stretch of the DNA double helix to pass through the break and, finally, reseals the break (Fig. 30.13). As a result, both negative and positive supercoils can be relieved by this ATP-requiring process. DNA gyrase, a type II topoisomerase found in bacteria and plants, has the unusual property of being able to introduce negative supercoils into circular DNA using energy from the hydrolysis of ATP. This facilitates the replication of DNA because the negative supercoils neutralize the positive supercoils introduced during opening of the double helix. It also aids in the transient strand separation required during transcription (see p. 436).
Anticancer agents, such as the camptothecins, target human type I topoisomerases, whereas etoposide targets human type II topoisomerases. Bacterial DNA gyrase is a unique target of a group of antimicrobial agents called fluoroquinolones (for example, ciprofloxacin).
C. Direction of DNA replication
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The DNA polymerases (DNA pols) responsible for copying the DNA templates are only able to read the parental nucleotide sequences in the 3′→5′ direction, and they synthesize the new DNA strands only in the 5′→3′ (antiparallel) direction. Therefore, beginning with one parental double helix, the two newly synthesized stretches of nucleotide chains must grow in opposite directions, one in the 5′→3′ direction toward the replication fork and one in the 5′→3′ direction away from the replication fork (Fig. 30.14). This feat is accomplished by a slightly different mechanism on each strand.
1.
Leading strand: The strand that is being copied in the direction of the advancing replication fork is synthesized continuously and is called the leading strand.
2.
Lagging strand: The strand that is being copied in the direction away from the replication fork is synthesized discontinuously, with small fragments of DNA being copied near the replication fork. These short stretches of discontinuous DNA, termed Okazaki fragments, are eventually joined (ligated) by ligase to become a single, continuous strand. The new strand of DNA produced by this mechanism is termed the lagging strand.
D. RNA primer
DNA pols cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. Rather, they require an RNA primer, which is a short piece of RNA base-paired to the DNA template, thereby forming a double-stranded DNA–RNA hybrid. The free hydroxyl group on the 3′end of the RNA primer serves as the first acceptor of a deoxynucleotide by action of a DNA pol (Fig. 30.15). [Note: Recall that glycogen synthase also requires a primer (see p. 126).] 1.
Primase: A specific RNA polymerase, called primase (DnaG), synthesizes the short stretches of RNA (~10 nucleotides long) that are complementary and antiparallel to the DNA template. In the resulting hybrid duplex, the U (uracil) in RNA pairs with A in DNA. As shown in Figure 30.16, these short RNA sequences are constantly being synthesized at the replication fork on the lagging strand, but only one RNA sequence at the origin of replication is required on the leading strand. The substrates for this process are 5′-ribonucleoside triphosphates, and pyrophosphate is released as each ribonucleoside monophosphate is added through formation of a 3′→5′-phosphodiester bond. [Note: The RNA primer is later removed, as described in F. below.] 2.
Primosome: The addition of primase converts the prepriming complex of proteins required for DNA strand separation (see p. 415) to a primosome. The primosome makes the RNA primer required for leading-strand synthesis and initiates Okazaki fragment formation in discontinuous lagging-strand synthesis. As with DNA synthesis, the direction of synthesis of the primer is 5′→3′.
E. Chain elongation
Prokaryotic (and eukaryotic) DNA pols elongate a new DNA strand by adding deoxyribonucleotides, one at a time, to the 3′-end of the growing chain (see Fig. 30.16). The sequence of nucleotides that are added is dictated by the base sequence of the template strand with which the incoming nucleotides are paired.
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1. DNA polymerase III: DNA chain elongation is catalyzed by the multisubunit enzyme, DNA pol III. Using the 3′-hydroxyl group of the RNA primer as the acceptor of the first deoxyribonucleotide, DNA pol III begins to add nucleotides along the single-stranded template that specifies the sequence of bases in the newly synthesized chain. DNA pol III is a highly processive enzyme (that is, it remains bound to the template strand as it moves along and does not diffuse away and then rebind before adding each new nucleotide). The processivity of DNA pol III is the result of the β subunits of the holoenzyme forming a ring that encircles and moves along the template strand of the DNA, thus serving as a sliding DNA clamp. [Note: Clamp formation is facilitated by a protein complex, the clamp loader, and ATP hydrolysis.] The new (daughter) strand grows in the 5′→3′ direction, antiparallel to the parental strand (see Fig. 30.16). The nucleotide substrates are 5′deoxyribonucleoside triphosphates. Pyrophosphate (PPi) is released when each new deoxynucleoside monophosphate is added to the free 3′hydroxyl group of the growing chain through a 3′→5′-phosphodiester bond (see Fig. 30.15). Hydrolysis of PPi to 2 Pi by pyrophosphatase means that a total of two high-energy bonds are used to drive the addition of each deoxynucleotide.
The production of PPi with subsequent hydrolysis to 2 Pi is a common theme in biochemistry. Removal of the PPi product drives a reaction in the forward direction, making it essentially irreversible.
All four substrates (deoxyadenosine triphosphate [dATP], deoxythymidine triphosphate [dTTP], deoxycytidine triphosphate [dCTP], and deoxyguanosine triphosphate [dGTP]) must be present for DNA elongation to occur. If one of the four is in short supply, DNA synthesis stops when that nucleotide is depleted.
2. Proofreading newly synthesized DNA: It is highly important for the survival of an organism that the nucleotide sequence of DNA be replicated with as few errors as possible. Misreading of the template sequence could result in deleterious, perhaps lethal, mutations. To insure replication fidelity, DNA pol III has a proofreading activity (3′→5′ exonuclease, Fig. 30.17) in addition to its 5′→3′ polymerase activity. As each nucleotide is added to the chain, DNA pol III checks to make certain the base of the newly added nucleotide is, in fact, the complement of the base on the template strand. If it is not, the 3′→5′ exonuclease activity removes the error in the direction opposite to polymerization. [Note: Because the enzyme requires an improperly base-paired 3′hydroxy terminus, it does not degrade correctly paired nucleotide sequences.] For example, if the template base is C and the enzyme inserts an A instead of a G into the new chain, the 3′→5′ exonuclease activity hydrolytically removes the misplaced nucleotide. The 5′→3′ polymerase activity then replaces it with the correct nucleotide containing G (see Fig. 30.17). [Note: The 5′→3′ polymerase and 3′→5′ exonuclease domains are located on different subunits of DNA pol III.]
F. RNA primer excision and replacement by DNA
DNA pol III continues to synthesize DNA on the lagging strand until it is blocked by proximity to an RNA primer. When this occurs, the RNA is excised and the gap filled by DNA pol I.
1.
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5′→3′ Exonuclease activity: In addition to having the 5′→3′ polymerase activity that synthesizes DNA and the 3′→5′ exonuclease activity that proofreads the newly synthesized DNA like DNA pol III, monomeric DNA pol I also has a 5′→3′ exonuclease activity that is able to hydrolytically remove the RNA primer. [Note: Exonucleases remove nucleotides from the end of the DNA chain, rather than cleaving the chain internally as do endonucleases (Fig. 30.18).] First, DNA pol I locates the space (nick) between the 3′-end of the DNA newly synthesized by DNA pol III and the 5′-end of the adjacent RNA primer. Next, DNA pol I hydrolytically removes the RNA nucleotides ahead of itself, moving in the 5′→3′ direction (5′→3′ exonuclease activity). As it removes ribonucleotides, DNA pol I replaces them with deoxyribonucleotides, synthesizing DNA in the 5′→3′ direction (5′→3′ polymerase activity). As it synthesizes the DNA, it also proofreads using its 3′→5′ exonuclease activity to remove errors. This removal/synthesis/proofreading continues until the RNA primer is totally degraded, and the gap is filled with DNA (Fig. 30.19). [Note: DNA pol I uses its 5′→3′ polymerase activity to fill in gaps generated during most types of DNA repair (see p. 428).] 2.
Comparison of 5′→3′ and 3′→5′ exonuclease activities: The 5′→3′ exonuclease activity of DNA pol I allows the polymerase, moving 5′→3′, to hydrolytically remove one or more nucleotides at a time from the 5′-end of the ~10 nucleotide–long RNA primer. In contrast, the 3′→5′ exonuclease activity of DNA pol I and pol III allows these polymerases, moving 3′→5′, to hydrolytically remove one misplaced nucleotide at a time from the 3′-end of a growing DNA strand, increasing the fidelity of replication such that newly replicated DNA has one error per 107 nucleotides.
G. DNA ligase
The final phosphodiester linkage between the 5′-phosphate group on the DNA synthesized by DNA pol III and the 3′-hydroxyl group on the DNA made by DNA pol I is catalyzed by DNA ligase (Fig. 30.20). The joining of these two stretches of DNA requires energy, which in most organisms is provided by the cleavage of ATP to adenosine monophosphate + PPi.
H. Termination
Replication termination in E. coli is mediated by sequence-specific binding of the protein Tus (terminus utilization substance) to replication termination (ter) sites on the DNA, stopping the movement of the replication fork.
IV. EUKARYOTIC DNA REPLICATION
The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been noted. Eukaryotic origin recognition proteins, ssDNA-binding proteins, and ATP-dependent DNA helicases have been identified, and their functions are analogous to those of the prokaryotic proteins previously discussed. In contrast, RNA primers are removed by RNase H and flap endonuclease 1 (FEN1) rather than by a DNA pol (Fig. 30.21).
A. Eukaryotic cell cycle
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The events surrounding eukaryotic DNA replication and cell division (mitosis) are coordinated to produce the cell cycle (Fig. 30.22). The period preceding replication is called the G1 phase (Gap 1). DNA replication occurs during the S (synthesis) phase. Following DNA synthesis, there is another phase (G2, or Gap 2) before mitosis (M). Cells that have stopped dividing, such as mature T lymphocytes, are said to have gone out of the cell cycle into the G0 phase. Such quiescent cells can be stimulated to reenter the G1 phase to resume division. [Note: The cell cycle is controlled at a series of checkpoints that prevent entry into the next phase of the cycle until the preceding phase has been completed. Two key classes of proteins that control the progress of a cell through the cell cycle are the cyclins and cyclin-dependent kinases (Cdk).]
B. Eukaryotic DNA polymerases
At least five high-fidelity eukaryotic DNA pols have been identified and categorized on the basis of molecular weight, cellular location, sensitivity to inhibitors, and the templates or substrates on which they act. They are designated by Greek letters rather than by Roman numerals (Fig. 30.23).
1.
Pol α: Pol α is a multisubunit enzyme. One subunit has primase activity, which initiates strand synthesis on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. The primase subunit synthesizes a short RNA primer that is extended by the 5′→3′ polymerase activity of pol α, generating a short piece of DNA. [Note: Pol α is also referred to as pol α/primase.] 2.
Pol ε and pol δ: Pol ε is recruited to complete DNA synthesis on the leading strand, whereas pol δ elongates the Okazaki fragments of the lagging strand, each using 3′→5′ exonuclease activity to proofread the newly synthesized DNA. [Note: DNA pol ε associates with proliferating cell nuclear antigen (PCNA), a protein that serves as a sliding DNA clamp in much the same way the β subunits of DNA pol III do in E. coli, thus insuring high processivity.] 3.
Pol β and pol γ: Pol β is involved in gap filling in DNA repair. Pol γ replicates mitochondrial DNA.
C. Telomeres
Telomeres are complexes of DNA plus proteins (collectively known as shelterin) located at the ends of linear chromosomes. They maintain the structural integrity of the chromosome, preventing attack by nucleases, and allow repair systems to distinguish a true end from a break in dsDNA. In humans, telomeric DNA consists of several thousand tandem repeats of a noncoding hexameric sequence, AGGGTT, base-paired to a complementary region containing C and A. The G-rich strand is longer than its C-rich complement, leaving ssDNA a few hundred nucleotides in length at the 3′end. The single-stranded region is thought to fold back on itself, forming a loop structure that is stabilized by protein.
1.
Telomere shortening: Eukaryotic cells face a special problem in replicating the ends of their linear DNA molecules. Following removal of the RNA primer from the extreme 5′-end of the lagging strand, there is no way to fill in the remaining gap with DNA. Consequently, in most normal human somatic cells, telomeres shorten with each successive cell division. Once telomeres are shortened beyond some critical length, the cell is no longer able to divide and is said to be senescent. In germ cells and stem cells, as well as in cancer cells, telomeres do not shorten and the cells do not senesce. This is a result of the ribonucleoprotein telomerase, which maintains telomeric length in these cells.
2.
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Telomerase: This complex contains a protein (Tert) that acts as a reverse transcriptase and a short piece of RNA (Terc) that acts as a template. The C-rich RNA template base-pairs with the G-rich, single-stranded 3′end of telomeric DNA (Fig. 30.24). The reverse transcriptase uses the RNA template to synthesize DNA in the usual 5′→3′ direction, extending the already longer 3′-end. Telomerase then translocates to the newly synthesized end, and the process is repeated. Once the G-rich strand has been lengthened, primase activity of DNA pol α can use it as a template to synthesize an RNA primer. The primer is extended by DNA pol α and then removed by nucleases.
Telomeres may be viewed as mitotic clocks in that their length in most cells is inversely related to the number of times the cells have divided. The study of telomeres provides insight into the biology of normal aging, diseases of premature aging (the progerias), and cancer.
D. Reverse transcriptases
As seen with telomerase, reverse transcriptases are RNA-directed DNA pols. A reverse transcriptase is involved in the replication of retroviruses, such as human immunodeficiency virus (HIV). These viruses carry their genome in the form of ssRNA molecules. Following infection of a host cell, the viral enzyme reverse transcriptase uses the viral RNA as a template for the 5′→3′ synthesis of viral DNA, which then becomes integrated into host chromosomes. Reverse transcriptase activity is also seen with transposons, DNA elements that can move about the genome (see p. 477). In eukaryotes, most transposons are transcribed to RNA, the RNA is used as a template for DNA synthesis by a reverse transcriptase encoded by the transposon, and the DNA is randomly inserted into the genome. [Note: Transposons that involve an RNA intermediate are called retrotransposons or retroposons.]
E. DNA replication inhibition by nucleoside analogs
DNA chain growth can be blocked by the incorporation of certain nucleoside analogs that have been modified on the sugar portion (Fig. 30.25). For example, removal of the hydroxyl group from the 3′-carbon of the deoxyribose ring as in 2′,3′-dideoxyinosine ([ddI] also known as didanosine), or conversion of the deoxyribose to another sugar, such as arabinose, prevents further chain elongation. By blocking DNA replication, these compounds slow the division of rapidly growing cells and viruses. Cytosine arabinoside (cytarabine, or araC) has been used in anticancer chemotherapy, whereas adenine arabinoside (vidarabine, or araA) is an antiviral agent. Substitution on the sugar moiety, as seen in azidothymidine (AZT), also called zidovudine (ZDV), also terminates DNA chain elongation. [Note: These drugs are generally supplied as nucleosides, which are then converted to nucleotides by cellular kinases.]
