Glucose Production

By convention, the basal or postabsorptive state is defined as the metabolic condition that prevails in the morning after an overnight (10 to 14 hours) fast. For most individuals, this time represents the longest period of fasting in everyday life. For the rest of the day, most people are more or less in the fed state. Maintenance of the fasting plasma glucose concentration is primarily the responsibility of the liver.9,10 The liver provides glucose for all tissues of the body, either by breaking down its own stores of glycogen or by synthesizing glucose from gluconeogenic precursors, of which the most important are lactate, pyruvate, glycerol, alanine, and other gluconeogenic amino acids. The central role of the liver in providing a constant supply of glucose to the body is related to the presence of G6Pase, which catalyzes the conversion of glucose 6-phosphate (G6P) to glucose within the hepatocyte. Although a number of tissues, including muscle and adipocytes, possess the enzymatic machinery necessary to degrade glycogen and synthesize G6P from lactate and amino acids, they either completely lack or possess too little of the key enzyme, G6Pase, to release significant amounts of free glucose into the circulation. The kidney, like the liver, also possesses the necessary enzymatic apparatus to produce glucose via the gluconeogenic pathway and to release it into the circulation. In normal subjects after an overnight fast, the kidney contributes ∼10% of total body glucose production.¹¹ During prolonged starvation and metabolic acidosis, renal gluconeogenesis is enhanced and may contribute as much as 25% of basal glucose production. Unlike the liver, the major gluconeogenic precursor for the kidney is glutamine.

Under postabsorptive conditions, glucose output in healthy adults averages 140 mg/min (778 µmol/min) or 2.0 mg/min per kg of body weight (11 µmol/min per kilogram) in a 70-kg individual (Fig. 10-2). The variation around this mean is significant (20% to 30%), with an unknown contribution of genetic and environmental factors. Little information is available concerning how much the fasting glucose output varies as a consequence of changes in dietary habits, caloric intake, or physical fitness. Intrafamilial covariance of this physiologic variable also is undetermined. Under standard nutritional conditions, the normal liver contains approximately 80 g of glycogen (see Table 10-1), and during fasting, liver glycogen stores decline at a rate of approximately 110 mg/min (611 µmol/min) or 8% per hour. From this it follows that hepatic glycogen depots would become empty after approximately 12 hours. Because fasting can be prolonged well beyond 12 hours, it is obvious that gluconeogenesis must progressively replace glycogenolysis as the fast continues.¹² In animals, the basal rate of glucose turnover is considerably higher than in humans—for example, dogs (3.6 mg/min per kilogram) and rats (7.2 mg/min per kilogram)—and the limited capacity of the liver to store glycogen confers an increasing role to gluconeogenesis for the maintenance of basal glycemia. This limitation on glycogen accumulation has an anatomic basis, because stuffing the cytoplasm with glycogen granules impairs cellular functions and results in liver damage, as seen in patients with glycogen storage diseases. In healthy subjects, after a 10- to 12-hour overnight fast, gluconeogenesis accounts for approximately 50% of total hepatic glucose release (see Fig. 10-2).¹² The substrates for this de novo glucose synthesis remain somewhat elusive. Circulating lactate, pyruvate, glycerol, alanine, and other gluconeogenic amino acids are natural candidate precursors and have been shown to transfer their carbons to newly synthesized glucose molecules, as documented by the incorporation of labeled lactate into glucose, that is, the Cori cycle. However, transsplanchnic catheterization in humans has shown that the net uptake of known circulating gluconeogenic precursors (lactate, pyruvate, glycerol, amino acids) can account for only 15% to 20% of total endogenous glucose production.¹³ The discrepancy between radioisotopic estimates of basal gluconeogenic rate and accountable circulating precursors suggests that the bloodborne substrates may not be the only source of gluconeogenic precursors. Within the splanchnic area, the intestine returns 10% to 20% of its glucose uptake to the liver as lactate, but this fills only part of the gap. It has been suggested that intrahepatic proteolysis and/or lipolysis could provide ample amounts of gluconeogenic precursors in addition to those entering from the systemic circulation.

The regulation of basal hepatic glucose production is controlled by the sum of multiple neural, hormonal, and metabolic stimuli, some stimulatory and others inhibitory.9,10 Fig. 10-3 portrays the control system as a simple balance between inhibition and stimulation. Insulin and glucagon provide the primary hormonal signals that regulate the production of glucose by the liver under postabsorptive conditions. Of the two, the action of insulin normally predominates. Hepatic glucose production is exquisitely sensitive to very small fluctuations in the circulating plasma insulin concentration. Increments in the plasma insulin concentration of as little as 5 to 10 µU/mL cause a marked, rapid suppression of glycogenolysis and decline in hepatic glucose output, whereas inhibition of gluconeogenesis is less sensitive.¹⁰

By restraining lipolysis and proteolysis, insulin also reduces the delivery of potential glucose precursors (glycerol and amino acids) from peripheral tissues (adipocytes and muscle) to the liver, and this further reduces hepatic glucose output. In its capacity as the inhibitory signal for glucose release, insulin is greatly favored by the anatomic connection between the pancreas and the liver. Because the pancreatic vein is a tributary of the portal vein, insulin, which is secreted by the β cells, reaches the liver in fasting humans at a concentration that is three to four times higher than the peripheral (arterial) concentration. This steep portosystemic gradient is maintained by the high rate of insulin degradation by hepatic tissues (fractional insulin extraction = 50%). Consequently, a small secretory stimulus to the β cells will disproportionately raise the portal insulin concentration, thereby selectively acting on glucose production rather than enhancing peripheral glucose utilization. In addition to short-circuiting the general circulation, pancreatic insulin release is potentiated by a number of gastrointestinal hormones (e.g., glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, secretin, cholecystokinin, pancreozymin, and others, which are released in response to meal ingestion).¹⁴ Therefore, anatomic and physiologic connections that comprise the gut-liver-pancreas axis ensure that the primary station for the handling of foodstuff, the liver, is under close control by a nearby, well-informed unit, the β cell.

