Regulation of Fuel Utilization in Response to Food Intake

Chapter 19

Regulation of Fuel Utilization in Response to Food Intake

Martha H. Stipanuk, PhD

Humans require food to maintain body tissues and health and to perform work. Food is a source of all of the nutrients, but the amount or dry weight of food that is required is largely determined by energy expenditure. The bulk of the carbohydrates, lipids, and proteins, as well as alcohol, consumed in the human diet are metabolized to provide the energy needed to support cellular and bodily functions and physical activity. Despite the need for continual metabolism of macronutrients as a source of energy, the intake of fuel from the diet is discontinuous. Vertebrates have the capacity to deal with the inevitable periods of food deprivation by storing fuel, first as glycogen and then as triacylglycerol, when food is available and then using these stored fuels during times of deprivation. To a limited extent, this same cycle functions over the feeding and fasting episodes that occur during a 24-hour period, with an important example being the use of liver glycogen stores to maintain plasma glucose levels during the overnight period.

Adults typically consume a diet of mixed foods that supplies 1,500 to 3,000 kcal per day. In the United States, approximately 15% of the energy is supplied by protein, 50% by carbohydrate, and 35% by fat, but the proportion provided by each macronutrient can vary considerably for different populations and for individuals within a population and still be adequate. The body must regulate the metabolism of the amino acids, sugars, and triacylglycerols taken up from the gastrointestinal tract during the absorptive phase to meet immediate energy needs and also to store fuel to meet energy needs during the postabsorptive period and potentially during longer periods of food deprivation. In addition, the use of some essential nutrients as fuels must be coordinately regulated with their use for other essential processes, such as the use of indispensable amino acids for protein synthesis or certain fatty acids for synthesis of membrane lipids.

In addition to the overall fuel needs of the body, the synthesis, oxidation, and storage of each of the individual macronutrients must be regulated to meet the specific needs of the various body tissues. In the context of fuel metabolism, different tissues have unique functions and metabolic pathways and also are exposed to circulating fuels in differing ways. For example, the portal blood draining the gastrointestinal tract is the major blood supply to the liver such that the glucose and amino acid supply to the liver during the absorptive period exceeds the fuel needs of the liver itself, and the liver plays a key role in modulating the mixture of circulating fuels that are supplied to the rest of the body. In addition, some tissues require specific types of fuel molecules. Red blood cells, which do not contain mitochondria, cannot obtain energy by oxidizing fatty acids, but they can generate ATP by converting glucose to lactate, which is released back into the circulation. If plasma glucose levels are not maintained, tissues dependent upon glucose as a fuel will suffer damage. Without energy, cells no longer function and eventually die, resulting in tissue injury. Lack of fuel can even result in permanent brain damage, stroke, and death. The brain is capable of using only glucose and ketone bodies as major fuels; although brain cells have mitochondria, the blood–brain barrier in key regions of the brain limits the rate of transfer of long-chain fatty acids. Thus, long-chain fatty acids are not a major fuel for the brain even when their level in the circulation is elevated (Vannucci and Hawkins, 1983). Ketone body production by the liver plays a key role in reducing brain glucose use during prolonged starvation. Thus the body must maintain a mixture of circulating fuel molecules that will meet the needs of all tissues, and this might be remarkably different from the mixture absorbed from the gastrointestinal tract following a meal or the mixture released from endogenous stores of fat and glycogen during starvation. Table 19-1 summarizes the main circulating fuels that are used and released by various tissues.

Specific macronutrients may be only partially catabolized within a particular tissue, with released partially oxidized compounds consequently serving as metabolic fuels for other tissues. The production of ketone bodies produced from fatty acids by the liver, the release of branched-chain keto acids by the skeletal muscle, and the production of lactate from glucose by red blood cells are examples. At the tissue level, the definition of a fuel includes both the macromolecules absorbed from the gastrointestinal tract or released from endogenous storage reserves and some partially oxidized forms of these molecules. Thus a metabolic fuel may be defined as a circulating compound that is taken up by tissues for energy production; the major circulating fuels include glucose, triacylglycerols in lipoprotein complexes, amino acids, fatty acids, ketone bodies, lactate, and glycerol.

In this chapter, fuel utilization in the postprandial or absorptive (fed) state is compared to that in the postabsorptive (fasted) and more extreme starved state. These contrasts illustrate various regulatory processes governing macronutrient metabolism and fuel utilization. A further discussion of fuel utilization by skeletal muscle in the context of rest and exercise is the topic of Chapter 20. In preparation for describing the changes in fuel utilization that occur under these conditions, this chapter begins with a brief overview of the mechanisms used by tissues to regulate metabolism in the face of changing fuel availability or energy need.

Regulation of Macronutrient Metabolism at the Whole-Body Level

Several whole-body mechanisms play an extremely important role in the regulation of fuel metabolism.