V. EUKARYOTIC DNA ORGANIZATION
A typical (diploid) human somatic cell contains 46 chromosomes, whose total DNA is ~2 m long! It is difficult to imagine how such a large amount of genetic material can be effectively packaged into a volume the size of a cell nucleus so that it can be efficiently replicated and its genetic information expressed. To do so requires the interaction of DNA with a large number of proteins, each of which performs a specific function in the ordered packaging of these long molecules of DNA. Eukaryotic DNA is associated with tightly bound basic proteins, called histones. These serve to order the DNA into fundamental structural units, called nucleosomes, which resemble beads on a string. Nucleosomes are further arranged into increasingly more complex structures that organize and condense the long DNA molecules into chromosomes that can be segregated during cell division. [Note: The complex of DNA and protein found inside the nuclei of eukaryotic cells is called chromatin.]
A. Histones and nucleosome formation
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There are five classes of histones, designated H1, H2A, H2B, H3, and H4. These small, evolutionally conserved proteins are positively charged at physiologic pH as a result of their high content of lysine and arginine. Because of their positive charge, they form ionic bonds with negatively charged DNA. Histones, along with ions such as Mg2+ , help neutralize the negatively charged DNA phosphate groups.
1. Nucleosomes: Two molecules each of H2A, H2B, H3, and H4 form the octameric core of the individual nucleosome “beads.” Around this structural core, a segment of dsDNA is wound nearly twice (Fig. 30.26). Winding eliminates a helical turn, causing negative supercoiling. [Note: The N-terminal ends of these histones can be acetylated, methylated, or phosphorylated. These reversible covalent modifications influence how tightly the histones bind to the DNA, thereby affecting the expression of specific genes. Histone modification is an example of epigenetics, or heritable changes in gene expression caused without alteration of the nucleotide sequence.] Neighboring nucleosomes are joined by linker DNA ~50 bp long. H1 is not found in the nucleosome core, but instead binds to the linker DNA chain between the nucleosome beads. H1 is the most tissue specific and species specific of the histones. It facilitates the packing of nucleosomes into more compact structures.
2. Higher levels of organization: Nucleosomes can be packed more tightly (stacked) to form a nucleofilament. This structure assumes the shape of a coil, often referred to as a 30-nm fiber. The fiber is organized into loops that are anchored by a nuclear scaffold containing several proteins. Additional levels of organization lead to the final chromosomal structure (Fig. 30.27).
B. Nucleosome fate during DNA replication
Parental nucleosomes are disassembled to allow access to DNA during replication. Once DNA is synthesized, nucleosomes form rapidly. Their histone proteins come both from de novo synthesis and from the transfer of parental histones.
VI. DNA REPAIR
Despite the elaborate proofreading system employed during DNA synthesis, errors (including incorrect base-pairing or insertion of one to a few extra nucleotides) can occur. In addition, DNA is constantly being subjected to environmental insults that cause the alteration or removal of nucleotide bases. The damaging agents can be either chemicals (for example, nitrous acid, which can deaminate bases) or radiation (for example, nonionizing ultraviolet [UV] radiation, which can fuse two pyrimidines adjacent to each other in the DNA, and high-energy ionizing radiation, which can cause double-strand breaks). Bases are also altered or lost spontaneously from mammalian DNA at a rate of many thousands per cell per day. If the damage is not repaired, a permanent change (mutation) is introduced that can result in any of a number of deleterious effects, including loss of control over the proliferation of the mutated cell, leading to cancer. Luckily, cells are remarkably efficient at repairing damage done to their DNA. Most of the repair systems involve recognition of the damage (lesion) on the DNA, removal or excision of the damage, replacement or filling the gap left by excision using the sister strand as a template for DNA synthesis, and ligation. These excision repair systems remove one to tens of nucleotides. [Note: Repair synthesis of DNA can occur outside of the S phase.]
A. Mismatch repair
Sometimes replication errors escape the proofreading activity during DNA synthesis, causing a mismatch of one to several bases. In E. coli, mismatch repair (MMR) is mediated by a group of proteins known as the Mut proteins (Fig. 30.28). Homologous proteins are present in humans. [Note: MMR occurs within minutes of replication and reduces the error rate of replication from 1 in 107 to 1 in 109 nucleotides.] 1.
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Mismatched strand identification: When a mismatch occurs, the Mut proteins that identify the mispaired nucleotide(s) must be able to discriminate between the correct strand and the strand with the mismatch. In prokaryotes, discrimination is based on the degree of methylation. GATC sequences, which are found once every thousand nucleotides, are methylated on the adenine (A) residue by DNA adenine methylase (DAM). This methylation is not done immediately after synthesis, so the DNA is hemimethylated (that is, the parental strand is methylated, but the daughter strand is not). The methylated parental strand is assumed to be correct, and it is the daughter strand that gets repaired. [Note: The exact mechanism by which the daughter strand is identified in eukaryotes is not yet known, but likely involves recognition of nicks in the newly synthesized strand.] 2.
Repair procedure: When the strand containing the mismatch is identified, an endonuclease nicks the strand, and the mismatched nucleotide(s) is/are removed by an exonuclease. Additional nucleotides at the 5′-and 3′-ends of the mismatch are also removed. The gap left by removal of the nucleotides is filled, using the sister strand as a template, by a DNA pol, typically DNA pol III. The 3′-hydroxyl of the newly synthesized DNA is joined to the 5′-phosphate of the remaining stretch of the original DNA strand by DNA ligase.
Mutation to the proteins involved in MMR in humans is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. Although HNPCC confers an increased risk for developing colon cancer (as well as other cancers), only about 5% of all colon cancer is the result of mutations in MMR.
B. Nucleotide excision repair
Exposure of a cell to UV radiation can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. These intrastrand cross-links prevent DNA pol from replicating the DNA strand beyond the site of dimer formation. Thymine dimers are excised in bacteria by UvrABC proteins in a process known as nucleotide excision repair (NER), as illustrated in Figure 30.29. A related pathway is present in humans (see 2. below). [Note: Transcription-coupled repair, a type of NER, fixes DNA lesions encountered during RNA synthesis.] 1.
Recognition and excision of UV-induced dimers: A UV-specific endonuclease (called uvrABC excinuclease) recognizes the bulky dimer and cleaves the damaged strand on both the 5′-side and 3′-side of the lesion. A short oligonucleotide containing the dimer is excised, leaving a gap in the DNA strand. This gap is filled in using a DNA pol I and DNA ligase. NER occurs throughout the cell cycle.
2.
UV radiation and cancer: Pyrimidine dimers can be formed in the skin cells of humans exposed to UV radiation in unfiltered sunlight. In the rare genetic disease xeroderma pigmentosum (XP), the cells cannot repair the damaged DNA, resulting in extensive accumulation of mutations and, consequently, early and numerous skin cancers (Fig. 30.30). XP can be caused by defects in any of the several genes that code for the XP proteins required for NER of UV damage in humans.
C. Base excision repair
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DNA bases can be altered, either spontaneously, as is the case with cytosine, which slowly undergoes deamination (the loss of its amino group) to form uracil, or by the action of deaminating or alkylating compounds. For example, nitrous acid, which is formed by the cell from precursors such as the nitrates, deaminates cytosine, adenine (to hypoxanthine), and guanine (to xanthine). Dimethyl sulfate can alkylate (methylate) adenine. Bases can also be lost spontaneously. For example, ~10,000 purine bases are lost this way per cell per day. Lesions involving base alterations or loss can be corrected by base excision repair ([BER], Fig. 30.31).
1.
Abnormal base removal: In BER, abnormal bases, such as uracil, which can occur in DNA by either deamination of cytosine or improper use of dUTP instead of dTTP during DNA synthesis, are recognized by specific DNA glycosylases that hydrolytically cleave them from the deoxyribosephosphate backbone of the strand. This leaves an apyrimidinic site, or apurinic if a purine was removed, both referred to as AP sites.
2.
AP site recognition and repair: Specific AP endonucleases recognize that a base is missing and initiate the process of excision and gap filling by making an endonucleolytic cut just to the 5′-side of the AP site. A deoxyribose phosphate lyase removes the single, base-free, sugar phosphate residue. DNA pol I and DNA ligase complete the repair process.
D. Double-strand break repair
Ionizing radiation, chemotherapeutic agents such as doxorubicin, and oxidative free radicals (see p. 148) can cause double-strand breaks in DNA that can be lethal to the cell. [Note: Such breaks also occur naturally during genetic recombination.] dsDNA breaks cannot be corrected by the previously described strategy of excising the damage on one strand and using the undamaged strand as a template for replacing the missing nucleotide(s). Instead, they are repaired by one of two systems. The first is nonhomologous end joining (NHEJ), in which a group of proteins mediates the recognition, processing, and ligation of the ends of two DNA fragments. However, some DNA is lost in the process. Consequently, NHEJ is error prone and mutagenic. Defects in NHEJ are associated with a predisposition to cancer and immunodeficiency syndromes. The second repair system, homologous recombination (HR), uses the enzymes that normally perform genetic recombination between homologous chromosomes during meiosis. This system is much less error prone (“error-free”) than NHEJ because any DNA that was lost is replaced using homologous DNA as a template. HR occurs in late S and G2 of the cell cycle, whereas NHEJ can occur anytime.
[Note: Mutations to the proteins BRCA1 or BRCA2 (breast cancer 1 or 2), which are involved in HR, increase the risk for developing breast and ovarian cancer.]
VII. CHAPTER SUMMARY
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DNA is a polymer of deoxynucleoside monophosphates covalently linked by 3′→5′-phosphodiester bonds (Fig. 30.32). The resulting long, unbranched chain has polarity, with both a 5′-end (free phosphate) and a 3′end (free hydroxyl). The sequence of nucleotides is read 5′→3′. DNA exists as a double-stranded molecule, in which the two chains are paired in an antiparallel manner and wind around each other, forming a double helix. Adenine pairs with thymine, and cytosine pairs with guanine. Each strand of the double helix serves as a template for constructing a complementary daughter strand (semiconservative replication). DNA replication occurs in the S phase of the cell cycle and begins at an origin of replication. As the two strands unwind and separate, synthesis occurs at two replication forks that move away from the origin in opposite directions (bidirectionally). Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in the region of DNA ahead of the replication fork and negative supercoils behind the fork. DNA topoisomerases types I and II remove supercoils. DNA polymerases (pols) synthesize new DNA strands only in the 5′→3′ direction. Therefore, one of the newly synthesized stretches of nucleotide chains must grow in the 5′→3′ direction toward the replication fork (leading strand) and one in the 5′→3′ direction away from the replication fork (lagging strand). DNA pols require a primer, a short stretch of RNA synthesized by primase. Leading-strand synthesis needs only one RNA primer (continuous synthesis), whereas the lagging strand needs many (discontinuous synthesis involving Okazaki fragments). In Escherichia coli (E. coli), DNA chain elongation is catalyzed by DNA pol III, using 5′-deoxyribonucleoside triphosphates as substrates. The enzyme proofreads the newly synthesized DNA, removing terminal mismatched nucleotides with its 3′→5′ exonuclease activity. RNA primers are removed by DNA pol I, using its 5′→3′ exonuclease activity. This enzyme fills the gaps with DNA, proofreading as it synthesizes. The final phosphodiester linkage is catalyzed by DNA ligase. There are at least five high-fidelity eukaryotic DNA pols. Pol α is a multisubunit enzyme, one subunit of which is a primase. Pol α 5′→3′ polymerase activity adds a short piece of DNA to the RNA primer. Pol ε completes DNA synthesis on the leading strand, whereas pol δ elongates each lagging strand fragment. Pol β is involved with DNA repair, and pol γ replicates mitochondrial DNA. Pols ε, δ, and γ use 3′→5′ exonuclease activity to proofread. Nucleoside analogs containing modified sugars can be used to block DNA chain growth. They are useful in anticancer and antiviral chemotherapy. Telomeres are stretches of highly repetitive DNA complexed with protein that protect the ends of linear chromosomes. As most cells divide and age, these sequences are shortened, contributing to senescence. In cells that do not senesce (for example, germline and cancer cells), the ribonucleoprotein telomerase employs its protein component reverse transcriptase to extend the telomeres, using its RNA component as a template. There are five classes of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting the nucleosomes, called linker DNA, is bound to H1. Nucleosomes can be packed more tightly to form a nucleofilament. Additional levels of organization create a chromosome. Most DNA damage can be corrected by excision repair involving recognition and removal of the damage by repair proteins, followed by replacement by DNA pols and joining by ligase. Ultraviolet radiation can cause thymine dimers that are recognized and removed in E. coli by uvrABC proteins of nucleotide excision repair. Defects in the XP proteins needed for nucleotide excision repair of thymine dimers in humans result in xeroderma pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes. Defective mismatch repair by homologous proteins in humans is associated with hereditary nonpolyposis colorectal cancer. Abnormal bases (such as uracil) are removed by DNA N-glycosylases in base excision repair, and the sugar phosphate at the apyrimidinic or apurinic site is cut out. Double-strand breaks in DNA are repaired by nonhomologous end joining (error prone) and template-requiring homologous recombination (“error-free”).
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Choose the ONE best answer.
0.1. A 10-year-old girl is brought by her parents to the dermatologist. She has many freckles on her face, neck, arms, and hands, and the parents report that she is unusually sensitive to sunlight. Two basal cell carcinomas are identified on her face. Based on the clinical picture, which of the following processes is most likely to be defective in this patient?
A. Repair of double-strand breaks by error-prone homologous recombination
B. Removal of mismatched bases from the 3′-end of Okazaki fragments by a methyl-directed process
C. Removal of pyrimidine dimers from DNA by nucleotide excision repair
D. Removal of uracil from DNA by base excision repair
Correct answer = C. The sensitivity to sunlight, extensive freckling on parts of the body exposed to the sun, and presence of skin cancer at a young age indicate that the patient most likely suffers from xeroderma pigmentosum (XP). These patients are deficient in any one of several XP proteins required for nucleotide excision repair of pyrimidine dimers in ultraviolet radiation– damaged DNA. Double-strand breaks are repaired by nonhomologous end joining (error prone) or homologous recombination (“error free”). Methylation is not used for strand discrimination in eukaryotic mismatch repair. Uracil is removed from DNA molecules by a specific glycosylase in base excision repair, but a defect in this process does not cause XP.