Conversely, small decrements in the peripheral plasma insulin concentration, as little as 1 to 2 µU/mL, lead to an increase in hepatic glucose production.¹⁵ This highly sensitive interaction between the liver and insulin plays a critical role in the maintenance of basal glucose levels when fasting is prolonged. As glycogen stores become depleted, there is a small decrease in the arterial glucose concentration, which in turn leads to a decline in pancreatic insulin secretion and stimulation of glucagon release. The resultant hypoinsulinemia removes the constraint on lipolysis, and plasma FFA levels rise. By mass action, FFAs enhance their own uptake by all cells in the body, including liver and muscle. Enhanced FFA oxidation by the hepatocytes provides an energy source to drive gluconeogenesis, and the end product of beta oxidation, acetyl-CoA, stimulates the first committed enzyme, pyruvate carboxylase, in the gluconeogenic pathway (Fig. 10-4).^1,2 The combination of hypoinsulinemia, hypoglycemia, and increased FFA and amino acid supply also stimulates hepatic gluconeogenesis. In peripheral tissues, enhanced FFA and ketone oxidation spare glucose utilization (Randle cycle; see subsequent discussion), thereby minimizing the need for carbohydrate as an energy source.^1,2

Another important consequence of the fasting-related decline in plasma insulin concentration is the stimulation of proteolysis.¹⁵ This augments the outflow of amino acids, especially alanine, which accounts for approximately 50% of total α-amino nitrogen release. The predominance of alanine in the amino acid efflux from muscle cannot be explained by its presence in cellular proteins, of which alanine accounts for only 7% to 10%. The major source of alanine outflow from muscle during starvation is derived from the transamination of glucose-derived (from muscle glycogen and circulating glucose) pyruvate. The branched-chain amino acids (valine, leucine, isoleucine) provide the amino groups for muscle alanine synthesis. The alanine that is released from muscle is transported via the bloodstream to the liver, where it is converted to glucose, thus completing the glucose-alanine cycle (see Fig. 10-4).¹³ The glucose-lactate cycle (Cori cycle) also provides an important source of three-carbon skeletons for gluconeogenesis during fasting.¹⁵ Insulinopenia enhances the breakdown of glycogen and leads to accumulation of pyruvate. Because the Krebs cycle has been inhibited by the accelerated rate of FFA oxidation (Randle cycle), the pyruvate can either be transaminated to alanine (glucose-alanine cycle) or converted to lactate and released into the circulation, where it is carried to the liver and synthesized into glucose (glucose-lactate cycle). From the quantitative standpoint, approximately twice as many carbon skeletons are recycled to glucose via the Cori cycle compared with the alanine cycle.

The counterregulatory hormones (glucagon, epinephrine, growth hormone, cortisol, thyroid hormones) all are capable of offsetting the action of insulin on the liver and work by stimulating both glycogenolysis and gluconeogenesis.¹⁶ Glucagon plays a major role in the tonic support of basal hepatic glucose release and, in humans and animals, experimental suppression of endogenous glucagon secretion with preservation of basal insulin levels causes a 30% to 40% decline in hepatic glucose production.¹⁷ This suppression of glucose production involves both the glycogenolytic and gluconeogenic pathways. The precise quantitative contribution of the other counterregulatory hormones to the maintenance of basal glucose output under normal conditions of fasting has not been assessed. However, it is likely that they (i.e., epinephrine, cortisol, and growth hormone) also exert a tonic effect on hepatic glucose production in the postabsorptive state, and the withdrawal of insulin (i.e., hypoinsulinemia) that occurs with prolonged fasting allows their stimulatory effect on glycogenolysis and gluconeogenesis to occur unopposed. The net result of their unopposed action is an increase in hepatic glucose and renal output.

During more pronounced hypoglycemia, as may occur with insulin administration, all the counterregulatory hormones are released and act synergistically to restore normoglycemia.¹⁶ However, they do so with different dose-response kinetics and time courses. Glucagon and catecholamines act rapidly, whereas cortisol, growth hormone, and thyroid hormones (in that order) are involved in the long-range control of hepatic glucose release. Small, acute increases in plasma glucagon and epinephrine concentrations markedly stimulate both glycogenolysis and gluconeogenesis. Acute elevations in plasma cortisol, growth hormone, and thyroid hormones have no stimulatory effect on total hepatic glucose release. However, both cortisol and growth hormone have been shown to markedly potentiate the effects of epinephrine and glucagon on hepatic glucose release.

In addition to hormonal regulation, the central nervous system has an important role in the maintenance of hepatic glucose production.¹⁸ Both parasympathetic and sympathetic fibers reach the liver via the splanchnic nerves, thereby supplying autonomic neural modulation of both glucose production and uptake. In animals, parasympathetic stimulation restrains glycogenolysis and enhances glycogen synthesis, whereas activation of the sympathetic nerves innervating the liver stimulates glucose output via potentiation of both glycogenolysis and gluconeogenesis. In humans, the influence of the sympathetic nervous system on hepatic glucose metabolism can be demonstrated under conditions of acute stimulation, but the contribution of the autonomic nervous system to the maintenance of basal hepatic glucose production remains undetermined.