Changes in Nutrient Supply

The body must rely on food intake or its own stores for a source of glucose, fatty acids, and amino acids for use as fuels. Because food intake is not continuous but occurs as discrete meals, with varying periods between meals, the body must have mechanisms to store excess fuel for later use. Meal size and composition will influence metabolism by establishing the exogenous nutrients the body must use or store. In the absence of food intake, the body must rely on stored fuels, mainly glycogen and triacylglycerols.

Release of endocrine hormones, particularly the pancreatic hormones insulin and glucagon, responds to meal size and composition and thus plays an over-arching role in communicating the body’s status with respect to influx of nutrients from the gastrointestinal tract to various tissues and in triggering metabolic remodeling of the tissue to respond appropriately to changes in nutrient supply. As discussed in Chapters 12 and 16, high insulin/low glucagon promotes lipid and carbohydrate storage, whereas low insulin/high glucagon results in stimulated adipose tissue lipolysis and hepatic glucose production. High insulin and low glucagon levels are characteristic of the postprandial state, whereas low insulin and high glucagon levels are characteristic of the fasted state.

The body has a complex system for regulation of food intake and energy expenditure that involves the central nervous system’s integration of numerous signals from the gastrointestinal tract, pancreas, and adipose tissue. Output from the central nervous system stimulates changes in food intake to maintain energy balance. These include the impetus to initiate eating and also the satiety/satiation effects that promote the ending of a meal; regulation of food intake is discussed in more detail in Chapter 22.

Underlying the use of macronutrients by tissues in response to changes in nutrient availability is the metabolic flexibility of most tissues to use more than one fuel. Glucose and fatty acids are the major fuels used by tissues, and most tissues can use either glucose or fatty acids. The “glucose–fatty acid cycle,” which was first proposed by Randle and associates (1963), is a helpful concept in understanding fuel flux between tissues and fuel selection by tissues. The primary tenet of the Randle cycle was that the increased provision of exogenous lipid fuels or the increased breakdown of endogenous triacylglycerol stores promotes the use of lipid fuels and, in so doing, blocks the utilization of glucose. This was based on studies with isolated heart and skeletal muscle preparations in which they found that the provision of lipid fuels (fatty acids, ketone bodies) suppressed glucose uptake and glycolysis whether or not hormones were added. Thus the glucose–fatty acid cycle drew attention to the competition between glucose and fatty acids for their oxidation in muscle. Although hormones such as insulin play important roles not only in determining nutrient availability but also in altering the metabolic capacities of tissues, Randle’s observation that the utilization of one nutrient could inhibit the use of the other nutrient directly and without hormonal mediation introduced the concept of nutrient-mediated fine-tuning of fuel utilization that occurs on top of the more coarse substrate availability/hormonal effects. These observations also led to the discovery of biochemical mechanisms by which fatty acids suppressed glucose utilization and, subsequently, of mechanisms by which glucose could suppress fatty acid utilization (Hue and Taegtmeyer, 2009). Some of these mechanisms are discussed in subsequent text and in Chapters 12 and 16.

Blood Flow Patterns

Because nutrients are carried to and from tissues via the blood, blood flow patterns and rates, as well as the form of nutrients in the blood, can affect nutrient availability. For example, the drainage of the splanchnic tissues into the portal vein, which becomes the major blood supply to the liver, exposes the liver to high concentrations of absorbed nutrients. During exercise, the increase in blood flow to the skeletal muscle results in the transport of more oxygen and blood-borne fuels to the working muscle. The transport of absorbed lipids as triacylglycerol in chylomicrons prevents the liver and other tissues from being exposed to high levels of free fatty acids during the fed state and facilitates their direction to adipose tissue for storage. Hemodynamics also affects tissue exposure to circulating hormones. For example, resting skeletal muscle is relatively insensitive to changes in circulating levels of insulin compared to working muscle.


The Glucose–Fatty Acid Cycle

In 1963 Philip Randle, Peter Garland, Nick Hales, and Eric Newsholme published a paper in Lancet in which they proposed a glucose–fatty acid cycle, which came to be commonly referred to as the “Randle cycle” (Randle et al., 1963). The primary tenet of the Randle cycle was that the increased provision of exogenous lipid fuels or the increased breakdown of endogenous triacylglycerol stores promotes the use of lipid fuels and, in so doing, blocks the utilization of glucose.


Randle and colleagues showed, in studies with isolated heart and skeletal muscle preparations, that the provision of lipid fuels (fatty acids, ketone bodies) suppressed glucose uptake and glycolysis whether or not hormones were added. Randle went on to demonstrate the role of several glycolytic enzymes in the “fine-tuning” of fuel utilization at the cellular level. In particular, Randle demonstrated the role of the products of β-oxidation in inhibition of the pyruvate dehydrogenase complex, thus blocking glucose/pyruvate oxidation when lipid fuels were being metabolized. Fatty acid or ketone body oxidation in the mitochondria results in increases in the mitochondrial ratios of acetyl-CoA/CoA and NADH/NAD+, both of which strongly inhibit pyruvate dehydrogenase complex activity (Randle, 1998).