0.2. Telomeres are complexes of DNA and protein that protect the ends of linear chromosomes. In most normal human somatic cells, telomeres shorten with each division. In stem cells and in cancer cells, however, telomeric length is maintained. In the synthesis of telomeres:
A. telomerase, a ribonucleoprotein, provides both the RNA and the protein needed for synthesis.
B. the RNA of telomerase serves as a primer.
C. the RNA of telomerase is a ribozyme.
D. the protein of telomerase is a DNA-directed DNA polymerase.
E. the shorter 3′→5′ strand gets extended.
F. the direction of synthesis is 3′→5′.
Correct answer = A. Telomerase is a ribonucleoprotein particle required for telomere maintenance. Telomerase contains an RNA that serves as the template, not the primer, for the synthesis of telomeric DNA by the reverse transcriptase of telomerase. Telomeric RNA has no catalytic activity. As a reverse transcriptase, telomerase synthesizes DNA using its RNA template and so is an RNA-directed DNA polymerase. The direction of synthesis, as with all DNA synthesis, is 5′→3′, and it is the 3′-end of the already longer 5′→3′ strand that gets extended.
0.3. While studying the structure of a small gene that was sequenced during the Human Genome Project, an investigator notices that one strand of the DNA molecule contains 20 A, 25 G, 30 C, and 22 T. How many of each base is found in the complete double-stranded molecule?
A. A=40,G =50,C =60,T =44
E. A=42,G =55,C =55,T =42
B. A=44,G =60,C =50,T =40
C. A=45,G =45,C =52,T =52
D. A=50,G =47,C =50,T =47
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Correct answer = B. The two DNA strands are complementary to each other, with A base-paired with T and G base-paired with C. So, for example, the 20 A on the first strand would be paired with 20 T on the second strand, the 25 G on the first strand would be paired with 25 C on the second strand, and so forth. When these are all added together, the correct numbers of each base are indicated in choice B. Notice that, in the correct answer, A = T and G = C.
0.4. List the order in which the following enzymes participate in prokaryotic replication.
A. Ligase
B. Polymerase I (3′→5′ exonuclease activity)
C. Polymerase I (5′→3′ exonuclease activity)
D. Polymerase I (5′→3′ polymerase activity)
E. Polymerase III
F. Primase
Correct answer: F, E, C, D, B, A. Primase makes the RNA primer; polymerase (pol) III extends the primer with DNA (and proofreads); pol I removes the primer with its 5′→3′ exonuclease activity, fills in the gap with its 5′→3′ polymerase activity, and removes errors with its 3′→5′ exonuclease activity; and ligase makes the 5′→3′-phosphodiester bond that links the DNA made by pols I and III.
0.5. Dideoxynucleotides lack a 3′-hydroxyl group. Why would incorporation of a dideoxynucleotide into DNA stop replication?
The lack of the 3′-OH group prevents formation of the 3′-hydroxyl → 5′phosphate bond that links one nucleotide to the next in DNA.
RNA Structure, Synthesis, and Processing 31
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I. OVERVIEW
The genetic master plan of an organism is contained in the sequence of deoxyribonucleotides in its DNA. However, it is through ribonucleic acid (RNA), the “working copies” of DNA, that the master plan is expressed (Fig. 31.1). The copying process, during which a DNA strand serves as a template for the synthesis of RNA, is called transcription. Transcription produces messenger RNA (mRNA), which are translated into sequences of amino acids (proteins), and ribosomal RNA (rRNA), transfer RNA (tRNA), and additional RNA molecules that perform specialized structural, catalytic, and regulatory functions and are not translated. That is, they are noncoding RNA (ncRNA). Therefore, the final product of gene expression can be RNA or protein, depending upon the gene. [Note: Only ~2% of the genome encodes proteins.] A central feature of transcription is that it is highly selective. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made. This selectivity is due, at least in part, to signals embedded in the nucleotide sequence of the DNA. These signals instruct the RNA polymerase where to start, how often to start, and where to stop transcription. Several regulatory proteins are also involved in this selection process. The biochemical differentiation of an organism’s tissues is ultimately a result of the selectivity of the transcription process. [Note: This selectivity of transcription is in contrast to the “all-or-none” nature of genomic replication.] Another important feature of transcription is that many RNA transcripts that initially are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the inactive primary transcript into a functional molecule. The transcriptome is the complete set of RNA transcripts expressed by a genome.
II. RNA STRUCTURE
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There are three major types of RNA that participate in the process of protein synthesis: rRNA, tRNA, and mRNA. Like DNA, these RNA are unbranched polymeric molecules composed of nucleoside monophosphates joined together by 3′→5′-phosphodiester bonds (see p. 412). However, they differ from DNA in several ways. For example, they are considerably smaller than DNA, contain ribose instead of deoxyribose and uracil instead of thymine, and exist as single strands that are capable of folding into complex structures. The three major types of RNA also differ from each other in size, function, and special structural modifications. [Note: In eukaryotes, additional small ncRNA molecules found in the nucleolus (snoRNA), nucleus (snRNA), and cytoplasm (microRNA [miRNA]) perform specialized functions as described on pp. 441, 442, and 475.]
A. Ribosomal RNA rRNA are found in association with several proteins as components of the ribosomes, the complex structures that serve as the sites for protein synthesis (see p. 451). Prokaryotic cells contain three distinct size species of rRNA (23S, 16S, and 5S, where S is the Svedberg unit for sedimentation rate that is determined by the size and shape of the particle), as shown in Figure 31.2. Eukaryotic cells contain four rRNA species (28S, 18S, 5.8S, and 5S). Together, rRNA make up ~80% of the total RNA in the cell. [Note: Some RNA function as catalysts, for example, an rRNA in protein synthesis (see p. 455). RNA with catalytic activity is termed a ribozyme.]
B. Transfer RNA tRNA are the smallest (4S) of the three major types of RNA molecules. There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins. Together, tRNA make up ~15% of the total RNA in the cell. The tRNA molecules contain a high percentage of unusual (modified) bases, for example, dihydrouracil (see Fig. 22.2, p. 292), and have extensive intrachain base-pairing (Fig. 31.3) that leads to characteristic secondary and tertiary structure. Each tRNA serves as an adaptor molecule that carries its specific amino acid, covalently attached to its 3′-end, to the site of protein synthesis. There, it recognizes the genetic code sequence on an mRNA, which specifies the addition of that amino acid to the growing peptide chain (see p. 447).
C. Messenger RNA mRNA comprises only ~5% of the RNA in a cell, yet is by far the most heterogeneous type of RNA in size and base sequence. mRNA is coding RNA in that it carries genetic information from DNA for use in protein synthesis. In eukaryotes, this involves transport of mRNA out of the nucleus and into the cytosol. An mRNA carrying information from more than one gene is polycistronic (cistron = gene). Polycistronic mRNA is characteristic of prokaryotes. An mRNA carrying information from only one gene is monocistronic and is characteristic of eukaryotes. In addition to the protein-coding regions that can be translated, mRNA contains untranslated regions at its 5′-and 3′-ends (Fig. 31.4). Special structural characteristics of eukaryotic (but not prokaryotic) mRNA include a long sequence of adenine nucleotides (a poly-A tail) on the 3′-end of the RNA, plus a cap on the 5′-end consisting of a molecule of 7-methylguanosine attached through an unusual (5′→5′) triphosphate linkage. The mechanisms for modifying mRNA to create these special structural characteristics are discussed on pp. 441–442.
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III. PROKARYOTIC GENE TRANSCRIPTION
The structure of magnesium-requiring RNA polymerase (RNA pol), the signals that control transcription, and the varieties of modification that RNA transcripts can undergo differ among organisms, particularly from prokaryotes to eukaryotes. Therefore, the discussions of prokaryotic and eukaryotic transcription are presented separately.
A. Prokaryotic RNA polymerase
In bacteria, one species of RNA pol synthesizes all of the RNA except for the short RNA primers needed for DNA replication [Note: RNA primers are synthesized by the specialized, monomeric enzyme primase (see p. 418).] RNA pol is a multisubunit enzyme that recognizes a nucleotide sequence (the promoter region) at the beginning of a length of DNA that is to be transcribed. It next makes a complementary RNA copy of the DNA template strand and then recognizes the end of the DNA sequence to be transcribed (the termination region). RNA is synthesized from its 5′-end to its 3′-end, antiparallel to its DNA template strand (see p. 415). The template is copied as it is in DNA synthesis, in which a guanine (G) on the DNA specifies a cytosine (C) in the RNA, a C specifies a G, a thymine (T) specifies an adenine (A), but an A specifies a uracil (U) instead of a T (Fig. 31.5). The RNA, then, is complementary to the DNA template (antisense, minus) strand and identical to the coding (sense, plus) strand, with U replacing T. Within the DNA molecule, regions of both strands can serve as templates for transcription. For a given gene, however, only one of the two DNA strands can be the template. Which strand is used is determined by the location of the promoter for that gene. Transcription by RNA pol involves a core enzyme and several auxiliary proteins.
1.
Core enzyme: Five of the enzyme’s peptide subunits, 2 α, 1 β, 1 β′, and 1 Ω, are required for enzyme assembly (α, Ω), template binding (β′), and the 5′→3′ polymerase activity (β) and together are referred to as the core enzyme (Fig. 31.6). However, this enzyme lacks specificity (that is, it cannot recognize the promoter region on the DNA template).
2.
Holoenzyme: The σ subunit (sigma factor) enables RNA pol to recognize promoter regions on the DNA. The σ subunit plus the core enzyme make up the holoenzyme. [Note: Different σ factors recognize different groups of genes, with σ70 predominating.]
B. Steps in RNA synthesis
The process of transcription of a typical gene of Escherichia coli (E. coli) can be divided into three phases: initiation, elongation, and termination. A transcription unit extends from the promoter to the termination region, and the initial product of transcription by RNA pol is termed the primary transcript.
1. Initiation: Transcription begins with the binding of the RNA pol holoenzyme to a region of the DNA known as the promoter, which is not transcribed. The prokaryotic promoter contains characteristic consensus sequences (Fig. 31.7). [Note: Consensus sequences are idealized sequences in which the base shown at each position is the base most frequently (but not necessarily always) encountered at that position.] Those that are recognized by prokaryotic RNA pol σ factors include the following.
a.
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–35 Sequence: A consensus sequence (5′-TTGACA-3′), centered about 35 bases to the left of the transcription start site (see Fig. 31.7), is the initial point of contact for the holoenzyme, and a closed complex is formed. [Note: By convention, the regulatory sequences that control transcription are designated by the 5′→3′ nucleotide sequence on the coding strand. A base in the promoter region is assigned a negative number if it occurs prior to (to the left of, toward the 5′-end of, or “upstream” of) the transcription start site. Therefore, the TTGACA sequence is centered at approximately base −35. The first base at the transcription start site is assigned a position of +1. There is no base designated “0”.] b.
Pribnow box: The holoenzyme moves and covers a second consensus sequence (5′-TATAAT-3′), centered at about −10 (see Fig. 31.7), which is the site of melting (unwinding) of a short stretch (~14 base pairs) of DNA. This initial melting converts the closed initiation complex to an open complex known as a transcription bubble. [Note: A mutation in either the −10 or the −35 sequence can affect the transcription of the gene controlled by the mutant promoter.] 2.
Elongation: Once the promoter has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues (Fig. 31.8), mediated by the polymerase. [Note: Unwinding generates supercoils in the DNA that can be relieved by DNA topoisomerases (see p. 417).] RNA pol begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase begins when the transcript (typically starting with a purine) exceeds 10 nucleotides in length. Sigma is then released, and the core enzyme is able to leave (clear) the promoter and move along the template strand in a processive manner, serving as its own sliding clamp. During transcription, a short DNA–RNA hybrid helix is formed (see Fig. 31.8). Like DNA pol, RNA pol uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In contrast to DNA pol, RNA pol does not require a primer and does not have a 3′→5′ exonuclease domain for proofreading. [Note: Misincorporation of a ribonucleotide causes RNA pol to pause, backtrack, cleave the transcript, and restart. Nonetheless, transcription has a higher error rate than does replication.] 3.
Termination: The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (occur without additional proteins) or dependent upon the participation of a protein known as the ρ (rho) factor.
a. ρ-Independent: Seen with most prokaryotic genes, this requires that a sequence in the DNA template generates a sequence in the nascent (newly made) RNA that is self-complementary (Fig. 31.9). This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized by hydrogen bonds) plus a loop. This structure is known as a “hairpin.” Additionally, just beyond the hairpin, the RNA transcript contains a string of Us at the 3′-end. The bonding of these Us to the complementary As of the DNA template is weak. This facilitates the separation of the newly synthesized RNA from its DNA template, as the double helix “zips up” behind the RNA pol.
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b. ρ-Dependent: This requires the participation of the additional protein rho, which is a hexameric ATPase with helicase activity. Rho binds a C-rich rho utilization (rut) site near the 5′-end of the nascent RNA and, using its ATPase activity, moves along the RNA until it reaches the RNA pol paused at the termination site. The ATP-dependent helicase activity of rho separates the RNA–DNA hybrid helix, causing the release of the RNA.
4. Antibiotics: Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis. For example, rifampin (rifampicin) inhibits transcription initiation by binding to the β subunit of prokaryotic RNA pol and preventing chain growth beyond three nucleotides (Fig. 31.10). Rifampin is important in the treatment of tuberculosis. Dactinomycin (actinomycin
D) was the first antibiotic to find therapeutic application in tumor chemotherapy. It inserts (intercalates) between the DNA bases and inhibits transcription initiation and elongation.
IV. EUKARYOTIC GENE TRANSCRIPTION
The transcription of eukaryotic genes is a far more complicated process than transcription in prokaryotes. Eukaryotic transcription involves separate polymerases for the synthesis of rRNA, tRNA, and mRNA. In addition, a large number of proteins called transcription factors (TF) are involved. TF bind to distinct sites on the DNA within the core promoter region, close (proximal) to it, or some distance away (distal). They are required for both the assembly of a transcription initiation complex at the promoter and the determination of which genes are to be transcribed. [Note: Each eukaryotic RNA pol has its own promoters and TF that bind core promoter sequences.] For TF to recognize and bind to their specific DNA sequences, the chromatin structure in that region must be decondensed (relaxed) to allow access to the DNA. The role of transcription in the regulation of gene expression is discussed in Chapter 33.