A number of metabolic signals play an important role in the control of hepatic glucose production in the postabsorptive state.9,10,19 Hyperglycemia per se inhibits liver glucose output. In normal adults, hyperglycemia and hyperinsulinemia occur concurrently, and in combination they provide a potent stimulus to suppress hepatic glucose release. As shown in Fig. 10-5, physiologic hyperglycemia (maintained using the hyperglycemic clamp technique), while maintaining basal insulinemia, is as effective as insulin in suppressing hepatic glucose production. More impressive is the observation that hyperglycemia in the presence of hypoinsulinemia (hyperglycemic clamp with somatostatin) causes a greater than 50% suppression of hepatic glucose release (see Fig. 10-5). Conversely, hypoglycemia by itself provides a trigger to increase hepatic glucose release. During insulin-induced hypoglycemia in humans and animals, an increase in plasma glucose occurs even when the counterregulatory hormonal response is inhibited (glucose autoregulation). Recent studies suggest that this effect of hypoglycemia is mediated via glucose sensors in the hypothalamic region of the brain, which activate hepatic glycogenolysis via sympathetic connections to the liver.²⁰

Altered substrate delivery to the liver also influences glucose release by the liver. In nondiabetic humans, it is difficult to demonstrate a detectable increase in hepatic glucose production by infusing large quantities of glycerol, lactate, or a mixture of amino acids, as long as there occurs a physiologic increase in plasma insulin concentration to balance out such gluconeogenic push. However, even though total hepatic glucose output does not increase, there is a marked stimulation of gluconeogenesis that is precisely counterbalanced by an inhibition of glycogenolysis. The increased provision of gluconeogenic precursors leads to an increase in the intrahepatic formation of G6P, but the eventual fate of this intermediate is glycogen rather than free glucose, because the rate-limiting enzyme for glucose production, G6Pase, is not simultaneously activated. FFAs play an important role in setting the level of hepatic glucose production. Only the odd-chain FFAs (i.e., propionate) can donate their carbon atoms to oxaloacetate in the tricarboxylic acid cycle and thus directly contribute to net gluconeogenesis. Most physiologic FFAs are of even chain, and although they can exchange their carbon moieties with tricarboxylic acid cycle intermediates, they do not contribute to de novo glucose synthesis. Nonetheless, when the perfusion medium of isolated rat liver is enriched with oleate or palmitate, new glucose formation from lactate or pyruvate is enhanced. The biochemical mechanisms involved in this stimulation of gluconeogenesis have been well worked out.²¹ The products of FFA oxidation, citrate and acetyl-CoA, activate the key enzymes that control gluconeogenesis, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and G6Pase (see Fig. 10-4). In addition, elevated plasma FFA concentrations in vivo are usually accompanied by raised glycerol levels because both result from the hydrolysis of triglycerides (see Fig. 10-4). Therefore, accelerated lipolysis supplies both the stimulus (FFA), the substrate (glycerol), and the energy source (ATP) to drive gluconeogenesis. In isolated hepatocytes, FFAs in micromolar amounts also have been shown to inhibit glycogen synthase. This suggests that an additional interaction of FFA metabolism with hepatic glucose production may be at the level of glycogen metabolism. In healthy volunteers, short-term infusion of triglycerides (with heparin to activate lipoprotein lipase) increases the plasma FFA concentration, leading to the stimulation of hepatic glucose output under conditions (i.e., hyperglycemia and insulinopenia induced by somatostatin plus glucose infusion) that mimic the diabetic state.²² A large part of this effect can be reproduced by infusing, under the same experimental circumstances, glycerol alone. On the other hand, when endogenous insulin is allowed to increase or when exogenous insulin is administered, the stimulatory effect of triglyceride infusion to increase the plasma FFA concentration on hepatic glucose release is easily overcome. In summary, the long-chain FFAs can regulate hepatic glucose production both by acting on the key enzymes of gluconeogenesis (i.e., through buildup of the products of FFA oxidation) and by virtue of the substrate push of glycerol. This regulatory loop is operative particularly when insulin secretion is not stimulated (i.e., in the basal state). Conversely, studies in both animals and humans have documented that a significant part of the suppressive action of insulin on hepatic glucose production is mediated via the hormone’s antilipolytic effect on adipocytes.^10,23 If the plasma FFA concentration is maintained during insulin infusion, the inhibitory effect of physiologic hyperinsulinemia on hepatic glucose production is impaired.