Although Randle and colleagues recognized the effects of glucose and insulin in promoting triacylglycerol storage and by blocking lipolysis and β-oxidation of fatty acids, it was more than a decade before a cellular mechanism for the reciprocal process by which glucose oxidation can block utilization of fatty acids as fuel was elucidated (McGarry et al., 1977). This mechanism involves the formation of malonyl-CoA by acetyl-CoA carboxylase when glucose is available as a fuel and the consequent inhibition of carnitine palmitoyltransferase 1 by malonyl-CoA (McGarry, 1998). In addition, metabolism of glucose by adipose tissue fosters the storage of fatty acids by providing glycerol 3-phosphate for triacylglycerol synthesis.

Today we know that the regulation of fuel utilization is complex and occurs at many levels in addition to the short-term regulation of pyruvate dehydrogenase and acetyl-CoA carboxylase (Hue and Taegtmeyer, 2009). Nevertheless, the fundamental concept of cellular coordination of metabolic fuel selection developed by Randle and colleagues made a large contribution to our current understanding of fuel utilization by various tissues.

Distributed Control

Consideration of the many factors that influence fuel utilization has led to the understanding that metabolic regulation of fuel utilization is under “distributed control” by several different processes, and that the steps that limit fuel utilization vary in response to physiological and biochemical changes. Both whole-body and cell-specific processes are involved. An excellent example of distributed control is provided by the work of Wasserman and colleagues on the regulation of glucose flux into muscle in vivo (Wasserman et al., 2011). Muscle glucose uptake is regulated by control of glucose delivery to muscle, control of membrane transport into muscle, and control of the phosphorylation of glucose within muscle. Glucose transport into muscle is regulated by translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane. In the fasted state GLUT4 limits glucose uptake, but in the insulin-stimulated or fed state the upregulated GLUT4 glucose uptake is such that other factors become more limiting. Carbohydrate intake and hepatic glucose production are important factors affecting glucose concentrations and hence muscle glucose delivery. Glucose is phosphorylated by hexokinase, which traps glucose in the myocyte. If glucose 6-phosphate accumulates, this feedback inhibits hexokinase and slows glucose uptake and utilization. Pathways downstream of glucose phosphorylation (i.e., glycogen synthesis and glycolysis) can play a role in control of glucose uptake through their effects on glucose 6-phosphate levels.

Regulation of Macronutrient Metabolism at the Cellular Level

Fuel metabolism occurs at the level of individual cells in various tissues. Different tissues and even various types of cells within tissues are differentiated to serve their particular functions, and this is associated with the expression, or lack of expression, of genes encoding enzymes of different pathways, receptors for various hormones, transporters for particular substrates, particular transcription factors, and other proteins. Most tissues have some flexibility to adapt to using either glucose or fatty acids as the major fuel, and both rapid and longer-term metabolic remodeling occurs within tissues to respond to short-term changes in food intake and to longer-term dietary changes, respectively.

Biochemical Mechanisms of Regulation of Metabolic Pathways

In describing metabolism, we often look at particular pathways as going from beginning substrate(s) through various steps to end product(s) of the pathway. Many reactions in pathways, however, are reversible and function at near equilibrium, and many intermediates or substrates are shared by various pathways and essentially link the pathways to each other. Intermediates may enter or leave pathways at any of the sequential steps. For example, oxaloacetate might be seen as a citric acid cycle intermediate that does not leave the cycle, but it also is used as a substrate for gluconeogenesis, as a partner with malate for movement of reducing equivalents out of the mitochondria, and as a precursor of aspartate by transamination. It can also be synthesized by transamination of aspartate, dehydrogenation of malate generated in the cytosol, or carboxylation of pyruvate.

Because tissues with different capabilities work together to maintain various bodily functions, it is difficult to view fuel metabolism as occurring simply within individual cells or individual tissues. For example, even the simple pathway of glycolysis in red blood cells, which appears to begin with glucose and end with lactate, actually requires the continual provision (from the intestine or liver) of glucose into the plasma and the continual removal of the lactate by the liver. Thus the utilization of glucose by red blood cells is directly linked to the control of such processes as hepatic glycogen metabolism and gluconeogenesis. The glucose–alanine cycle similarly functions to recover the carbon skeleton (pyruvate) of alanine for gluconeogenesis, recycling the glucose carbon back to muscle or other tissues. As an additional example, during prolonged starvation, the upregulation of lipolysis in adipose tissue is accompanied by increased synthesis of ketone bodies from these fatty acids by the liver and consequently increased uptake and oxidation of ketone bodies by the muscle and brain. Thus metabolic remodeling in one tissue needs to be coordinated with metabolic changes in other tissues.