A. Chromatin structure and gene expression
The association of DNA with histones to form nucleosomes (see p. 425) affects the ability of the transcription machinery to access the DNA to be transcribed. Most actively transcribed genes are found in a relatively decondensed form of chromatin called euchromatin, whereas most inactive segments of DNA are found in highly condensed heterochromatin. The interconversion of these forms is called chromatin remodeling. A major component of chromatin remodeling is the covalent modification of histones (for example, the acetylation of lysine residues at the amino terminus of histone proteins), as shown in Figure 31.11. Acetylation, mediated by histone acetyltransferases (HAT), eliminates the positive charge on the lysine, thereby decreasing the interaction of the histone with the negatively charged DNA. Removal of the acetyl group by histone deacetylases (HDAC) restores the positive charge and fosters stronger interactions between histones and DNA. [Note: The ATP-dependent repositioning of nucleosomes is also required to access DNA.]
B. Nuclear RNA polymerases
There are three distinct types of RNA pol in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each type of RNA pol recognizes particular genes. [Note: Mitochondria contain a single RNA pol that resembles the bacterial enzyme.] 1.
RNA polymerase I: This enzyme synthesizes the precursor of the 28S, 18S, and 5.8S rRNA in the nucleolus.
2.
RNA polymerase II: This enzyme synthesizes the nuclear precursors of mRNA that are processed and then translated to proteins. RNA pol II also synthesizes certain small ncRNA, such as snoRNA, snRNA, and miRNA.
a.
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Promoters for RNA polymerase II: In some genes transcribed by RNA pol II, a sequence of nucleotides (TATAAA) that is nearly identical to that of the Pribnow box (see p. 436) is found centered ~25 nucleotides upstream of the transcription start site. This core promoter consensus sequence is called the TATA, or Hogness, box. In the majority of genes, however, no TATA box is present. Instead, different core promoter elements such as Inr (initiator) or DPE (downstream promoter element) are present (Fig. 31.12). [Note: No one consensus sequence is found in all core promoters.] Because these sequences are on the same molecule of DNA as the gene being transcribed, they are cis-acting. The sequences serve as binding sites for proteins known as general transcription factors (GTF), which in turn interact with each other and with RNA pol II.
b.
General transcription factors: GTF are the minimal requirements for recognition of the promoter, recruitment of RNA pol II to the promoter, formation of the preinitiation complex, and initiation of transcription at a basal level (Fig. 31.13A). GTF are encoded by different genes, synthesized in the cytosol, and diffuse (transit) to their sites of action, and so are trans-acting. [Note: In contrast to the prokaryotic holoenzyme, eukaryotic RNA pol II does not itself recognize and bind the promoter. Instead, TFIID, a GTF containing TATA-binding protein and TATA-associated factors, recognizes and binds the TATA box (and other core promoter elements). TFIIF, another GTF, brings the polymerase to the promoter. The helicase activity of TFIIH melts the DNA, and its kinase activity phosphorylates polymerase, allowing it to clear the promoter.] c. Regulatory elements and transcriptional activators: Additional consensus sequences lie upstream of the core promoter (see Fig. 31.12). Those close to the core promoter (within ~200 nucleotides) are the proximal regulatory elements, such as the CAAT and GC boxes. Those farther away are the distal regulatory elements such as enhancers (see d. below). Proteins known as transcriptional activators or specific transcription factors (STF) bind these regulatory elements. STF bind to promoter proximal elements to regulate the frequency of transcription initiation and to distal elements to mediate the response to signals such as hormones (see p. 472) and regulate which genes are expressed at a given point in time. A typical protein-coding eukaryotic gene has binding sites for many such factors. STF have two binding domains. One is a DNA-binding domain, the other is a transcription activation domain that recruits the GTF to the core promoter as well as coactivator proteins such as the HAT enzymes involved in chromatin modification. [Note: Mediator, a multisubunit coactivator of RNA pol II–catalyzed transcription, binds the polymerase, the GTF, and the STF and regulates transcription initiation.]
Transcriptional activators bind DNA through a variety of motifs, such as the helix-loop-helix, zinc finger, and leucine zipper (see p. 18).
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d. Role of enhancers: Enhancers are special DNA sequences that increase the rate of initiation of transcription by RNA pol II. Enhancers are typically on the same chromosome as the gene whose transcription they stimulate (Fig. 31.13B). However, they can 1) be located upstream (to the 5′-side) or downstream (to the 3′-side) of the transcription start site, 2) be close to or thousands of base pairs away from the promoter (Fig. 31.14), and 3) occur on either strand of the DNA. Enhancers contain DNA sequences called response elements that bind STF. By bending or looping the DNA, STF can interact with other TF bound to a promoter and with RNA pol II, thereby stimulating transcription (see Fig. 31.13B). Mediator also binds enhancers. [Note: Although silencers are similar to enhancers in that they also can act over long distances, they reduce gene expression.] e. RNA polymerase II inhibitor: α-Amanitin, a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called the “death cap”), binds RNA pol II tightly and slows its movement, thereby inhibiting mRNA synthesis.
3. RNA polymerase III: This enzyme synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA.
V. POSTTRANSCRIPTIONAL MODIFICATION OF RNA
A primary transcript is the initial, linear, RNA copy of a transcription unit (the segment of DNA between specific initiation and termination sequences). The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified by cleavage of the original transcripts by ribonucleases. tRNA are further modified to help give each species its unique identity. In contrast, prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is extensively modified both co-and posttranscriptionally.
A. Ribosomal RNA rRNA of both prokaryotic and eukaryotic cells are generated from long precursor molecules called pre-rRNA. The 23S, 16S, and 5S rRNA of prokaryotes are produced from a single pre-rRNA molecule, as are the 28S, 18S, and 5.8S rRNA of eukaryotes (Fig. 31.15). [Note: Eukaryotic 5S rRNA is synthesized by RNA pol III and modified separately.] The prerRNA are cleaved by ribonucleases to yield intermediate-sized pieces of rRNA, which are further processed (trimmed by exonucleases and modified at some bases and riboses) to produce the required RNA species. [Note: In eukaryotes, rRNA genes are found in long, tandem arrays. rRNA synthesis and processing occur in the nucleolus, with base and sugar modifications facilitated by snoRNA.]
B. Transfer RNA
Both eukaryotic and prokaryotic tRNA are also made from longer precursor molecules that must be modified (Fig. 31.16). Sequences at both ends of the molecule are removed, and, if present, an intron is removed from the anticodon loop by nucleases. Other posttranscriptional modifications include addition of a –CCA sequence by nucleotidyltransferase to the 3′terminal end of tRNA and modification of bases at specific positions to produce the unusual bases characteristic of tRNA (see p. 291).
C. Eukaryotic messenger RNA
The collection of all the primary transcripts synthesized in the nucleus by RNA pol II is known as heterogeneous nuclear RNA (hnRNA). The premRNA components of hnRNA undergo extensive co-and posttranscriptional modification in the nucleus and become mature mRNA. These modifications usually include the following. [Note: Pol II itself recruits the proteins required for the modifications.] 1.
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Addition of a 5′-cap: This is the first of the processing reactions for premRNA (Fig. 31.17). The cap is a 7-methylguanosine attached to the 5′terminal end of the mRNA through an unusual 5′→5′-triphosphate linkage that is resistant to most nucleases. Creation of the cap requires removal of the γ phosphoryl group from the 5′-triphosphate of the premRNA, followed by addition of guanosine monophosphate (from guanosine triphosphate) by the nuclear enzyme guanylyltransferase. Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7-methyltransferase. S-Adenosylmethionine is the source of the methyl group (see p. 263). Additional methylation steps may occur. The addition of this 7-methylguanosine cap helps stabilize the mRNA and permits efficient initiation of translation (see p. 455).
2.
Addition of a 3′-poly-A tail: Most eukaryotic mRNA (with several exceptions, including those for the histones) have a chain of 40–250 adenylates (adenosine monophosphates) attached to the 3′-end (see Fig. 31.17). This poly-A tail is not transcribed from the DNA but rather is added by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate. The pre-mRNA is cleaved downstream of a consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3′-end of the RNA, and the poly-A tail is added to the new 3′end. Tailing terminates eukaryotic transcription. Tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid in translation. After the mRNA enters the cytosol, the poly-A tail is gradually shortened.
3.
Splicing: Maturation of eukaryotic mRNA usually involves removal from the primary transcript of RNA sequences (introns or intervening sequences) that do not code for protein. The remaining coding (expressed) sequences, the exons, are joined together to form the mature mRNA. The process of removing introns and joining exons is called splicing. The molecular complex that accomplishes these tasks is known as the spliceosome. A few eukaryotic primary transcripts contain no introns (for example, those from histone genes). Others contain a few introns, whereas some, such as the primary transcripts for the α chains of collagen, contain >50 introns that must be removed.
a. Role of small nuclear RNA: In association with multiple proteins, uracil-rich snRNA form five small nuclear ribonucleoprotein particles (snRNP, or “snurp”) designated as U1, U2, U4, U5, and U6 that mediate splicing. They facilitate the removal of introns by forming base pairs with the consensus sequences at each end of the intron (Fig. 31.18). [Note: In systemic lupus erythematosus, an autoimmune disease, patients produce antibodies against their own nuclear proteins such as snRNP.] b.
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Mechanism: The binding of snRNP brings the sequences of neighboring exons into the correct alignment for splicing, allowing two transesterification reactions (catalyzed by the RNA of U2, U5, and U6) to occur. The 2′-OH group of an adenine nucleotide (known as the branch site A) in the intron attacks the phosphate at the 5′-end of the intron (splice-donor site), forming an unusual 2′→5′-phosphodiester bond and creating a “lariat” structure (see Fig. 31.18). The newly freed 3′-OH of exon 1 attacks the 5′-phosphate at the spliceacceptor site, forming a phosphodiester bond that joins exons 1 and 2. The excised intron is released as a lariat, which is typically degraded but may be a precursor for ncRNA such as snoRNA. [Note: The GU and AG sequences at the beginning and end, respectively, of introns are invariant. However, additional sequences are critical for splice-site recognition.] After introns have been removed and exons joined, the mature mRNA molecules pass into the cytosol through pores in the nuclear membrane. [Note: The introns in tRNA (see Fig. 31.16) are removed by a different mechanism.] c.
Effect of splice site mutations: Mutations at splice sites can lead to improper splicing and the production of aberrant proteins. It is estimated that at least 20% of all genetic diseases are a result of mutations that affect RNA splicing. For example, mutations that cause the incorrect splicing of β-globin mRNA are responsible for some cases of β-thalassemia, a disease in which the production of the βglobin protein is defective (see p. 38). Splice site mutations can result in exons being skipped (removed) or introns retained. They can also activate cryptic splice sites, which are sites that contain the 5′ or 3′ consensus sequence but are not normally used.
4. Alternative splicing: The pre-mRNA molecules from >90% of human genes can be spliced in alternative ways in different tissues. Because this produces multiple variations of the mRNA and, therefore, of its protein product (Fig. 31.19), it is a mechanism for producing a large, diverse set of proteins from a limited set of genes. For example, the mRNA for tropomyosin (TM), an actin filament–binding protein of the cytoskeleton (and of the contractile apparatus in muscle cells), undergoes extensive tissue-specific alternative splicing with production of multiple isoforms of the TM protein.
VI. CHAPTER SUMMARY
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Three major types of RNA participate in the process of protein synthesis: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA), as shown in Figure 31.20. They are unbranched polymers of nucleotides but differ from DNA by containing ribose instead of deoxyribose and uracil instead of thymine. rRNA is a component of the ribosomes. tRNA serves as an adaptor molecule that carries a specific amino acid to the site of protein synthesis. mRNA (coding RNA) carries genetic information from DNA for use in protein synthesis. The process of RNA synthesis is called transcription, and the substrates are ribonucleoside triphosphates. The enzyme that synthesizes RNA is RNA polymerase (RNA pol). In prokaryotic cells, the core enzyme has five subunits (2 α, 1 β, 1 β′, and 1 Ω) and possesses 5′→3′ polymerase activity needed for transcription. The core enzyme requires an additional subunit, sigma (σ) factor, to recognize the nucleotide sequence (promoter region) at the beginning of the DNA to be transcribed. This region contains consensus sequences that are highly conserved and include the −10 Pribnow box and the −35 sequence. Another protein, rho (ρ), is required for termination of transcription of some genes. There are three distinct types of RNA pol in the nucleus of eukaryotic cells. RNA pol I synthesizes the precursor of rRNA in the nucleolus. In the nucleoplasm, RNA pol II synthesizes the precursors for mRNA and some noncoding RNA, and RNA pol III synthesizes the precursors of tRNA and 5S rRNA. In both prokaryotes and eukaryotes, RNA pol does not require a primer. Proofreading involves the polymerase backtracking and cleaving the transcript. Core promoters for genes transcribed by RNA pol II contain cis-acting consensus sequences, such as the TATA (Hogness) box, which serve as binding sites for transacting general transcription factors. Upstream of these are proximal regulatory elements, such as the CAAT and GC boxes, and distal regulatory elements, such as enhancers. Specific transcription factors (transcriptional activators) and Mediator complex bind these elements and regulate the frequency of transcription initiation, the response to signals such as hormones, and which genes are expressed at any given time. Eukaryotic transcription requires that the chromatin be relaxed (decondensed) in a process known as chromatin remodeling. A primary transcript is a linear copy of a transcription unit, the segment of DNA between specific initiation and termination sequences. The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified. The rRNA are synthesized from long precursor molecules called pre-rRNA. These precursors are cleaved and trimmed by ribonucleases, producing the three largest rRNA, and bases and sugars are modified. Eukaryotic 5S rRNA is synthesized by RNA pol III and is modified separately. Prokaryotic and eukaryotic tRNA are also made from longer precursor molecules (pre-tRNA). If present, an intron is removed by nucleases, and both ends of the molecule are trimmed by ribonucleases. A 3′-CCA sequence is added, and bases at specific positions are modified. Prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic pre-mRNA is extensively modified co-and posttranscriptionally. For example, a 7-methylguanosine cap is attached to the 5′-end of the mRNA through a 5′→5′ linkage. A long poly-A tail, not transcribed from the DNA, is attached by polyadenylate polymerase to the 3′-end of most mRNA. Most eukaryotic mRNA also contains intervening sequences (introns) that must be removed for the mRNA to be functional. Their removal, as well as the joining of expressed sequences (exons), requires a spliceosome composed of small nuclear ribonucleoprotein particles (“snurps”) that mediate the process of splicing. Eukaryotic mRNA is monocistronic, containing information from just one gene, whereas prokaryotic mRNA is polycistronic.
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Choose the ONE best answer.