Glucose Disposal

In the basal (postabsorptive) state, the rate of whole-body glucose disposal equals the rate of hepatic glucose production, and the plasma glucose concentration remains constant.1,2 Information about the contribution of individual organs and tissues to total glucose uptake has been obtained in regional catheterization studies performed in combination with glucose tracers and indirect calorimetry.²⁴ By collating the available information, the organ circulation model depicted in Fig. 10-6 can be constructed.⁵ In this synthesis, steady-state interorgan exchanges of glucose, tissue blood flow, and regional glucose gradients are calculated based on a rate of hepatic glucose production of 140 mg/min (778 µmol/min). For a 70-kg man, this equals 2.0 mg/min per kilogram or 11 µmol/min per kilogram. In the postabsorptive state, approximately 70% of basal glucose disposal takes place in insulin-independent tissues (brain, liver, kidney, intestine, RBC). Of these, the brain predominates and accounts for almost half of the total hepatic glucose production. The liver plus gastrointestinal (splanchnic) tissues account for an additional 20%. It also can be appreciated that the fractional extraction of glucose (as defined earlier) is quite low everywhere in the body (ranging from 1.0% to 3.5%) except in the brain (9%) (Table 10-2). Because skeletal muscle represents 40% of total body weight and receives 16% of the cardiac output, one can calculate that it accounts for one third of the overall glucose disposal in the basal state (i.e., ∼245 µmol/min or 44 mg/min) (see Fig. 10-6).^1,2,5 As shown in Table 10-2, the muscle glucose clearance averages 1.3 mL/min per kilogram of tissue. Glucose clearance is a useful metabolic concept and is defined as the amount of plasma that is completely cleared of glucose in a given period; as such, it provides an index of the efficiency of tissue glucose removal. In the rank of efficiency of tissue glucose clearance in the basal state, resting muscle is last, being 10 times less active than the liver and 50 times less avid than the brain. It is noteworthy that tissues (brain, liver, kidneys) that have a high glucose clearance in the basal state are insulin independent. Increasing the plasma insulin concentration above fasting values has no effect on glucose clearance by these tissues (i.e., brain, liver, kidneys), whereas in muscle, glucose clearance increases by a factor of 10 or greater over the physiologic range of insulin concentrations. The intermediate position of heart muscle in the list is accounted for by its constant working state.

These organ-specific glucose clearance characteristics (see Table 10-2) represent the physiologic equivalent of the type and abundance of specific glucose transporters (GLUTs) the various tissues express (Table 10-3).^2,6 They also help to define the concept of an insulin-independent tissue. Thus in tissues in which an increase in the plasma insulin concentration does not accelerate the glucose clearance, the GLUT is not responsive to acute changes in the plasma insulin concentration. At present, several GLUT isoforms have been isolated.^2,6 A non-insulin-regulatable GLUT (GLUT-1) effects facilitated glucose transport in RBCs. The abundance of this transporter in RBCs ensures rapid diffusion of glucose across the RBC membrane, and this characteristic confers on RBCs an important role in the interorgan exchange of glucose. The same GLUT-1 transporter is present in the brain. Because of its low K_m (∼1 mmol/L), it saturates at plasma glucose concentrations well below the normal fasting plasma glucose concentration (∼5 mmol/L), thereby ensuring a constant flux of glucose into the brain cells. This is an important adaptive mechanism that provides the cerebral tissues an adequate supply of fuel even in the face of hypoglycemia. Another unique feature of GLUT-1 is its low V_max (∼3 mmol/L). This protects the brain against acute fluid shifts and cerebral edema that otherwise would accompany hyperglycemia. Thus GLUT-1 is well suited for its physiologic function, especially in the individual with insulin-dependent diabetes in whom extreme shifts in plasma glucose concentration (from hypoglycemia to hyperglycemia) are common. Another important corollary of GLUT-1 is that an increase in plasma glucose concentration above fasting levels (i.e., >5 mmol/L) will necessarily lead to a decline in brain glucose clearance because the transporter saturates at approximately 3 mmol/L. Moreover, because under postabsorptive conditions the brain is responsible for approximately half of the total body glucose disposal, it also follows that an increase in fasting plasma glucose concentration (with or without an increase in plasma insulin) will be associated with a decline in whole-body glucose clearance.

A totally distinct (from the physiologic standpoint) GLUT, GLUT-2, is present in liver and pancreatic β cells.2,6 It has a high K_m (∼15 to 20 mmol/L), and as a consequence, the free glucose concentration in cells expressing this transporter increases in direct proportion to the increase in plasma glucose concentration. This characteristic allows these cells to respond as “glucose sensors.”^1,2 As the ambient glucose concentration increases, more glucose enters the β cell, which responds by appropriately augmenting its secretion of insulin, whereas the liver reads the rising plasma glucose level and decreases its output of glucose. As a corollary of this, an increase in the plasma glucose concentration is associated with a proportional increase in glucose uptake by these tissues with an unchanging glucose clearance. Because GLUT-2 does not respond to insulin, hyperinsulinemia is not associated with an increase in hepatic or β-cell glucose clearance.

It is noteworthy that each GLUT is associated with a specific hexokinase, which has a K_m that parallels that of its associated GLUT.²⁵ For liver and β cells, the phosphorylating enzyme is hexokinase IV or glucokinase. Its high K_m constant has led investigators to propose glucokinase as the β-cell sensor. Consistent with this, recent studies have demonstrated that some forms of maturity-onset diabetes of the young (MODY) are associated with mutations in the glucokinase gene, and the physiologic counterpart of this is a defect in insulin secretion. Insulin-sensitive tissues, muscle, and adipocytes contain GLUT-4 and its physiologic coupler, hexokinase II. GLUT-4 has a K_m constant of approximately 5 mmol/L, which is close to that of the plasma glucose concentration. In the basal state, the majority of GLUT-4 are not located in the plasma membrane but reside in vesicles within the cell. After exposure to insulin, the concentration of GLUT-4 in the plasma membrane of adipocytes and muscle increases markedly, and there is a reciprocal decline in the intracellular GLUT-4 pool.²⁶ Insulin not only enhances their translocation and insertion into the plasma membrane but also augments their intrinsic activity. Thus, muscle glucose clearance increases markedly, 10-fold or greater, in response to increments in plasma insulin concentration within the physiologic range.^1,2,27 GLUT-3, the other major GLUT, is present in the gut and kidney.^2,6 It is sodium-dependent and does not respond to insulin. In the gut, it mediates unidirectional gastrointestinal absorption of glucose in the small intestine, whereas in the kidney, it regulates unidirectional glucose absorption in the proximal tubule. Although this transporter is insulin insensitive, it plays a crucial role in glucose homeostasis by regulating its entry into the body and preventing its loss via the kidney.