Regulation of Flux through a Metabolic Pathway

The regulation of flux through a pathway is usually regulated by multiple factors, and each of those factors may in turn be regulated by multiple factors. The ultimate flux through a particular pathway under a given circumstance in the cell reflects the integrated regulation of many different steps in the pathway, with each catalyzed by a different protein, perhaps with each protein being regulated by multiple mechanisms. Flux also depends upon regulation of the uptake of substrates and the removal of intermediates or products. In most cases of macronutrient metabolism, cells have opposing pathways, as, for example, glycogenesis and glycogenolysis or glycolysis and gluconeogenesis. In general, both pathways are operating all the time and the ability to upregulate one while downregulating the other allows the cell to fine-tune the overall metabolic flux to the needs of the body. Such reciprocal and coordinated regulation is common in macronutrient metabolism. The coordinated regulation of opposing pathways is further facilitated by spatial segregation of some opposing pathways in different tissues (e.g., ketone body synthesis in liver and ketone body oxidation in extrahepatic tissues) or in different subcellular compartments (e.g., cholesterol synthesis from acetyl-CoA in the cytosol and ketone body synthesis from acetyl-CoA in the mitochondria; de novo synthesis of fatty acids in the cytosol and β-oxidation of fatty acids in the mitochondria).

Although regulation of metabolism generally involves many types of regulation at many different steps in a pathway and related pathways and processes, some changes have large effects on flux through a pathway, while other changes have little or no effect. Obviously, those that result in changes in flux are more significant physiologically, but it is not always easy to discern the consequence of an individual change in the context of the whole body. One example of a highly regulated enzyme with substantial physiological consequences for cholesterol synthesis is 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (Goldstein et al., 2006). This is an early step in the overall cholesterol synthetic pathway that is highly regulated and can exhibit a many-fold change in activity. HMG-CoA reductase is the target of the statin drugs used to treat patients with hypercholesterolemia.

Short-Term and Long-Term Regulation

In describing the regulation of the cellular capacity to perform various enzymatic reactions or other processes, we often speak of short-term and long-term regulation. As their names imply, they may be distinguished by the length of time required for bringing about changes, but this distinction does not apply absolutely. In reality, these two terms refer to two distinct mechanisms of regulation. Short-term control refers to changes in the specific activity of an enzyme (or transporter, transcription factor, etc.), with no change in its concentration. Long-term control refers to changes in the amount of enzyme (or transporter, transcription factor, etc.), with no change in its kinetic properties. The two mechanisms are not exclusive, and some proteins are subject to both short-term and long-term control in response to the same stimulus or different stimuli. In some cases, a long-term response can occur as rapidly as a short-term response; control of protein concentration is highly dependent on the half-life of the protein and new steady state concentrations are reached quickly for proteins with very short half-lives (see Chapter 13). Regardless of whether they are short-term or long-term, regulatory mechanisms must be reversible if they are to be of physiological importance. In other words, the concentration or the activity state of the protein must be able to be both upregulated and downregulated.

Short-Term Control of Metabolism

Short-term control can be brought about by a variety of mechanisms. Many specific examples are given in Chapters 12, 14, 16, and 17.

Changes in Levels of Key Metabolites or Regulators

Changes in fuel availability may bring about changes in flux through metabolic pathways. Either an altered concentration of a substrate or an altered concentration of a metabolite in the pathway can result in a change in the concentration of allosteric effectors. Short-term regulation is often related to small changes in the levels of key intermediates such as ATP and NADH. This may result simply from changes in cosubstrate or coenzyme supply. For example, a change in NADH concentration will usually be mirrored by an opposite change in NAD+, and a change in acyl-CoA concentration likewise will result in an opposite change in the free coenzyme A (CoA) concentration. Any change in these ratios will affect flux through enzymatic reactions utilizing one of these couplets. Both allosteric activation and inhibition are also important short-term mechanisms that involve the binding of a regulatory molecule to a distinct site on an enzyme. Several examples of short-term control can be seen in the pathway for glycolysis. For example, ATP is a strong inhibitor of 6-phosphofructo-1-kinase, and this inhibition can be relieved by a second allosteric factor, such as fructose 2,6-bisphosphate in liver or AMP in muscle (see Chapter 12, Figure 12-8). Hexokinase is inhibited by glucose 6-phosphate, restricting uptake of glucose in tissues that do not express the glucokinase isoform, which is insensitive to the inhibitory effects of glucose 6-phosphate. Hepatic pyruvate kinase is stimulated by fructose 1,6-bisphosphate, which is elevated when glucose is being metabolized, and inhibited by alanine, which is an important gluconeogenic substrate during starvation (see Chapter 12, Figure 12-8). In addition, hepatic pyruvate kinase is subject to covalent modification (phosphorylation) by 5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA); this makes it more sensitive to allosteric inhibition. The inhibition of mitochondrial uptake of long-chain fatty acids by the effects of malonyl CoA on the carnitine palmitoyltransferase 1 (CPT1) is another example of regulation by allosteric effects of small molecules; in this case a substrate or intermediate in a competing pathway, fatty acid synthesis, acts to inhibit the opposing pathway, fatty acid oxidation.