1.1. An 8-month-old male with severe anemia is found to have β-thalassemia. Genetic analysis shows that one of his β-globin genes has a mutation that creates a new splice-acceptor site 19 nucleotides upstream of the normal splice-acceptor site of the first intron. Which of the following best describes the new messenger RNA molecule that can be produced from this mutant gene?
A. Exon 1 will be too short.
B. Exon 1 will be too long.
C. Exon 2 will be too short.
D. Exon 2 will be too long.
E. Exon 2 will be missing.
Correct answer = D. Because the mutation creates an additional splice-acceptor site (the 3′-end) upstream of the normal acceptor site of intron 1, the 19 nucleotides that are usually found at the 3′-end of the excised intron 1 lariat can remain behind as part of exon 2. The presence of these extra nucleotides in the coding region of the mutant messenger RNA (mRNA) molecule will prevent the ribosome from translating the message into a normal β-globin protein molecule. Those mRNA for which the normal splice site is used to remove the first intron will be normal, and their translation will produce normal β-globin protein.
1.2. A 4-year-old child who easily tires and has trouble walking is diagnosed with Duchenne muscular dystrophy, an X-linked recessive disorder. Genetic analysis shows that the patient’s gene for the muscle protein dystrophin contains a mutation in its promoter region. Of the choices listed, which would be the most likely effect of this mutation?
A. Initiation of dystrophin transcription will be defective.
B. Termination of dystrophin transcription will be defective.
C. Capping of dystrophin messenger RNA will be defective.
D. Splicing of dystrophin messenger RNA will be defective.
E. Tailing of dystrophin messenger RNA will be defective.
Correct answer = A. Mutations in the promoter typically prevent formation of the RNA polymerase II transcription initiation complex, resulting in a decrease in the initiation of messenger RNA (mRNA) synthesis. A deficiency of dystrophin mRNA will result in a deficiency in the production of the dystrophin protein. Capping, splicing, and tailing defects are not a consequence of promoter mutations. They can, however, result in mRNA with decreased stability (capping and tailing defects) or an mRNA in which exons have been skipped (lost) or introns retained (splicing defects).
1.3. A mutation to this sequence in eukaryotic messenger RNA (mRNA) will affect the process by which the 3′-end polyadenylate (poly-A) tail is added to the mRNA.
A. AAUAAA
B. CAAT
C. CCA
D. GU… A…AG
E. TATAAA
Correct answer = A. An endonuclease cleaves mRNA just downstream of this polyadenylation signal, creating a new 3′-end to which polyadenylate polymerase adds the poly-A tail using ATP as the substrate in a template-independent process. CAAT and TATAAA are sequences found in promoters for RNA polymerase II. CCA is added to the 3′-end of pre-transfer RNA by nucleotidyltransferase. GU…A…AG denotes an intron in eukaryotic premRNA.
1.4. This protein factor identifies the promoter of protein-coding genes in eukaryotes.
A. Pribnow box
B. Rho
C. Sigma
D. TFIID E. U1
Correct answer = D. The general transcription factor TFIID recognizes and binds core promoter elements such as the TATA-like box in eukaryotic protein-coding genes. These genes are transcribed by RNA polymerase II. The
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Pribnow box is a cis-acting element in prokaryotic promoters. Rho is involved in the termination of prokaryotic transcription. Sigma is the subunit of prokaryotic RNA polymerase that recognizes and binds the prokaryotic promoter. U1 is a ribonucleoprotein involved in splicing of eukaryotic premRNA.
1.5. What is the sequence (conventionally written) of the RNA product of the DNA template sequence, GATCTAC, also conventionally written?
Correct answer = 5′-GUAGAUC-3′. Nucleic acid sequences are conventionally written 5′ to 3′. The template strand (5′-GATCTAC-3′) is used as 3′CATCTAG-5′. The RNA product is complementary to the template strand (and identical to the coding strand), with U replacing T.
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I. OVERVIEW
Genetic information, stored in the chromosomes and transmitted to daughter cells through DNA replication, is expressed through transcription to RNA and, in the case of messenger RNA (mRNA), subsequent translation into proteins (polypeptides) as shown in Figure 32.1. [Note: The proteome is the complete set of proteins expressed in a cell.] The process of protein synthesis is called translation because the “language” of the nucleotide sequence on the mRNA is translated into the language of an amino acid sequence. Translation requires a genetic code, through which the information contained in the nucleotide sequence is expressed to produce a specific amino acid sequence. Any alteration in the nucleotide sequence may result in an incorrect amino acid being inserted into the protein, potentially causing disease or even death of the organism. Newly made immature (nascent) proteins undergo a number of processes to achieve their functional form. They must fold properly, and misfolding can result in aggregation or degradation of the protein. Many proteins are covalently modified to alter their activities. Lastly, proteins are targeted to their final intra-or extracellular destinations by signals present in the proteins themselves.
II. THE GENETIC CODE
The genetic code is a “dictionary” that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual “word” in the code is composed of three nucleotide bases. These genetic words are called codons.
A. Codons
Codons are presented in the mRNA language of adenine (A), guanine (G), cytosine (C), and uracil (U). Their nucleotide sequences are always written from the 5′-end to the 3′-end. The four nucleotide bases are used to produce the three-base codons. Therefore, 64 different combinations of bases exist, taken three at a time (a triplet code), as shown in the table in Figure 32.2.
many common amino acids are shown as examples.
1.
How to translate a codon: This table can be used to translate any codon and, thus, to determine which amino acids are coded for by an mRNA sequence. For example, the codon AUG codes for methionine ([Met] see Fig. 32.2). [Note: AUG is the initiation (start) codon for translation.] Sixty-one of the 64 codons code for the 20 standard amino acids (see p. 1).
2.
Termination codons: Three of the codons, UAA, UAG, and UGA, do not code for amino acids but, rather, are termination (also called stop, or nonsense) codons. When one of these codons appears in an mRNA sequence, synthesis of the polypeptide coded for by that mRNA stops.
B. Characteristics
Usage of the genetic code is remarkably consistent throughout all living organisms. It is assumed that once the standard genetic code evolved in primitive organisms, any mutation (a permanent change in DNA sequence) that altered its meaning would have caused the alteration of most, if not all, protein sequences, resulting in lethality. Characteristics of the genetic code include the following.
1.
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Specificity: The genetic code is specific (unambiguous), because a particular codon always codes for the same amino acid.
2.
Universality: The genetic code is virtually universal insofar as its specificity has been conserved from very early stages of evolution, with only slight differences in the manner in which the code is translated. [Note: An exception occurs in mitochondria, in which a few codons have meanings different than those shown in Figure 32.2. For example, UGA codes for tryptophan (Trp).] 3.
Degeneracy: The genetic code is degenerate (sometimes called redundant). Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. For example, arginine (Arg) is specified by six different codons (see Fig. 32.2). Only Met and Trp have just one coding triplet.
4.
Nonoverlapping and commaless: The genetic code is nonoverlapping and commaless, meaning that the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time without any punctuation between codons. For example, AGCUGGAUACAU is read as AGC UGG AUA CAU.
C. Consequences of altering the nucleotide sequence
Changing a single nucleotide base (a point mutation) in the coding region of an mRNA can lead to any one of three results (Fig. 32.3).
1.
Silent mutation: The codon containing the changed base may code for the same amino acid. For example, if the serine (Ser) codon UCA is changed at the third base and becomes UCU, it still codes for Ser. This is termed a silent mutation.
2.
Missense mutation: The codon containing the changed base may code for a different amino acid. For example, if the Ser codon UCA is changed at the first base and becomes CCA, it will code for a different amino acid (in this case, proline [Pro]). This is termed a missense mutation.
3.
Nonsense mutation: The codon containing the changed base may become a termination codon. For example, if the Ser codon UCA is changed at the second base and becomes UAA, the new codon causes premature termination of translation at that point and the production of a shortened (truncated) protein. This is termed a nonsense mutation. [Note: The nonsense-mediated degradation pathway can degrade mRNA containing premature stops.] 4.
Other mutations: These can alter the amount or structure of the protein produced by translation.
a. Trinucleotide repeat expansion: Occasionally, a sequence of three bases that is repeated in tandem will become amplified in number so that too many copies of the triplet occur. If this happens within the coding region of a gene, the protein will contain many extra copies of one amino acid. For example, expansion of the CAG codon in exon 1 of the gene for huntingtin protein leads to the insertion of many extra glutamine residues in the protein, causing the neurodegenerative disorder Huntington disease (Fig. 32.4). The additional glutamines result in an abnormally long protein that is cleaved, producing toxic fragments that aggregate in neurons. If the trinucleotide repeat expansion occurs in an untranslated region (UTR) of a gene, the result can be a decrease in the amount of protein produced, as seen in fragile X syndrome and myotonic dystrophy. Over 20 triplet expansion diseases are known. [Note: In fragile X syndrome, the most common cause of intellectual disability in males, the expansion results in gene silencing through DNA hypermethylation (see p. 476).] b.
Splice site mutations: Mutations at splice sites (see p. 443) can alter the way in which introns are removed from pre-mRNA molecules, producing aberrant proteins. [Note: In myotonic dystrophy, a muscle disorder, gene silencing is the result of splicing alterations due to triplet expansion.] c.
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Frameshift mutations: If one or two nucleotides are either deleted from or added to the coding region of an mRNA, a frameshift mutation occurs, altering the reading frame. This can result in a product with a radically different amino acid sequence or a truncated product due to the eventual creation of a termination codon (Fig. 32.5). If three nucleotides are added, a new amino acid is added to the peptide. If three are deleted, an amino acid is lost. Loss of three nucleotides maintains the reading frame but can result in serious pathology. For example, cystic fibrosis (CF), a chronic, progressive, inherited disease that primarily affects the pulmonary and digestive systems, is most commonly caused by deletion of three nucleotides from the coding region of a gene, resulting in the loss of phenylalanine (Phe, or F; see p. 5) at the 508th position (∆F508) in the CF transmembrane conductance regulator (CFTR) protein encoded by that gene. This ∆F508 mutation prevents normal folding of CFTR, leading to its destruction by the proteasome (see p. 247). CFTR normally functions as a chloride channel in epithelial cells, and its loss results in the production of thick, sticky secretions in the lungs and pancreas, leading to lung damage and digestive deficiencies (see p. 174). The incidence of CF is highest (1 in 3,300) in those of Northern European origin. In >70% of individuals with CF, the ∆F508 mutation is the cause of the disease.
= guanine; U = uracil.
III. COMPONENTS REQUIRED FOR TRANSLATION
A large number of components are required for the synthesis of a protein. These include all the amino acids that are found in the finished product, the mRNA to be translated, transfer RNA (tRNA) for each of the amino acids, functional ribosomes, energy sources, and enzymes as well as noncatalytic protein factors needed for the initiation, elongation, and termination steps of polypeptide chain synthesis.
A. Amino acids
All the amino acids that eventually appear in the finished protein must be present at the time of protein synthesis. If one amino acid is missing, translation stops at the codon specifying that amino acid. [Note: This demonstrates the importance of having all the essential amino acids (see p.
262) in sufficient quantities in the diet to insure continued protein synthesis.]
B. Transfer RNA
At least one specific type of tRNA is required for each amino acid. In humans, there are at least 50 species of tRNA, whereas bacteria contain at least 30 species. Because there are only 20 different amino acids commonly carried by tRNA, some amino acids have more than one specific tRNA molecule. This is particularly true of those amino acids that are coded for by several codons.
1. Amino acid attachment site: Each tRNA molecule has an attachment site for a specific (cognate) amino acid at its 3′-end (Fig. 32.6). The carboxyl group of the amino acid is in an ester linkage with the 3′-hydroxyl of the ribose portion of the A nucleotide in the –CCA sequence at the 3′-end of the tRNA. [Note: A tRNA with a covalently attached (activated) amino acid is charged. Without an attached amino acid, it is uncharged.] 2. Anticodon: Each tRNA molecule also contains a three-base nucleotide sequence, the anticodon, which pairs with a specific codon on the mRNA (see Fig. 32.6). This codon specifies the insertion into the growing polypeptide chain of the amino acid carried by that tRNA.
C. Aminoacyl-tRNA synthetases
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This family of 20 different enzymes is required for attachment of amino acids to their corresponding tRNA. Each member of this family recognizes a specific amino acid and all the tRNA that correspond to that amino acid (isoaccepting tRNA, up to five per amino acid). Aminoacyl-tRNA synthetases catalyze a two-step reaction that results in the covalent attachment of the α-carboxyl group of an amino acid to the A in the –CCA sequence at the 3′-end of its corresponding tRNA. The overall reaction requires ATP, which is cleaved to adenosine monophosphate and inorganic pyrophosphate (PPi), as shown in Figure 32.7. The extreme specificity of the synthetases in recognizing both the amino acid and its cognate tRNA contributes to the high fidelity of translation of the genetic message. In addition to their synthetic activity, the aminoacyl-tRNA synthetases have a proofreading, or editing activity that can remove an incorrect amino acid from the enzyme or the tRNA molecule.
RNA (tRNA) by an aminoacyl-tRNA synthetase. PPi = pyrophosphate; Pi = monophosphate; ~ = high-energy bond.
D. Messenger RNA
The specific mRNA required as a template for the synthesis of the desired polypeptide must be present. [Note: In eukaryotes, mRNA is circularized for use in translation.]
E. Functionally competent ribosomes
As shown in Figure 32.8, ribosomes are large complexes of protein and ribosomal RNA (rRNA), in which rRNA predominates. They consist of two subunits (one large and one small) whose relative sizes are given in terms of their sedimentation coefficients, or S (Svedberg) values. [Note: Because the S values are determined by both shape and size, their numeric values are not strictly additive. For example, the prokaryotic 50S and 30S ribosomal subunits together form a 70S ribosome. The eukaryotic 60S and 40S subunits form an 80S ribosome.] Prokaryotic and eukaryotic ribosomes are similar in structure and serve the same function, namely, as the macromolecular complexes in which the synthesis of proteins occurs.
The small ribosomal subunit binds mRNA and determines the accuracy of translation by insuring correct base-pairing between the mRNA codon and the tRNA anticodon. The large ribosomal subunit catalyzes formation of the peptide bonds that link amino acid residues in a protein.
1.
Ribosomal RNA: As discussed on p. 434, prokaryotic ribosomes contain three size species of rRNA, whereas eukaryotic ribosomes contain four (see Fig. 32.8). The rRNA are generated from a single pre-rRNA by the action of ribonucleases, and some bases and riboses are modified.
2.
Ribosomal proteins: Ribosomal proteins are present in greater numbers in eukaryotic ribosomes than in prokaryotic ribosomes. These proteins play a variety of roles in the structure and function of the ribosome and its interactions with other components of the translation system.