The intracellular disposition of transported glucose can be studied in vivo by using glucose tracers and then measuring the appearance of the label in specific metabolic products such as lactate (i.e., anaerobic glycolysis) and carbon dioxide (i.e, complete oxidation).⁸ These techniques, even when correctly applied, provide only estimates of the metabolic fate of plasma glucose. For example, should glycogen in muscle be oxidized directly, the plasma glucose–specific activity would miss it completely because plasma glucose does not equilibrate with the intracellular free-glucose pool. To circumvent this problem, investigators have employed indirect calorimetry, which measures total carbon dioxide production from all carbohydrate sources, both intracellular and extracellular.²⁸ Although indirect calorimetry depends on a number of assumptions, these are reasonable and have been largely validated. Moreover, this technique is easy to apply and noninvasive. Indirect calorimetry also provides a good estimate of the rate of energy expenditure and complements information obtained by tracer methods. In the basal state and under ordinary nutritionally circumstances, oxygen consumption averages 250 mL/min, whereas carbon dioxide production is 200 mL/min—that is, the whole-body respiratory quotient equals 0.8 (respiratory quotient = carbon dioxide production/oxygen consumption). From the equations depicted in Table 10-4, whole-body carbohydrate oxidation can be estimated to account for approximately 60% of total glucose uptake in the postabsorptive state.²⁴ Because the brain uses 46% of the total glucose turnover (see Table 10-2), and because essentially all brain glucose uptake is accounted for by oxidation, it follows that three fourths (i.e., 46/0.6 or 77%) of basal glucose oxidation occurs in the brain. Little is left for other tissues, which preferentially derive their metabolic energy from the oxidation of FFAs and other lipids under postabsorptive conditions. Skeletal muscle, for example, has a respiratory quotient of 0.75 and relies on fat oxidation for the production of 80% of the energy that it needs in the resting state. Thus the basal state is characterized by parsimonious use of glucose as a metabolic fuel.¹⁵ Moreover, the glucose is selectively channeled to organs that cannot rely on alternative energy sources. In the postabsorptive state, more than half of the total energy production is generated via oxidation of fat, of which there are plentiful stores (see Table 10-1). Insulin is the principal regulator that determines the metabolic mix of fuels in the basal state. A small decrement in the circulating hormone level releases the brake on lipolysis, and the plasma FFA concentration increases, thereby allowing fat to override glucose in the competition between the two substrates. Although these very small changes in plasma insulin concentration are sufficient to promote a shift in fuel metabolism from carbohydrate to fat, the plasma insulin level is still sufficiently elevated to maintain glucose transport and metabolism in target tissues at minimal rates and to restrain protein breakdown, which contributes only approximately 15% to basal energy metabolism.¹⁵ The role that counterregulatory (glucagon, epinephrine, cortisol, growth hormone, thyroid hormones) hormones play in basal glucose uptake is less well defined but probably centers on the maintenance of lipolysis, because all the insulin antagonistic hormones are more or less potent lipolytic stimuli.

Glucose Cycles

After entry of glucose into the cell through a specific GLUT, the sugar does not necessarily follow a direct path to its eventual fate, be it glycogen, lactate, carbon dioxide, or pentoses. Rather, it may in part reach its destination via a number of circuitous routes that have become known as futile cycles. A metabolic futile cycle is one in which a precursor is converted into a product by a forward reaction and then resynthesized to the precursor. In such a reaction, there is no net product accumulation, but energy (ATP) is used. There are multiple examples of such futile cycles in the glucose metabolic pathway.29,30 The first involves the conversion of glucose to G6P by glucokinase and its subsequent reconversion to intracellular free glucose by G6Pase in the liver. Each turn of this cycle uses one molecule of ATP. Another example of a futile cycle is represented by the conversion of G6P to fructose-6-phosphate and back through the phosphoglucoisomerase reaction. Perhaps the best-studied futile cycle that is under the control of insulin is the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate in the liver. The reverse reaction is regulated by fructose-1,6-bisphosphatase, whereas the forward reaction is catalyzed by phosphofructokinase (PFK). The latter enzyme is controlled by the energy status of the cell and key intracellular metabolites. High levels of ATP, acidosis, and citrate inhibit PFK, whereas ADP and alkalosis stimulate PFK. The most potent activator of PFK is fructose-2,6-bisphosphate, whose synthesis is stimulated by the enzyme fructose-2,6-bisphosphate kinase. This latter enzyme is under the control of insulin, and the PFK step, therefore, represents an important regulatory control point for insulin action.³¹

In general terms, whenever bidirectional flux through a metabolic pathway is simultaneously operative, there exists a cycle, regardless of the number of intermediate reactions and regardless of whether one or more tissues are involved. In the examples cited previously, the cycles occurred within individual cells. However, cycles also can exist between organs. In this regard, lipolysis in adipose tissue followed by partial reesterification of FFAs in the liver is a complete cycle. Another important cycle is the breakdown of proteins in the liver or other tissues. The glucose-alanine and glucose-lactate (Cori) loops¹³ also represent important cycles (see previous discussion) that provide conservation of carbon skeletons and transfer of α-amino groups between muscle and liver.