Reversible Covalent Modification of Proteins

Reversible covalent modification of proteins is another important short-term regulatory mechanism. A common type of regulatory modification is phosphorylation–dephosphorylation, catalyzed by kinases and phosphatases. Phosphorylation of proteins usually occurs on particular serine, threonine, or tyrosine residues of the proteins, resulting in activation of some proteins and in inhibition of others. Phosphorylation may affect the target protein by changing its kinetic properties, its ability to associate with other proteins, its sensitivity to allosteric effectors, or its intracellular localization. Important examples of enzymes that are controlled by phosphorylation–dephosphorylation include hormone-sensitive lipase in adipose tissue, which is activated by phosphorylation in response to catecholamines and inactivated by dephosphorylation in response to insulin (see Chapter 16, Figure 16-9); acetyl-CoA carboxylase in liver, which is activated by dephosphorylation in response to insulin, so that cytosolic acetyl-CoA is converted to malonyl-CoA (see Chapter 16, Figure 16-3); glycogen synthase, which is inactivated by phosphorylation in response to glucagon in liver (see Chapter 12, Figure 12-15); and 6-phosphofructo-2-kinase, a bifunctional enzyme that loses kinase activity while its fructose 2,6-bisphosphatase activity is enhanced in response to glucagon in liver (see Chapter 12, Figure 12-10).

Another very important phosphorylation is that of the pyruvate dehydrogenase complex, which is inactivated by phosphorylation catalyzed by a specific kinase bound to the enzyme complex. Phosphorylation (inactivation) of the pyruvate dehydrogenase complex is stimulated by acetyl-CoA and NADH and inhibited by high CoA, NAD+, or pyruvate (see Chapter 12, Figure 12-18). These effectors influence the activity of the pyruvate dehydrogenase kinase that catalyzes the covalent modification and inactivation of the pyruvate dehydrogenase complex. The tissue-specific expression of different isoforms of pyruvate dehydrogenase kinase and phosphatase allows different tissues to respond differently in terms of pyruvate dehydrogenase complex inactivation during starvation (Sugden and Holness, 2006; Harris et al., 2002).

Thus the oxidative decarboxylation of pyruvate to acetyl-CoA is inhibited when the cell is oxidizing fatty acids or ketone bodies as fuel, playing a significant role in the suppression of glucose oxidation when fatty acids are available as a fuel (i.e., the Randle or glucose-fatty acid cycle). The mitochondrial β-oxidation of these lipid fuels generates high acetyl-CoA and NADH levels during starvation, and this is a condition in which glucose carbon needs to be conserved. In the presence of high acetyl-CoA and NADH and in the absence of insulin or excess pyruvate, the pyruvate dehydrogenase kinase will be activated and its phosphorylation of the pyruvate dehydrogenase complex will cause the complex to be inactivated. Inactivation of the pyruvate dehydrogenase complex blocks conversion of pyruvate to acetyl-CoA, thus conserving the pyruvate carbon and reducing net glucose oxidation. On the other hand, in the fed state when glucose is available as a fuel, the sensitivity of pyruvate dehydrogenase kinases to pyruvate allows regulation of the pyruvate dehydrogenase complex in response to glucose and insulin availability. When the cell is exposed to a high level of glucose, the kinase is inhibited by pyruvate and, in addition, the dephosphorylation of pyruvate dehydrogenase complex by its phosphatase is stimulated by insulin. Hence the pyruvate dehydrogenase is in its active, dephosphorylated form. This allows conversion of pyruvate to acetyl-CoA and its entrance into the citric acid cycle for oxidative metabolism as a fuel. In the liver, a lipogenic tissue, the higher activity of the pyruvate dehydrogenase complex in the fed state allows more entrance of acetyl-CoA into the citric acid cycle, with the accumulation of citrate and its ultimate use for de novo lipogenesis.