3.
A, P, and E sites: The ribosome has three binding sites for tRNA molecules: the A, P, and E sites, each of which extends over both subunits. Together, they cover three neighboring codons. During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The P site is occupied by peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome. (See Fig. 32.13 for an illustration of the role of the A, P, and E sites in translation.) 4.
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Cellular location: In eukaryotic cells, the ribosomes either are free in the cytosol or are in close association with the endoplasmic reticulum (which is then known as the rough endoplasmic reticulum, or RER). RER-associated ribosomes are responsible for synthesizing proteins (including glycoproteins; see p. 166) that are to be exported from the cell, incorporated into membranes, or imported into lysosomes (see p. 169 for an overview of the latter process). Cytosolic ribosomes synthesize proteins required in the cytosol itself or destined for the nucleus, mitochondria, or peroxisomes. [Note: Mitochondria contain their own ribosomes (55S) and their own unique, circular DNA. Most mitochondrial proteins, however, are encoded by nuclear DNA, synthesized completely in the cytosol, and then targeted to mitochondria.]
F. Protein factors
Initiation, elongation, and termination (or, release) factors are required for polypeptide synthesis. Some of these protein factors perform a catalytic function, whereas others appear to stabilize the synthetic machinery. [Note: A number of the factors are small, cytosolic G proteins and thus are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). See p. 95 for a discussion of the membrane-associated G proteins.]
G. Energy sources
Cleavage of four high-energy bonds (see p. 73) is required for the addition of one amino acid to the growing polypeptide chain: two from ATP in the aminoacyl-tRNA synthetase reaction, one in the removal of PPi and one in the subsequent hydrolysis of the PPi, to two Pi by pyrophosphatase, and two from GTP, one for binding the aminoacyl-tRNA to the A site and one for the translocation step (see Fig. 32.13, p. 457). [Note: Additional ATP and GTP molecules are required for initiation in eukaryotes, whereas an additional GTP molecule is required for termination in both eukaryotes and prokaryotes.] Translation, then, is a major consumer of energy.
IV. CODON RECOGNITION BY TRANSFER RNA
Correct pairing of the codon in the mRNA with the anticodon of the tRNA is essential for accurate translation (see Fig. 32.6). Most tRNA (isoaccepting tRNA) recognize more than one codon for a given amino acid.
A. Antiparallel binding between codon and anticodon
Binding of the tRNA anticodon to the mRNA codon follows the rules of complementary and antiparallel binding, that is, the mRNA codon is read 5′→3′ by an anticodon pairing in the opposite (3′→5′) orientation (Fig. 32.9). [Note: Nucleotide sequences are always written in the 5′ to 3′ direction unless otherwise noted. Two nucleotide sequences orient in an antiparallel manner.]
B. Wobble hypothesis
The mechanism by which a tRNA can recognize more than one codon for a specific amino acid is described by the wobble hypothesis, which states that codon–anticodon pairing follows the traditional Watson-Crick rules (G pairs with C and A pairs with U) for the first two bases of the codon but can be less stringent for the last base. The base at the 5′-end of the anticodon (the first base of the anticodon) is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the 3′-base of the codon (the last base of the codon). This movement is called wobble and allows a single tRNA to recognize more than one codon. Examples of these flexible pairings are shown in Figure 32.9. The result of wobble is that 61 tRNA species are not required to read the 61 codons that code for amino acids.
V. STEPS IN TRANSLATION
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The process of protein synthesis translates the 3-letter alphabet of nucleotide sequences on mRNA into the 20-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5′-end to its 3′-end, producing a protein synthesized from its amino (N)-terminal end to its carboxyl (C)-terminal end. Prokaryotic mRNA often have several coding regions (that is, they are polycistronic; see p. 434). Each coding region has its own initiation and termination codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA has only one coding region (that is, it is monocistronic). The process of translation is divided into three separate steps: initiation, elongation, and termination. Eukaryotic translation resembles that of prokaryotes in most aspects. Individual differences are noted in the text.
One important difference is that translation and transcription are temporally linked in prokaryotes, with translation starting before transcription is completed as a consequence of the lack of a nuclear membrane in prokaryotes.
A. Initiation
Initiation of protein synthesis involves the assembly of the components of the translation system before peptide-bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP, and initiation factors that facilitate the assembly of this initiation complex (see Fig. 32.13). [Note: In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukaryotes, there are many (designated eIF to indicate eukaryotic origin). Eukaryotes also require ATP for initiation.] The following are two mechanisms by which the ribosome recognizes the nucleotide sequence (AUG) that initiates translation.
1. Shine-Dalgarno sequence: In Escherichia coli (E. coli), a purine-rich sequence of nucleotide bases, known as the Shine-Dalgarno (SD) sequence, is located six to ten bases upstream of the initiating AUG codon on the mRNA molecule (that is, near its 5′-end). The 16S rRNA component of the small (30S) ribosomal subunit has a nucleotide sequence near its 3′-end that is complementary to all or part of the SD sequence. Therefore, the 5′-end of the mRNA and the 3′-end of the 16S rRNA can form complementary base pairs, facilitating the positioning of the 30S subunit on the mRNA in close proximity to the initiating AUG codon (Fig. 32.10).
2.
5′-Cap: Eukaryotic mRNA do not have SD sequences. In eukaryotes, the small (40S) ribosomal subunit (aided by members of the eIF-4 family of proteins) binds close to the cap structure at the 5′-end of the mRNA and moves 5′→3′ along the mRNA until it encounters the initiator AUG. This scanning process requires ATP. Cap-independent initiation can occur if the 40S subunit binds to an internal ribosome entry site close to the start codon. [Note: Interactions between the cap-binding eIF-4 proteins and the poly-A tail–binding proteins on eukaryotic mRNA mediate circularization of the mRNA and likely prevent the use of incompletely processed mRNA in translation.] 3.
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Initiation codon: The initiating AUG is recognized by a special initiator tRNA (tRNAi). Recognition is facilitated by IF-2-GTP in prokaryotes and eIF-2-GTP (plus additional eIF) in eukaryotes. The charged tRNAi is the only tRNA recognized by (e)IF-2 and the only tRNA to go directly to the P site on the small subunit. [Note: Base modifications distinguish tRNAi from the tRNA used for internal AUG codons.] In bacteria and mitochondria, tRNAi carries an N-formylated methionine (fMet), as shown in Figure 32.11. After Met is attached to tRNAi, the formyl group is added by the enzyme transformylase, which uses N10-formyl tetrahydrofolate (see p. 267) as the carbon donor. In eukaryotes, tRNAi carries a Met that is not formylated. In both prokaryotic and eukaryotic cells, this N-terminal Met is usually removed before translation is completed. The large ribosomal subunit then joins the complex, and a functional ribosome is formed with the charged tRNAi in the P site. The A site is empty. [Note: Specific (e)IF function as anti-association factors and prevent premature addition of the large subunit.] The GTP on (e)IF-2 gets hydrolyzed to GDP. In eukaryotes, the guanine nucleotide exchange factor eIF-2B facilitates the reactivation of eIF-2-GDP through replacement of GDP by GTP.
B. Elongation
Elongation of the polypeptide involves the addition of amino acids to the carboxyl end of the growing chain. Delivery of the aminoacyl-tRNA whose codon appears next on the mRNA template in the ribosomal A site (a process known as decoding) is facilitated in E. coli by elongation factors EF-Tu-GTP and EF-Ts and requires GTP hydrolysis. [Note: In eukaryotes, comparable elongation factors are EF-1α-GTP and EF-1βγ. Both EF-Ts and EF-1βγ function in guanine nucleotide exchange.] Peptide-bond formation between the α-carboxyl group of the amino acid in the P site and the αamino group of the amino acid in the A site is catalyzed by peptidyltransferase, an activity intrinsic to an rRNA of the large subunit (Fig. 32.12). [Note: Because this rRNA catalyzes the reaction, it is a ribozyme (see p. 54).] After the peptide bond has been formed, the peptide on the tRNA at the P site is transferred to the amino acid on the tRNA at the A site, a process known as transpeptidation. The ribosome then advances three nucleotides toward the 3′-end of the mRNA. This process is known as translocation and, in prokaryotes, requires the participation of EF-G-GTP (eukaryotes use EF-2-GTP) and GTP hydrolysis. Translocation causes movement of the uncharged tRNA from the P to the E site for release and movement of the peptidyl-tRNA from the A to the P site. The process is repeated until a termination codon is encountered. [Note: Because of the length of most mRNA, more than one ribosome at a time can translate a message. Such a complex of one mRNA and a number of ribosomes is called a polysome, or polyribosome.]
C. Termination
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Termination occurs when one of the three termination codons moves into the A site. These codons are recognized in E. coli by release factors: RF-1, which recognizes UAA and UAG, and RF-2, which recognizes UGA and UAA. The binding of these release factors results in hydrolysis of the bond linking the peptide to the tRNA at the P site, causing the nascent protein to be released from the ribosome. A third release factor, RF-3-GTP, then causes the release of RF-1 or RF-2 as GTP is hydrolyzed (see Fig. 32.13). [Note: Eukaryotes have a single release factor, eRF, which recognizes all three termination codons. A second factor, eRF-3, functions like the prokaryotic RF-3. See Figure 32.14 for a summary of the factors used in translation.] The steps in prokaryotic protein synthesis, as well as some antibiotic inhibitors of the process, are summarized in Figure 32.13. The newly synthesized polypeptide may undergo further modification as described below, and the ribosomal subunits, mRNA, tRNA, and protein factors can be recycled and used to synthesize another polypeptide. [Note: In prokaryotes, ribosome recycling factors mediate separation of the subunits. In eukaryotes, eRF and ATP hydrolysis are required.]
D. Translation regulation
Gene expression is most commonly regulated at the transcriptional level, but translation may also be regulated. An important mechanism by which this is achieved in eukaryotes is by covalent modification of eIF-2: Phosphorylated eIF-2 is inactive (see p. 476). In both eukaryotes and prokaryotes, regulation can also be achieved through proteins that bind mRNA and inhibit its use by blocking translation.
E. Protein folding
Proteins must fold to assume their functional, native state. Folding can be spontaneous (as a result of the primary structure) or facilitated by proteins known as chaperones (see p. 20).
F. Protein targeting
Although most protein synthesis in eukaryotes is initiated in the cytoplasm, many proteins perform their functions within subcellular organelles or outside of the cell. Such proteins normally contain amino acid sequences that direct the proteins to their final locations. For example, secreted proteins are targeted during synthesis (cotranslational targeting) to the RER by the presence of an N-terminal hydrophobic signal sequence. The sequence is recognized by the signal recognition particle (SRP), a ribonucleoprotein that binds the ribosome, halts elongation, and delivers the ribosome–peptide complex to an RER membrane channel (the translocon) via interaction with the SRP receptor. Translation resumes, the protein enters the RER lumen, and its signal sequence is cleaved (Fig. 32.15). The protein moves through the RER and the Golgi, is processed, packaged into vesicles, and secreted. Proteins targeted after synthesis (posttranslational) include nuclear proteins that contain an internal, short, basic nuclear localization signal; mitochondrial matrix proteins that contain an N-terminal, amphipathic, α-helical mitochondrial entry sequence; and peroxisomal proteins that contain a C-terminal tripeptide signal.
VI. CO-AND POSTTRANSLATIONAL MODIFICATIONS
Many polypeptides are covalently modified, either while they are still attached to the ribosome (cotranslational) or after their synthesis has been completed (posttranslational). These modifications may include removal of part of the translated sequence or the covalent addition of one or more chemical groups required for protein activity.
A. Trimming
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Many proteins destined for secretion are initially made as large, precursor molecules that are not functionally active. Portions of the protein must be removed by specialized endoproteases, resulting in the release of an active molecule. The cellular site of the cleavage reaction depends on the protein to be modified. Some precursor proteins are cleaved in the RER or the Golgi; others are cleaved in developing secretory vesicles (for example, insulin; see Fig. 23.4, p. 309); and still others, such as collagen (see p. 47), are cleaved after secretion.
B. Covalent attachments
Protein function can be affected by the covalent attachment of a variety of chemical groups (Fig. 32.16). Examples include the following.
1.
Phosphorylation: Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less frequently, tyrosine residues in a protein. It is catalyzed by one of a family of protein kinases and may be reversed by the action of protein phosphatases. The phosphorylation may increase or decrease the functional activity of the protein. Several examples of phosphorylation reactions have been previously discussed (for example, see Chapter 11, p. 132, for the regulation of glycogen synthesis and degradation).
2.
Glycosylation: Many of the proteins that are destined to become part of a membrane or to be secreted from a cell have carbohydrate chains added en bloc to the amide nitrogen of an asparagine (N-linked) or built sequentially on the hydroxyl groups of a serine, threonine, or hydroxylysine (O-linked). N-glycosylation occurs in the RER and Oglycosylation in the Golgi. (The process of producing such glycoproteins was discussed on p. 165.) N-glycosylated acid hydrolases are targeted to the matrix of lysosomes by the phosphorylation of mannose residues at carbon 6 (see p. 169).
3.
Hydroxylation: Proline and lysine residues of the α chains of collagen are extensively hydroxylated by vitamin C–dependent hydroxylases in the RER (see p. 47).
4.
Other covalent modifications: These may be required for the functional activity of a protein. For example, additional carboxyl groups can be added to glutamate residues by vitamin K–dependent carboxylation (see p. 393). The resulting γ-carboxyglutamate (Gla) residues are essential for the activity of several of the blood-clotting proteins. (See online Chapter 35.) Biotin is covalently bound to the ε-amino groups of lysine residues of biotin-dependent enzymes that catalyze carboxylation reactions such as pyruvate carboxylase (see Fig. 10.3 on p. 119). Attachment of lipids, such as farnesyl groups, can help anchor proteins to membranes (see p. 221). Many eukaryotic proteins are cotranslationally acetylated at the N-end. [Note: Reversible acetylation of histone proteins influences gene expression (see p. 476).]
C. Protein degradation
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Proteins that are defective (for example, misfolded) or destined for rapid turnover are often marked for destruction by ubiquitination, the covalent attachment of chains of a small, highly conserved protein called ubiquitin (see Fig. 19.3 on p. 247). Proteins marked in this way are rapidly degraded by the proteasome, which is a macromolecular, ATP-dependent, proteolytic system located in the cytosol. For example, misfolding of the CFTR protein (see p. 450) results in its proteasomal degradation. [Note: If folding is impeded, unfolded proteins accumulate in the RER causing stress that triggers the unfolded protein response, in which the expression of chaperones is increased; global translation is decreased by eIF-2 phosphorylation; and the unfolded proteins are sent to the cytosol, ubiquitinated, and degraded in the proteasome by a process called ER-associated degradation.]