The derogatory connotation of futility has traditionally been reserved for those cycles that go on in the same cell. These cycles are, however, anything but futile. As elegantly discussed by Newsholme and Leech,²⁹ a metabolic cycle with a reverberating internal loop provides the best kinetic stratagem to maintain the enzymes of a dormant pathway at a minimum of activity, while at the same time ensuring a high sensitivity gain for rapid amplification of incoming signals. The ATP cost of these cycles is itself a means of increasing the efficiency of energy dissipation. The fact that the activity of these cycles is under hormonal control (e.g., catecholamines, glucagon, and thyroid hormones enhance the cycling rate) establishes a mechanism for rapid modulation. In this way, these cycles become components of facultative thermogenesis. Equally important, the operation of these futile or substrate cycles allows the generation of metabolic intermediates that can modulate the activities of key enzymes and allow allosteric regulation.

Quantitation of Insulin Sensitivity and Insulin Secretion

Because of the difficulties involved in following the gastrointestinal absorption of glucose and the continuously changing plasma glucose and insulin concentrations, the regulation of glucose homeostasis during the fed state has classically been investigated using intravenous glucose, which can be administered in formats that are more suitable for some formal analysis. The most detailed information concerning glucose utilization by the whole body, organs, and specific intracellular pathways has come from studies employing the insulin/glucose clamp technique³³ in combination with indirect calorimetry, isotope turnover methodology, and limb (forearm and leg) catheterization. Because the insulin-glucose clamp technique has become the reference method for the study of glucose metabolism, this procedure is described briefly. The euglycemic insulin version of the clamp technique is shown in Fig. 10-7. An exogenous infusion of regular insulin is started at time zero and is given as a priming dose followed by a constant infusion (usually at a rate of 1 mU/min per kilogram or 40 mU/min per square meter or 240 pmol/min per square meter). Such an infusion quickly establishes a hyperinsulinemic plateau of approximately 70 to 80 µU/mL. A few minutes after starting the insulin, an infusion of 20% glucose is begun. Based on the plasma glucose concentration, which is measured every 5 to 10 minutes, and using the negative feedback principle, the glucose infusion rate is adjusted periodically to maintain the plasma glucose concentration constant at basal level. In response to the hyperinsulinemic stimulus, there is an initial delay in the onset of insulin-stimulated glucose disposal that lasts approximately 15 to 20 minutes.²⁷ After this delay, there is a rapid increase in glucose utilization from 20 to 80 minutes, and this reaches a near-steady-state value of 5 to 10 mg/min per kilogram of body weight (27 to 54 µmol/min per kilogram) during the last 40 minutes of the insulin clamp in healthy young subjects.²⁷ Because endogenous glucose production is completely or nearly completely (>90%) suppressed by insulin, and the plasma glucose concentration is clamped at the basal level, the rate of exogenous glucose infusion must equal the rate of glucose uptake by all tissues of the body and provides a quantitative measure of the amount of glucose metabolized (M). In insulin-resistant conditions (e.g., obesity and type 2 diabetes mellitus^1,2), hepatic glucose production (measured with radiolabeled glucose or a stable isotope of glucose) is not completely suppressed and must be added to the rate of exogenous glucose infusion to obtain the true rate of total body glucose utilization. The higher the glucose metabolic rate (M), the more sensitive the individual is to insulin. The euglycemic insulin clamp technique has the following advantages: (1) any desired combination of plasma glucose and insulin levels can be achieved and maintained; (2) the time course of insulin action can be determined with a time resolution of approximately 10 minutes; (3) other techniques, such as tracer glucose infusion, indirect calorimetry, limb catheterization, magnetic resonance imaging/spectroscopy, and muscle biopsy, can be combined with the clamp protocol; (4) because hypoglycemia is avoided, the release of counterregulatory hormones (which antagonize insulin action) is prevented, and one can derive a pure measure of tissue sensitivity to insulin; (5) the interaction of other hormones or substrates with insulin action can be quantitated by simultaneously infusing them during a clamp study; (6) the achievement of constant or nearly constant levels of insulin, glucose, tracer glucose-specific activity (or enrichment), and glucose metabolic rate allows one to make quantitative measurements under steady-state conditions and thus avoid interpretive problems encountered when plasma glucose and insulin concentrations and glucose flux rates are constantly changing (i.e., during an oral glucose tolerance test or intravenous glucose tolerance test). The hyperglycemic version of the glucose clamp is depicted in Fig. 10-8.³³ In this procedure, the plasma glucose concentration is acutely increased by a priming infusion of glucose that is administered in a logarithmically decreasing manner over 15 minutes. Thereafter, the plasma glucose concentration is clamped at the designed plateau by periodically adjusting an exogenous glucose infusion as described in the euglycemic version. The hyperglycemic step evokes an endogenous insulin response that is typically biphasic. During the initial 10 minutes, there is an early burst of insulin release which is followed by a gradual, continuous increase in the plasma insulin concentration. The initial (0 to 10 minutes) peak of insulin represents the release of preformed hormone that is stored within β-cell granules. The late (10 to 120 minutes) phase, which is believed to represent the release of insulin packaged in immature granules or newly synthesized insulin, lasts until the glucose stimulus is withdrawn. By analogy with the euglycemic insulin clamp counterpart, the hyperglycemic clamp also provides a quantitative measure of the total amount of glucose taken up and metabolized (M) by the body in response to the combined stimuli of endogenous hyperinsulinemia plus hyperglycemia.