Changes in Intracellular Localization of Proteins

A somewhat different form of short-term control is the physical movement of proteins within a cell. Sequestering a protein in a subcellular compartment, which is often facilitated by other proteins that associate with the regulated protein, can remove it from the spatial location where it carries out its function. In contrast, processes that facilitate the movement of a protein to the location where it carries out its function (e.g., the movement of a transcription factor to the nucleus or a transporter to the plasma membrane) will upregulate its physiological effect. A well-known example of this is the sequestering of GLUT4 glucose transporters in intracellular vesicles until insulin activates the movement of these insulin-sensitive glucose transporters to the cell surface of myocytes and adipocytes. The increase in GLUT4 transporters in the plasma membrane increases the glucose transport capacity of these cells and can result in increased uptake and utilization of glucose by the tissue. Sometimes a combination of methods of short-term regulation are involved as, for example, in the phosphorylation of forkhead box class O (FoxO) transcription factors by Akt, which results in exclusion of FoxO from the nucleus where it would act as a transcriptional activator. Another example is the movement and activation of glucokinase and glycogen synthase within the liver in response to hormones, a process that results in these proteins becoming physically close to or removed from their substrates (Watford, 2002). In the postabsorptive state glucokinase is present within the nucleus where it is bound by a glucokinase regulatory protein together with fructose 6-phosphate. During the absorptive period in the presence of fructose 1-phosphate or high glucose, glucokinase is released from the regulatory protein and translocates to the cytosol where it is active in hepatic glucose uptake and metabolism (see Chapter 12, Figure 12-9).

Long-Term Control of Metabolism

Changes in protein expression are important in the metabolic remodeling of tissues, allowing the body to adapt to sustained changes in nutrient availability or physiological state. Long-term changes can alter the overall capacity of the metabolic system to respond to change and may improve or diminish the short-term regulatory response to subsequent physiological stressors.

Changes in the amount of protein (long-term control) are brought about by changes in the synthesis and/or degradation of the protein and consequently are rather slow, often taking hours or days to occur. However, some enzymes can show these long-term changes within relatively short time-periods (less than 1 hour), which can be comparable to the timescale over which some short-term covalent modifications take place. The steady state concentrations of proteins are functions of the rates of their synthesis and degradation. Different proteins exhibit half-lives that range from a few minutes to many days. Proteins with very short half-lives not only will be degraded rapidly but also will respond rapidly to changes in the synthesis or degradation rate. Thus enzymes with short half-lives are able to establish new steady state concentrations much more quickly than proteins with long half-lives. Such proteins are ideal candidates for regulation.

In recent decades, much emphasis has been given to predicting changes in metabolism from changes in the abundance of messenger RNA (mRNA) transcripts of genes. Regulation of levels of functional proteins, however, is a much more complex process. The synthesis of a protein depends upon gene transcription, mRNA processing, mRNA stability and/or localization, and mRNA translation. Furthermore, the function or activity of a translated protein depends upon proper posttranslational modifications, folding, and trafficking to its correct location, as well as its subsequent regulation by covalent modification, allosteric effectors, or other mechanisms. The concentration of the protein also depends on the rate of degradation of the protein. Protein degradation is regulated, and most cellular proteins are degraded in a regulated manner by the ubiquitin–proteasome system. Damage to cellular proteins, such as cross-linking or oxidation of functional groups, also may affect their functions and their degradation rates.

An example of a highly regulated key enzyme of hepatic glucose metabolism is phosphoenolpyruvate carboxykinase (PEPCK). This protein has a half-life of about 6 hours and is regulated exclusively by long-term mechanisms. Most of this regulation is brought about by changes in the rate of transcription of the PEPCK gene and hence changes in the overall level of enzyme protein (see Chapter 12, Figure 12-8). The fall in circulating insulin during starvation allows the FoxO transcription factors to enter the nucleus and activate transcription of the PEPCK gene. In addition, the rise in circulating glucagon during starvation acts via cAMP to stimulate transcription of the PEPCK gene. Thus, as starvation slowly develops and as liver glycogen stores are gradually depleted, the liver is synthesizing new PEPCK and thereby increasing its capacity to carry out gluconeogenesis. Such a mechanism is highly suited to a situation such as starvation in which changes in metabolism occur over hours or days. On the other hand, this mechanism would not be suitable for rapid responses such as those needed for muscle contraction, during which short-term regulatory mechanisms are necessary to allow for compensatory responses within fractions of a second.

Long-term changes can improve or diminish short-term regulatory responses to subsequent physiological stressors. An example of how long-term adaptation can affect short-term fuel availability is illustrated by a study in which aerobically trained men were adapted to a 75% fat/5% carbohydrate diet or to a 30% fat/50% carbohydrate control diet (Pehleman et al., 2005). Adaptation to the high-fat/low-carbohydrate diet increased muscle pyruvate dehydrogenase kinase activity, which decreased pyruvate dehydrogenase activation to the “a” form, and decreased the oxidative disposal of glucose by skeletal muscle. Subsequent administration of an oral glucose load to these men resulted in decreases in pyruvate kinase activity and increases in pyruvate dehydrogenase activity, in both subjects fed the control diet and in subjects fed the high-fat/low-carbohydrate diet. However, the absolute level of pyruvate dehydrogenase “a” remained lower in men adapted to the control diet.