VII. CHAPTER SUMMARY
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Codons are composed of three nucleotide bases presented in the messenger RNA (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U). They are always written 5′→3′. Of the 64 possible three-base combinations, 61 code for the 20 standard amino acids and 3 signal termination of protein synthesis (translation). Altering the nucleotide sequence in a codon can cause silent mutations (the altered codon codes for the original amino acid), missense mutations (the altered codon codes for a different amino acid), or nonsense mutations (the altered codon is a termination codon). Characteristics of the genetic code include specificity, universality, and degeneracy, and it is nonoverlapping and commaless (Fig. 32.17). Requirements for protein synthesis include all the amino acids that eventually appear in the finished protein; at least one specific type of transfer RNA (tRNA) for each amino acid; one aminoacyl-tRNA synthetase for each amino acid; the mRNA coding for the protein to be synthesized; fully competent ribosomes (70S in prokaryotes, 80S in eukaryotes); protein factors needed for initiation, elongation, and termination of protein synthesis; and ATP and guanosine triphosphate (GTP) as energy sources. tRNA has an attachment site for a specific amino acid at its 3′-end and an anticodon region that can recognize the codon specifying the amino acid the tRNA is carrying. Ribosomes are large complexes of protein and ribosomal RNA (rRNA). They consist of two subunits, 30S and 50S in prokaryotes and 40S and 60S in eukaryotes. Each ribosome has three binding sites for tRNA molecules: the A, P, and E sites that cover three neighboring codons. The A site binds an incoming aminoacyl-tRNA, the P site is occupied by peptidyl-tRNA, and the E site is occupied by the empty tRNA as it is about to exit the ribosome. Recognition of an mRNA codon is accomplished by the tRNA anticodon, which binds to the codon following the rules of complementarity and antiparallel binding. The wobble hypothesis states that the first (5′) base of the anticodon is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the last (3′) base of the codon, thus allowing a single tRNA to recognize more than one codon for a specific amino acid. For initiation of protein synthesis, the components of the translation system are assembled, and mRNA associates with the small ribosomal subunit. The process requires initiation factors (IF). In prokaryotes, a purine-rich region of the mRNA (the Shine-Dalgarno sequence) base-pairs with a complementary sequence on 16S rRNA, resulting in the positioning of the small subunit on the mRNA so that translation can begin. The 5′-cap (bound by proteins of the eIF-4 family) on eukaryotic mRNA is used to position the small subunit on the mRNA. The initiation codon is AUG, and N-formylmethionine is the initiating amino acid in prokaryotes, whereas methionine is used in eukaryotes. The charged initiating tRNA (tRNAi) is brought to the P site by (e)IF-2. In elongation, the polypeptide chain is lengthened by the addition of amino acids to the carboxyl end of its growing chain. The process requires elongation factors that facilitate the binding of the aminoacyl-tRNA to the A site as well as the movement of the ribosome along the mRNA. The formation of the peptide bond is catalyzed by peptidyltransferase, which is an activity intrinsic to the rRNA of the large subunit and, therefore, is a ribozyme. Following peptide-bond formation, the ribosome advances along the mRNA in the 5′→3′ direction to the next codon (translocation). Because of the length of most mRNA, more than one ribosome at a time can translate a message, forming a polysome. Termination begins when one of the three termination codons moves into the A site. These codons are recognized by release factors. The newly synthesized protein is released from the ribosomal complex, and the ribosome is dissociated from the mRNA. Initiation, elongation, and termination are driven by the hydrolysis of GTP. Initiation in eukaryotes also requires ATP for scanning. Numerous antibiotics interfere with the process of protein synthesis. Many polypeptide chains are covalently modified during or after translation. Such modifications include amino acid removal; phosphorylation, which may activate or inactivate the protein; glycosylation, which plays a role in protein targeting; and hydroxylation such as that seen in collagen. Protein targeting can be either cotranslational (as with secreted proteins) or posttranslational (as with mitochondrial matrix proteins). Proteins must fold to achieve their functional form. Folding can be spontaneous or facilitated by chaperones. Proteins that are defective (for example, misfolded) or destined for rapid turnover are marked for destruction by the attachment of chains of a small, highly conserved protein called ubiquitin. Ubiquitinated proteins are rapidly degraded by a cytosolic complex known as the proteasome.
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Choose the ONE best answer.
2.1. A 20-year-old man with a microcytic anemia is found to have an abnormal form of β-globin (Hemoglobin Constant Spring) that is 172 amino acids long, rather than the 141 found in the normal protein. Which of the following point mutations is consistent with this abnormality? Use Figure 32.2 to answer the question.
A. CGA→UGA
B. GAU→GAC
C. GCA→GAA
D. UAA→CAA
D. UAA→UAG
Correct answer = D. Mutating the normal termination (stop) codon from UAA to CAA in β-globin messenger RNA causes the ribosome to insert a glutamine at that point. It will continue extending the protein chain until it comes upon the next stop codon farther down the message, resulting in an abnormally long protein. The replacement of CGA (arginine) with UGA (stop) would cause the protein to be too short. GAU and GAC both code for aspartate and would cause no change in the protein. Changing GCA (alanine) to GAA (glutamate) would not change the size of the protein product. A change from UAA to UAG would simply change one termination codon for another and would have no effect on the protein.
2.2. A pharmaceutical company is studying a new antibiotic that inhibits bacterial protein synthesis. When this antibiotic is added to an in vitro protein synthesis system that is translating the messenger RNA sequence AUGUUUUUUUAG, the only product formed is the dipeptide fMet-Phe. What step in protein synthesis is most likely inhibited by the antibiotic?
A. Initiation
B. Binding of a charged transfer RNA to the ribosomal A site
C. Peptidyltransferase activity
D. Ribosomal translocation
E. Termination
Correct answer = D. Because fMet-Phe (formylated methionyl-phenylalanine) is made, the ribosomes must be able to complete initiation, bind Phe-tRNA to the A site, and use peptidyltransferase activity to form the first peptide bond. Because the ribosome is not able to proceed any further, ribosomal movement (translocation) is most likely the inhibited step. Therefore, the ribosome is stopped before it reaches the termination codon of this message.
2.3. A transfer RNA (tRNA) molecule that is supposed to carry cysteine (tRNAcys) is mischarged, so that it actually carries alanine (ala-tRNAcys). Assuming no correction occurs, what will be the fate of this alanine residue during protein synthesis? It will:
A. be incorporated into a protein in response to a codon for alanine.
B. be incorporated into a protein in response to a codon for cysteine.
C. be incorporated randomly at any codon.
D. remain attached to the tRNA because it cannot be used for protein synthesis.
E. be chemically converted to cysteine by cellular enzymes.
Correct answer = B. Once an amino acid is attached to a tRNA molecule, only the anticodon of that tRNA determines the specificity of incorporation. Therefore, the incorrectly activated alanine will be incorporated into the protein at a position determined by a cysteine codon.
2.4. In a patient with cystic fibrosis (CF) caused by the ∆F508 mutation, the mutant CF transmembrane conductance regulator (CFTR) protein folds incorrectly. The patient’s cells modify this abnormal protein by attaching ubiquitin molecules to it. What is the fate of this modified CFTR protein?
A. It performs its normal function because the ubiquitin largely corrects for the effect of the mutation.
B. It is degraded by the proteasome.
C. It is placed into storage vesicles.
D. It is repaired by cellular enzymes.
E. It is secreted from the cell.
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Correct answer = B. Ubiquitination usually marks old, damaged, or misfolded proteins for destruction by the cytosolic proteasome. There is no known cellular mechanism for repair of damaged proteins.
2.5. Many antimicrobials inhibit translation. Which of the following antimicrobials is correctly paired with its mechanism of action?
A. Erythromycin binds to the 60S ribosomal subunit.
B. Puromycin inactivates elongation factor-2.
C. Streptomycin binds to the 30S ribosomal subunit.
D. Tetracyclines inhibit peptidyltransferase.
Correct answer = C. Streptomycin binds the 30S subunit and inhibits translation initiation. Erythromycin binds the 50S ribosomal subunit (60S denotes a eukaryote) and blocks the tunnel through which the peptide leaves the ribosome. Puromycin has structural similarity to aminoacyl-transfer RNA. It is incorporated into the growing chain, inhibits elongation, and results in premature termination in both prokaryotes and eukaryotes. Tetracyclines bind the 30S ribosomal subunit and block access to the A site, inhibiting elongation.
2.6. Translation of a synthetic polyribonucleotide containing the repeating sequence CAA in a cell-free protein-synthesizing system produces three homopolypeptides: polyglutamine, polyasparagine, and polythreonine. If the codons for glutamine and asparagine are CAA and AAC, respectively, which of the following triplets is the codon for threonine?
A. AAC
B. ACA
C. CAA
D. CAC
E. CCA
Correct answer = B. The synthetic polynucleotide sequence of CAACAACAACAA … could be read by the in vitro protein-synthesizing system starting at the first C, the first A, or the second A (that is, in any one of three reading frames). In the first case, the first triplet codon would be CAA, which codes glutamine; in the second case, the first triplet codon would be AAC, which codes for asparagine; in the last case, the first triplet codon would be ACA, which codes for threonine.
2.7. Which of the following is required for both prokaryotic and eukaryotic protein synthesis?
A. Binding of the small ribosomal subunit to the Shine-Dalgarno sequence
B. Formylated methionyl-transfer (t)RNA
C. Movement of the messenger RNA out of the nucleus and into the cytoplasm
D. Recognition of the 5′-cap by initiation factors
E. Translocation of the peptidyl-tRNA from the A site to the P site
Correct answer = E. In both prokaryotes and eukaryotes, continued translation (elongation) requires movement of the peptidyl-tRNA from the A to the P site to allow the next aminoacyl-tRNA to enter the A site. Only prokaryotes have a Shine-Dalgarno sequence and use formylated methionine and only eukaryotes have a nucleus and co-and posttranscriptionally process their mRNA.
2.8. α1-Antitrypsin (AAT) deficiency can result in emphysema, a lung pathology, because the action of elastase, a serine protease, is unopposed. Deficiency of AAT in the lungs is the consequence of impaired secretion from the liver, the site of its synthesis. Proteins such as AAT that are destined to be secreted are best characterized by which of the following statements?
A. Their synthesis is initiated on the smooth endoplasmic reticulum.
B. They contain a mannose 6-phosphate targeting signal.
C. They always contain methionine as the N-terminal amino acid.
D. They are produced from translation products that have an N-terminal hydrophobic signal sequence.
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E. They contain no sugars with O-glycosidic linkages because their synthesis does not involve the Golgi.
Correct answer = D. Synthesis of secreted proteins is begun on free (cytosolic) ribosomes. As the N-terminal signal sequence of the peptide emerges from the ribosome, it is bound by the signal recognition particle, taken to the rough endoplasmic reticulum (RER), threaded into the lumen, and cleaved as translation continues. The proteins move through the RER and the Golgi and undergo processing such as N-glycosylation (RER) and O-glycosylation (Golgi). In the Golgi, they are packaged in secretory vesicles and released from the cell. The smooth endoplasmic reticulum is associated with synthesis of lipids, not proteins, and has no ribosomes attached. Phosphorylation at carbon 6 of terminal mannose residues in glycoproteins targets these proteins (acid hydrolases) to lysosomes. The N-terminal methionine is removed from most proteins during processing.
2.9. Why is the genetic code described as both degenerate and unambiguous?
A given amino acid can be coded for by more than one codon (degenerate code), but a given codon codes for just one particular amino acid (unambiguous code).
Regulation of Gene Expression 33
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I. OVERVIEW
Gene expression refers to the multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein. The first step in gene expression, the use of deoxyribonucleic acid (DNA) for the synthesis of RNA (transcription), is the primary site of regulation in both prokaryotes and eukaryotes. In eukaryotes, however, gene expression also involves extensive posttranscriptional and posttranslational processes as well as actions that influence access to particular regions of the DNA. Each of these steps can be regulated to provide additional control over the kinds and amounts of functional products that are produced.
Not all genes are tightly regulated. For example, genes described as constitutive encode products required for basic cellular functions and so are expressed at essentially a constant level. They are also known as “housekeeping” genes. Regulated genes, however, are expressed only under certain conditions. They may be expressed in all cells or in only a subset of cells, for example, hepatocytes. The ability to regulate gene expression (that is, to determine if, how much, and when particular gene products will be made) gives the cell control over structure and function. It is the basis for cellular differentiation, morphogenesis, and adaptability of any organism. Control of gene expression is best understood in prokaryotes, but many themes are repeated in eukaryotes. Figure 33.1 shows some of the sites where gene expression can be controlled.
II. REGULATORY SEQUENCES AND MOLECULES
Regulation of transcription, the initial step in all gene expression, is controlled by regulatory sequences of DNA that are usually embedded in the noncoding regions of the genome. The interaction between these DNA sequences and regulatory molecules, such as transcription factors, can induce or repress the transcriptional machinery, influencing the kinds and amounts of products that are produced. The regulatory DNA sequences are called cis-acting because they influence expression of genes on the same chromosome as the regulatory sequence (see p. 439). The regulatory molecules are called trans-acting because they can diffuse (transit) through the cell from their site of synthesis to their DNA-binding sites (Fig. 33.2). For example, a protein transcription factor (a trans-acting molecule) that regulates a gene on chromosome 6 might itself have been produced from a gene on chromosome 11. The binding of proteins to DNA is through structural motifs such as the zinc finger (Fig. 33.3), leucine zipper, or helix-turn-helix in the protein.
III. REGULATION OF PROKARYOTIC GENE EXPRESSION
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In prokaryotes such as the bacterium Escherichia coli (E. coli), regulation of gene expression occurs primarily at the level of transcription and, in general, is mediated by the binding of trans-acting proteins to cis-acting regulatory elements on their single DNA molecule (chromosome). [Note: Regulating the first step in the expression of a gene is an efficient approach, insofar as energy is not wasted making unneeded gene products.] Transcriptional control in prokaryotes can involve the initiation or premature termination of transcription.
A. Messenger RNA transcription from bacterial operons
In bacteria, the structural genes that encode proteins involved in a particular metabolic pathway are often found sequentially grouped on the chromosome along with the cis-acting elements that regulate the transcription of these genes. The transcription product is a single polycistronic messenger RNA ([mRNA] see p. 434). The genes are, thus, coordinately regulated (that is, turned on or off as a unit). This entire package is referred to as an operon.