One disadvantage of the hyperglycemic clamp or any study in which glucose is administered intravenously is the inability to examine the effect of incretins on insulin secretion.¹⁴ Thus, when glucose is administered orally, the insulin response is significantly greater than when the same arterial glucose profile is created by intravenous glucose administration (Fig. 10-9). This phenomenon is generally known as the incretin effect. The size of the incretin effect has been shown to be directly related to the strength of the oral stimulus; thus, ascending doses of oral glucose induce progressively larger incretin effects in the healthy subject.³⁴ A defective incretin effect has been demonstrated in nondiabetic obese subjects as well as in subjects with type 2 diabetes.³⁵ Two incretins, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide, which are secreted by the L and K cells of the small intestine, respectively, in response to nutrient ingestion, partake of the incretin effect on account of their ability to potentiate glucose-induced insulin secretion.^14,35

Dynamic Interaction Between Insulin Sensitivity and Insulin Secretion

In normal, healthy individuals, the euglycemic insulin clamp technique has demonstrated that insulin sensitivity declines with age,³⁶ in part because of the age-related increase in adiposity.³⁷ More importantly, within the normal population insulin sensitivity ranges widely. Among young, healthy, normal, glucose-tolerant subjects, insulin-mediated glucose disposal varies up to fivefold (Fig. 10-10).³⁸ A number of factors are known to influence insulin sensitivity. In addition to age, adipose tissue mass, fat topography, and degree of physical fitness all are powerful determinants of insulin-mediated glucose disposal.^1,2 Increased total body fat content and especially increased visceral fat,³ as well as decreased Vo₂max,³⁹ are associated with impaired insulin action. Increased metabolites of triglyceride and FFAs (fatty acyl-coenzyme A, diacylglycerol, and ceramide) within muscle and liver cells are associated with insulin resistance in these organs.³ Diet composition (increased fat and reduced carbohydrate) also has been shown to impair insulin sensitivity. However, even when these factors are taken into account, one cannot fully explain the wide variation in insulin sensitivity among healthy adult individuals.³⁸ Studies in whites, Pima Indians, and Mexican Americans^1,2,40 have demonstrated that genetic factors play an important role in the distribution of insulin sensitivity (as measured by the glucose disposal rate during a euglycemic insulin clamp).

Glucose homeostasis results from a fine balance between tissue sensitivity to insulin and insulin secretion.⁴¹ This implies that the β cell must be able to sense the defect in insulin action and precisely augment its secretion of insulin to offset the insulin resistance. In fact, insulin resistance is neither necessary nor sufficient so long as the β-cell functional reserve is large enough to cope even with severe insulin resistance. The typical case is the morbidly obese subject with normal glucose tolerance, in whom insulin output can be 10-fold higher than in lean individuals³⁸ and decrease to near-normal levels following major weight loss.⁴² β-Cell dysfunction, on the other hand, is necessary and sufficient to produce hyperglycemia; type 1 diabetes is the prototype of primary β-cell failure. In recent years, clinically significant advances have also been made in the area of in vivo β-cell function.⁴³ Firstly, it has been recognized that absolute insulin secretion (whether fasting or post-meal) reflects the set-point of β-cell secretory function (i.e., secretory capacity) of which insulin resistance and presumably β-cell mass are positive determinants. However, the strongest determinant of glucose tolerance is the ability of the secretory machinery to rapidly and adequately cope with the acute changes in glucose induced by feeding. This aspect of β-cell function, called glucose sensitivity, is largely independent of insulin resistance. Recent studies serve to emphasize the importance of this dynamic interaction between insulin sensitivity and insulin secretion.^44,45 In these studies, both insulin sensitivity (by the euglycemic insulin clamp technique) and β-cell function (by mathematical modeling of C-peptide concentrations⁴⁶) were measured in lean and obese subjects with and without type 2 diabetes. As shown in Fig. 10-10, in nondiabetic subjects, both fasting insulin secretion and total insulin output in response to an oral glucose tolerance test (OGTT) are inversely related to insulin sensitivity. When including type 2 diabetic patients with progressively worse glycemic control, the relation of the different parameters of glucose metabolism to glucose tolerance is strikingly different (Fig. 10-11). Insulin output shows a biphasic pattern, initially increasing as glucose tolerance deteriorates, to eventually fall below normal values in severe diabetes. Insulin sensitivity drops by 35% in the passage between lean and obese nondiabetic subjects (first two dots to the left of the panels) and continues to fall slowly in the transition from the following two tertiles of glucose tolerance, through impaired glucose tolerance (IGT), to progressively more severe hyperglycemia. In contrast, β-cell glucose sensitivity declines monotonically in parallel with progressively higher 2-hour plasma glucose concentrations. Thus, individuals in the highest tertile of what would be considered to represent normal glucose tolerance (i.e., 2-hour plasma = 120 to 139 mg/dL) have lost ∼50% of β-cell glucose sensitivity, whereas IGT individuals have lost ∼65% of β-cell function (see Fig. 10-11). Log-transformation of these variables demonstrates that the glucose sensitivity is continuously and linearly related to the 2-hour plasma glucose concentration in a reciprocal fashion throughout the range from low-normal to very high (Fig. 10-12), confirming that glucose sensing is a critical determinant of glucose tolerance. The separate quantitative contribution of insulin sensitivity and β-cell glucose sensitivity to glucose tolerance (as the 2-hour plasma glucose level) is shown in Figure 10-13, which also illustrates the complex, nonlinear interaction of these two main physiologic determinants of glucose tolerance.