Many Proteins are Regulated by Multiple Mechanisms

A number of key proteins/enzymes are regulated by many different mechanisms, each having a contribution to the fine-tuning of the protein’s concentration, activity, or both. For example, acetyl-CoA carboxylase, which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, is regulated by changes in gene transcription, with enzyme concentration being increased in fed animals due to the influence of insulin and/or glucose on the activity of transcription factors such as sterol response element binding protein 1c (SREBP1c) and carbohydrate response element binding protein (ChREBP). Acetyl-CoA carboxylase is also regulated by phosphorylation/dephosphorylation, being


PPARs as Therapeutic Drug Targets: Fibrates and Thiazolidinediones

Certain lipids exert transcriptional effects through binding to peroxisome proliferator–activated receptors (PPARs). The PPARs are nuclear receptors (ligand-activated transcription factors) that contribute to the regulation of energy homeostasis via their long-term transcriptional effects. The first PPAR was first discovered during the search for the molecular target of agents that increased peroxisomal numbers in rodent liver. The three members of this family are PPARα, PPARγ, and PPARδ (also known as PPARβ). Through their distinct but overlapping functions and tissue distributions, they mediate some of the long-term adaptations of the glucose−fatty acid cycle. The physiological ligands for the PPARs have been elusive. Polyunsaturated fatty acids (n−3 and n−6) and their eicosanoid oxidation products can act as ligands for PPARs. Recent work identified 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (a phospholipid) as a physiologically relevant ligand for PPARα (Chakravarthy et al., 2009). Because of their effects on lipid storage, lipid oxidation, and prevention of oxidative stress, the PPARs have been of interest as therapeutic targets (Wang, 2010).

PPARα is the target of the hypolipidemic fibrate drugs that induce transcription of genes involved in fatty acid uptake and mitochondrial and peroxisomal β-oxidation of fatty acids, particularly in liver, kidney, heart, and skeletal muscle. Studies in mice have shown that PPARα is induced by fasting and is required for hepatic ketogenesis (Kersten et al., 1999). This action probably requires peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1α) as a coactivator. Fibrates, acting as PPARα agonists, also inhibit the hepatic synthesis of triacylglycerols and VLDL, increase expression of lipoprotein lipase, and decrease expression of apolipoprotein A-I, all of which contribute to a reduction in plasma triacylglycerol levels. By increasing expression of other proteins such as apolipoprotein A-I, fibrates also favor a rise in HDL concentrations and reverse cholesterol transport.

PPARγ is the target of thiazolidinediones (TDZs), drugs used for management of type 2 diabetes. PPARγ is expressed mainly in adipose tissue and is necessary for adipocyte differentiation. TDZs improve insulin sensitivity of obese type 2 diabetic patients in parallel with changes in fat metabolism. It is thought that TDZs cause expansion of the subcutaneous adipose tissue, allowing lipid repartitioning from the skeletal muscle and liver to adipose tissue, thus eliminating the deleterious effects of lipid on insulin signaling and glucose metabolism. TDZs may also act by influencing adipokine secretion.

phosphorylated and converted to a less active form by AMP-activated protein kinase (AMPK). In addition, acetyl-CoA carboxylase is regulated by allosteric effectors. The activity of acetyl-CoA carboxylase is increased by citrate, the cytosolic source of acetyl-CoA substrate that is present when glucose is abundant, and decreased by long-chain fatty acyl-CoAs, which are elevated during active lipolysis and β-oxidation when production of malonyl-CoA and palmitate from glucose would be undesirable. The presence of two genes encoding acetyl-CoA carboxylase 1 and 2 allow tissue-specific regulation of their expression. The first form is predominantly expressed in lipogenic tissues and is cytosolic. The second form is expressed in heart, skeletal muscle, and liver and has a unique N-terminal sequence that targets acetyl-CoA carboxylase 2 for insertion in the mitochondrial membrane. Its location near CTP1 facilitates its role in regulation of fatty acid transport into mitochondria for β-oxidation. Thus in liver of subjects consuming a high-carbohydrate diet, acetyl-CoA carboxylase 1 concentration would be high, and the enzyme would be active due to lack of phosphorylation by AMPK, to allosteric inactivation by citrate, and to lack of inactivation by long-chain acyl-CoAs.

Integrative Pathways for Regulation of Macronutrient Metabolism at the Cellular Level

Over the past decade our understanding of how metabolism is regulated at the cellular level has greatly increased. In particular, several integrative pathways have been elucidated that serve to integrate various signals and outputs. The AMP-activated protein kinase senses fuel (ATP) depletion and phosphorylates many downstream targets to correct the ATP deficit. The mammalian target of rapamycin (mTOR) pathway is involved in regulating protein synthesis, autophagy, and growth. This pathway integrates insulin and growth factor signaling, sensing of the fuel supply, and sensing of amino acid substrate availability to ensure that all of these are present before protein synthesis and growth are positively regulated by mTOR.