B. Operators in bacterial operons
Bacterial operons contain an operator, a segment of DNA that regulates the activity of the structural genes of the operon by reversibly binding a protein known as the repressor. If the operator is not bound by the repressor, RNA polymerase (RNA pol) binds the promoter, passes over the operator, and reaches the protein-coding genes that it transcribes to mRNA. If the repressor is bound to the operator, the polymerase is blocked and does not produce mRNA. As long as the repressor is bound to the operator, no mRNA (and, therefore, no proteins) are made. However, when an inducer molecule is present, it binds to the repressor, causing the repressor to change shape so that it no longer binds the operator. When this happens, RNA pol can initiate transcription. One of the best-understood examples is the inducible lactose (lac) operon of E. coli that illustrates both positive and negative regulation (Fig. 33.4).
C. Lactose operon
The lac operon contains the genes that code for three proteins involved in the catabolism of the disaccharide lactose: the lacZ gene codes for βgalactosidase, which hydrolyzes lactose to galactose and glucose; the lacY gene codes for a permease, which facilitates the movement of lactose into the cell; and the lacA gene codes for thiogalactoside transacetylase, which acetylates lactose. [Note: The physiologic function of this acetylation is unknown.] All of these proteins are maximally produced only when lactose is available to the cell and glucose is not. [Note: Bacteria use glucose, if available, as a fuel in preference to any other sugar.] The regulatory portion of the operon is upstream of the three structural genes and consists of the promoter region where RNA pol binds and two additional sites, the operator (O) and the catabolite activator protein (CAP) sites, where regulatory proteins bind. The lacZ, lacY, and lacA genes are maximally expressed only when the O site is empty and the CAP site is bound by a complex of cyclic adenosine monophosphate ([cAMP] see p. 94) and the CAP, sometimes called the cAMP regulatory protein (CRP). A regulatory gene, the lacI gene, codes for the repressor protein (a trans-acting factor) that binds to the O site with high affinity. [Note: The lacI gene has its own promoter and is not part of the lac operon.] 1.
When only glucose is available: In this case, the lac operon is repressed (turned off). Repression is mediated by the repressor protein binding via a helix-turn-helix motif (Fig. 33.5) to the O site, which is downstream of the promoter (see Fig. 33.4A). Binding of the repressor interferes with the binding of RNA pol to the promoter, thereby inhibiting transcription of the structural genes. This is an example of negative regulation.
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2.
When only lactose is available: In this case, the lac operon is induced (maximally expressed, or turned on). A small amount of lactose is converted to an isomer, allolactose. This compound is an inducer that binds to the repressor protein, changing its conformation so that it can no longer bind to the O site. In the absence of glucose, adenylyl cyclase is active, and cAMP is made and binds to the CAP. The cAMP–CAP transacting complex binds to the CAP site, causing RNA pol to initiate transcription with high efficiency at the promoter site (see Fig. 33.4B). This is an example of positive regulation. The transcript is a single polycistronic mRNA molecule that contains three sets of start and stop codons. Translation of the mRNA produces the three proteins that allow lactose to be used for energy production by the cell. [Note: In contrast to the inducible lacZ, lacY, and lacA genes, whose expression is regulated, the lacI gene is constitutive. Its gene product, the repressor protein, is always made and is active unless the inducer is present.] 3.
When both glucose and lactose are available: In this case, the lac operon is uninduced, and transcription is negligible, even if lactose is present at a high concentration. Adenylyl cyclase is inhibited in the presence of glucose (a process known as catabolite repression) so no cAMP–CAP complex forms, and the CAP site remains empty. Therefore, the RNA pol is unable to effectively initiate transcription, even though the repressor is not bound to the O site. Consequently, the three structural genes of the operon are expressed only at a very low (basal) level (see Fig. 33.4C). [Note: Induction causes a 50-fold enhancement over basal expression.]
D. Tryptophan operon
The tryptophan (trp) operon contains five structural genes that code for enzymes required for the synthesis of the amino acid tryptophan. As with the lac operon, the trp operon is subject to negative control. However, for the repressible trp operon, negative control includes Trp itself binding to a repressor protein and facilitating the binding of the repressor to the operator: Trp is a corepressor. Because repression by Trp is not always complete, the trp operon, unlike the lac operon, is also regulated by a process known as attenuation. With attenuation, transcription is initiated but is terminated well before completion (Fig. 33.6). If Trp is plentiful, transcription initiation that escaped repression by Trp is attenuated (stopped) by the formation of an attenuator, a hairpin (stem-loop) structure in the mRNA similar to that seen in rho-independent termination (see p. 437). [Note: Because transcription and translation are temporally linked in prokaryotes (see p. 454), attenuation also results in the formation of a truncated, nonfunctional peptide product that is rapidly degraded.] If Trp becomes scarce, the operon is expressed. The 5′-end of the mRNA contains two adjacent codons for Trp. The lack of Trp causes ribosomes to stall at these codons, covering regions of the mRNA required for formation of the attenuation hairpin. This prevents attenuation and allows transcription to continue.
Transcriptional attenuation can occur in prokaryotes because translation of an mRNA begins before its synthesis is complete. This does not occur in eukaryotes because the presence of a membrane-bound nucleus spatially and temporally separates transcription and translation.
E. Coordination of transcription and translation
Although transcriptional regulation of mRNA production is primary in bacteria, regulation of ribosomal RNA (rRNA) and protein synthesis plays important roles in adaptation to environmental stress.
1.
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Stringent response: E. coli has seven operons that synthesize the rRNA needed for ribosome assembly, and each is regulated in response to changes in environmental conditions. Regulation in response to amino acid starvation is known as the stringent response. The binding of an uncharged transfer RNA (tRNA) to the A site of a ribosome (see p. 452) triggers a series of events that leads to the production of the alarmone guanosine tetraphosphate (ppGpp). The synthesis of this unusual derivative of guanosine diphosphate (GDP) is catalyzed by stringent factor (RelA), an enzyme physically associated with ribosomes. Elevated levels of ppGpp result in inhibition of rRNA synthesis (Fig. 33.7). [Note: In addition to rRNA synthesis, tRNA synthesis and some mRNA synthesis (for example, for ribosomal proteins) are also inhibited. However, synthesis of mRNA for enzymes required for amino acid biosynthesis is not inhibited. ppGpp binds RNA pol and alters promoter 2.
Regulatory ribosomal proteins: Operons for ribosomal proteins (rproteins) can be inhibited by an excess of their own protein products. For each operon, one specific r-protein functions in the repression of selection through use of different sigma factors for the polymerase (see p. 435).] translation of the polycistronic mRNA from that operon (Fig. 33.8). The r-protein does so by binding to the Shine-Dalgarno (SD) sequence located on the mRNA just upstream of the first initiating AUG codon (see p. 448) and acting as a physical impediment to the binding of the small ribosomal subunit to the SD sequence. Thus, one r-protein inhibits synthesis of all the r-proteins of the operon. This same r-protein also binds to rRNA and with a higher affinity than for mRNA. If the concentration of rRNA falls, the r-protein then is available to bind its own mRNA and inhibit its translation. This coordinated regulation keeps the synthesis of r-proteins in balance with the transcription of rRNA, so that each is present in appropriate amounts for the formation of ribosomes.
IV. REGULATION OF EUKARYOTIC GENE EXPRESSION
The higher degree of complexity of eukaryotic genomes, as well as the presence of a nuclear membrane, necessitates a wider range of regulatory processes. As with the prokaryotes, transcription is the primary site of regulation. Again, the theme of trans-acting factors binding to cis-acting elements is seen. Operons, however, are not found in eukaryotes, which must use alternate strategies to solve the problem of how to coordinately regulate all the genes required for a specific response. In eukaryotes, gene expression is also regulated at multiple levels other than transcription. For example, the major modes of posttranscriptional regulation at the mRNA level are alternative mRNA splicing and polyadenylation, control of mRNA stability, and control of translational efficiency. Additional regulation at the protein level occurs by mechanisms that modulate stability, processing, or targeting of the protein.
A. Coordinate regulation
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The need to coordinately regulate a group of genes to cause a particular response is of key importance in organisms with more than one chromosome. An underlying theme occurs repeatedly: A trans-acting protein functions as a specific transcription factor (STF) that binds to a cisacting regulatory consensus sequence (see p. 415) on each of the genes in the group even if they are on different chromosomes. [Note: The STF has a DNA-binding domain (DBD) and a transcription activation domain (TAD). The TAD recruits coactivators, such as histone acetyltransferases (see p. 438), and the general transcription factors (see p. 439) that, along with RNA pol, are required for formation of the transcription initiation complex at the promoter. Although the TAD recruits a variety of proteins, the specific effect of any one of them is dependent upon the protein composition of the complex. This is known as combinatorial control.] Examples of coordinate regulation in eukaryotes include the galactose circuit and the hormone response system.
1. Galactose circuit: This regulatory scheme allows for the use of galactose when glucose is not available. In yeast, a unicellular organism, the genes required to metabolize galactose are on different chromosomes. Coordinated expression is mediated by the protein Gal4 (Gal = galactose), a STF that binds to a short regulatory DNA sequence upstream of each of the genes. The sequence is called the upstream activating sequence Gal (UASGal). Binding of Gal4 to UASGal through zinc fingers in its DBD occurs in both the absence and presence of galactose. When the sugar is absent, the regulatory protein Gal80 binds Gal4 at its TAD, thereby inhibiting gene transcription (Fig. 33.9A). When present, galactose activates the Gal3 protein. Gal3 binds Gal80, thereby allowing Gal4 to activate transcription (Fig. 33.9B). [Note: Glucose prevents the use of galactose by inhibting expression of Gal4 protein.]
B. presence of galactose. [Note: Target genes, whether on the same or a different chromosome, each have an upstream activating sequence galactose (UASGal).] TAD = transcription activation domain; DBD = DNA-binding domain; mRNA = messenger RNA.
2. Hormone response system: Hormone response elements (HRE) are DNA sequences that bind trans-acting proteins and regulate gene expression in response to hormonal signals in multicellular organisms. Hormones bind to either intracellular (nuclear) receptors (for example, steroid hormones; see p. 240) or cell-surface receptors (for example, the peptide hormone glucagon; see p. 314).
a.
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Intracellular receptors: Members of the nuclear receptor superfamily, which includes the steroid hormone (glucocorticoids, mineralocorticoids, androgens, and estrogens), vitamin D, retinoic acid, and thyroid hormone receptors, function as STF. In addition to domains for DNA-binding and transcriptional activation, these receptors also contain a ligand-binding domain. For example, the steroid hormone cortisol (a glucocorticoid) binds intracellular receptors at the ligand-binding domain (Fig. 33.10). Binding causes a conformational change in the receptor that activates it. The receptor– hormone complex enters the nucleus, dimerizes, and binds via a zinc finger motif to DNA at a regulatory element, the glucocorticoid response element (GRE) that is an example of a HRE. Binding allows recruitment of coactivators to the TAD and results in expression of cortisol-responsive genes, each of which is under the control of its own GRE. Binding of the receptor–hormone complex to the GRE allows coordinate expression of a group of target genes, even though these genes are on different chromosomes. The GRE can be located upstream or downstream of the genes it regulates and at great distances from them. The GRE, then, can function as a true enhancer (see p. 440). [Note: If associated with repressors, hormone–receptor complexes inhibit transcription.] b.
Cell-surface receptors: These receptors include those for insulin, epinephrine, and glucagon. Glucagon, for example, is a peptide hormone that binds its G protein–coupled plasma membrane receptor on glucagon-responsive cells. This extracellular signal is then transduced to intracellular cAMP, a second messenger (Fig. 33.11; also see Fig. 8.7 on p. 95), which can affect protein expression (and activity) through protein kinase A–mediated phosphorylation. In response to a rise in cAMP, a trans-acting factor (cAMP response element–binding [CREB] protein) is phosphorylated and activated. Active CREB protein binds via a leucine zipper motif to a cis-acting regulatory element, the cAMP response element (CRE), resulting in transcription of target genes with CRE in their promoters. [Note: The genes for phosphoenolpyruvate carboxykinase and glucose 6phosphatase, key enzymes of gluconeogenesis (see p. 122), are examples of genes upregulated by the cAMP/CRE/CREB system.]
B. Messenger RNA processing and use
Eukaryotic mRNA undergoes several processing events before it is exported from the nucleus to the cytoplasm for use in protein synthesis. Capping at the 5′-end (see p. 441), polyadenylation at the 3′-end (see p. 442), and splicing (see p. 442) are essential for the production of a functional eukaryotic messenger from most pre-mRNA. Variations in splicing and polyadenylation can affect gene expression. In addition, messenger stability also affects gene expression.
1.
Alternative splicing: Tissue-specific protein isoforms can be made from the same pre-mRNA through alternative splicing, which can involve exon skipping (loss), intron retention, and use of alternative splice-donor or -acceptor sites (Fig. 33.12). For example, the pre-mRNA for tropomyosin (TM) undergoes tissue-specific alternative splicing to yield a number of TM isoforms (see p. 443). [Note: Over 90% of all human genes undergo alternative splicing.] 2.
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Alternative polyadenylation: Some pre-mRNA transcripts have more than one site for cleavage and polyadenylation. Alternative polyadenylation (APA) generates mRNA with different 3′-ends, altering the untranslated region (UTR) or the coding (translated) sequence. [Note: APA is involved in the production of the membrane-bound and secreted forms of immunoglobulin M.]
The use of alternative splicing and polyadenylation sites, as well as alternative transcription start sites explains, at least in part, how the ~20,000 to 25,000 genes in the human genome can give rise to well over 100,000 proteins.
3. Messenger RNA editing: Even after mRNA has been fully processed, it may undergo an additional posttranscriptional modification in which a base in the mRNA is altered. This is known as RNA editing. An important example in humans occurs with the transcript for apolipoprotein (apo) B, an essential component of chylomicrons (see p.
228) and very-low-density lipoproteins ([VLDL] see p. 230). Apo B mRNA is made in the liver and the small intestine. However, in the intestine only, the cytosine (C) base in the CAA codon for glutamine is enzymatically deaminated to uracil (U), changing the sense codon to the nonsense or stop codon UAA, as shown in Figure 33.13. This results in a shorter protein (apo B-48, representing 48% of the message) being made in the intestine (and incorporated into chylomicrons) than is made in the liver (apo B-100, full-length, incorporated into VLDL).
U = uracil.
4. Messenger RNA stability: How long an mRNA remains in the cytosol before it is degraded influences how much protein product can be produced from it. Regulation of iron metabolism and the gene-silencing process of RNA interference (RNAi) illustrate the importance of mRNA stability in the regulation of gene expression.
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