Role of Amp-Activated Protein Kinase in the Regulation of Cellular ATP Levels

Each cell must regulate its rate of ATP production (ADP phosphorylation) versus ATP consumption (ATP hydrolysis to ADP) and maintain ATP and ADP levels within a limited range. A drop in ATP serves as a signal that the cell needs to diminish its consumption of ATP and increase its production of ATP. When ADP begins to accumulate, adenylate kinase stimulates the conversion of 2 ADP to ATP + AMP. In the case of a cellular energy deficit, the ratio of AMP to ATP changes more dramatically than does the ratio of ATP to ADP, and thus the ratio of AMP to ATP is a useful indicator of the cell’s energy status.





A protein kinase that responds to changes in AMP levels is an important regulator of ATP levels at the cellular level. This kinase is called 5′-AMP-activated protein kinase, usually referred to as AMPK, or somewhat inappropriately as “AMP kinase.” As shown in Figure 19-1, AMPK is phosphorylated by various upstream kinases (AMPKKs such as LKB1 and calmodulin-dependent protein kinase kinase β) and dephosphorylated by a phosphatase. The AMPKKs specifically phosphorylate residue Thr172 of the α-subunit of AMPK. When the energy status of the cell is low, as indicated by a high AMP/ATP ratio, AMPK is in its active phosphorylated state. Phosphorylation of AMPK may be regulated both by activation of an upstream AMPKK, although some of these are thought to be constitutively active, and by the allosteric regulation of the phosphorylation state of AMPK by AMP (Witczak et al., 2008; Carling et al., 2011). The allosteric regulation of AMPK by AMP involves the binding of AMP to the γ-subunit of AMPK; this appears to make AMPK a better substrate for the upstream kinases and a poorer substrate for the phosphatase. Binding of AMP at another site may also activate AMPK directly.

FIGURE 19-1 Role of AMP-activated protein kinase (AMPK) in the regulation of cellular ATP levels.
AMP-activated protein kinase (AMPK) is phosphorylated by various kinases (AMPKK) and dephosphorylated by a phosphatase (PP2C) that is inhibited by AMP. When energy status of the cell is low, as indicated by a high AMP/ATP ratio, AMPK is in its active state (phosphorylated at Thr172). AMPK acts on a number of downstream substrates to stimulate processes that lead to increased ATP production and to inhibit processes that lead to increased ATP consumption. The net effect is a return of ATP and AMP levels to the normal range. Specific examples of AMPK targets are shown with — = inhibition/deactivation and ⟶ = stimulation/activation. The direction of the arrows inside of boxes indicates the final effect on the pathway or process listed in the box. Examples include the activation of NPY/AgRP hypothalamic neurons, leading to increased food intake and decreased peripheral energy expenditure; inhibition of Rab GTPase-activating proteins (i.e., Akt substrate 160), thereby permitting GLUT4 translocation to increase glucose uptake in muscle; activation of the heart isoform of 6-phosphofructo-2-kinase (6PF2K), increasing glycolysis; enhanced secretion of lipoprotein lipase (LPL) and enhanced translocation of CD36 fatty acid transporter to plasma membrane, which increases fatty acid uptake by muscle; inhibition of acetyl-CoA carboxylase (ACC2) to prevent malonyl-CoA formation which would block transport of fatty acids into the mitochondria. Other examples include the inhibition of ACC1 in liver, leading to decreased hepatic fatty acid, triacylglycerol and VLDL synthesis; inhibition of SREBP1c and HNF4 transcription factors, which prevents their activation of lipogenic gene transcription; inhibition of cytosolic HMG-CoA reductase, resulting in decreased cholesterol/isoprenoid synthesis; inhibition of glycogen synthase (GS), limiting the storage of glucose as glycogen; inhibition of cAMP element binding protein (CREB) regulated transcription coactivator 2 (CRTC2) which in turn inhibits cAMP response element (CRE)-mediated transcription of gluconeogenic genes; stimulatory phosphorylation of tuberous sclerosis complex 2 (TSC2) or inhibitory phosphorylation of the mammalian target of rapamycin complex 1 (mTORC1), either of which will inhibit mTORC1 activity and lead to inhibition of protein synthesis and activation of autophagy (not shown); and activation of eukaryotic elongation factor 2 kinase (eEF2K) which will lead to inhibition of eEF2 required for protein synthesis.

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Feb 26, 2017 | Posted by in PHARMACY | Comments Off on Regulation of Fuel Utilization in Response to Food Intake

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