Recent genome-wide scan association analyses in diabetic⁴⁷ and nondiabetic cohorts⁴⁸ have consistently identified common mutations in genes more or less directly related to β-cell glucose sensitivity. Although the risk conferred by such variants is small, it is conceivable that individuals carrying one or, more likely, several of these gene polymorphisms start with an inherently poor β-cell glucose sensitivity, which may be compounded by a degree of “essential” insulin resistance. Obesity and insulin resistance initially provide compensation by raising the secretory set-point; eventually, however, ageing and glucose toxicity—even that engendered by slight chronic elevations in plasma glucose levels—wear out β-cells (especially intrinsically defective β-cells), leading to diabetes. This paradigm of the evolution of hyperglycemia from normoglycemia under the separate pressures of insulin resistance and β-cell glucose sensitivity, schematized in Fig. 10-14, has received preliminary validation in longitudinal studies in humans.⁴⁹

Effect of Insulin on Hepatic and Peripheral Glucose Metabolism

The maintenance of normal glucose homeostasis requires the closely coordinated effects of insulin and hyperglycemia to simultaneously (1) suppress endogenous (primarily hepatic) glucose production; (2) stimulate glucose uptake by peripheral tissues, primarily muscle; (3) stimulate glucose uptake by the liver; and (4) inhibit lipolysis and reduce the plasma FFA concentration.1,2 The decrease in circulating plasma FFA levels plays an important role in enhancing the suppression of hepatic glucose production and augmenting muscle glucose uptake in response to a physiologic increase in plasma insulin concentration.³

Using the euglycemic insulin clamp technique, insulin can be shown to exert a potent suppressive action on hepatic glucose production such that portal insulin concentrations of less than 100 µU/mL completely abolish glucose entry into the circulation.9,19,50 Fig. 10-15 shows the typical time course for suppression of endogenous (hepatic) glucose production after an acute increase in plasma insulin to levels of 60 to 70 µU/mL in healthy subjects.⁵¹ Dose-response curves relating the calculated portal plasma insulin concentration to inhibition of hepatic glucose production (Fig. 10-16) indicate a half-maximal effect at a level of approximately 30 µU/mL, corresponding to an increment in the portal insulin concentration of only 5 to 10 µU/mL.^19,50 These results indicate the exquisite sensitivity of the liver to very small increments in the circulating plasma insulin concentration. Note that in its capacity of a glucose-producing organ, the liver is extremely sensitive to insulin, whereas the ability of insulin to augment hepatic glucose uptake under conditions of euglycemia is quite modest. In the presence of hyperglycemia, insulin has a small stimulatory effect on hepatic glucose uptake.^52,53 Hyperglycemia, induced by intravenous glucose administration, strongly synergizes this inhibitory action of insulin on hepatic glucose production (see Fig. 10-5). Under euglycemic conditions, the apparent maximal stimulation is approximately 15 mg/min per kilogram (∼80 µmol/min per kilogram) in healthy adult subjects and occurs with plasma insulin concentrations of approximately 250 µU/mL; the half-maximal stimulation of glucose uptake occurs with a plasma insulin concentration of 70 to 110 µU/mL. A dose-response curve of similar shape is derived when progressively higher insulin doses are infused locally into the forearm or leg tissues, approximately 70% of which consists of skeletal muscle. By extrapolating from forearm or leg muscle to total body muscle mass, it can be estimated that with prevailing peripheral plasma insulin concentrations in the high physiologic range (60 to 90 µU/mL), approximately 70% of total glucose disposal occurs in muscle tissue.²⁴ Obviously, this percentage increases further with progressively higher insulin levels, because the contribution of insulin-independent tissues declines. By combining the insulin clamp technique (plasma insulin concentration, ∼70 to 80 µU/mL) with leg and hepatic vein catheterization, a composite picture of whole-body glucose disposal can be generated (Fig. 10-17). Brain (∼1.2 mg/min per kilogram) and splanchnic (liver plus gastrointestinal tissues) (∼0.5 mg/min per kilogram) glucose uptake are unaffected by insulin infusion.^1,2 Adipose tissue in adult humans is relatively inert. Although it represents an insulin-dependent tissue, it accounts for no more than 4% to 5% of an infused glucose load. Consequently, muscle represents the primary tissue responsible for insulin-mediated glucose uptake under euglycemic conditions.²⁴ When hyperglycemia (plasma glucose increased from 90 to 180 mg/dL) is superimposed on the same level of hyperinsulinemia (70 to 80 µU/mL), a doubling of total body glucose utilization occurs (Fig. 10-18); consequently, the glucose clearance remains unchanged. As can be seen in Fig. 10-18, essentially all the additional increase in glucose disposal above that observed under euglycemic conditions occurs in muscle. In the presence of hyperglycemia, insulin has a small stimulatory effect on hepatic glucose uptake, which amounts to approximately 10% of the total body glucose disposal.⁵³

The regulation of glucose production and utilization by insulin is dependent on both the hormone concentration and time. At any given insulin concentration, there is a finite period before the effect of the hormone is seen and reaches its maximum. Such onset time is the sum of a circulatory delay (delivery of insulin from arterial blood to cell surface membrane) and a cellular lag (insulin receptor binding and effector activation). Similarly, insulin’s effect on glucose metabolism remains for some time (offset) after the circulating concentration has returned to basal levels. Fig. 10-19 shows the activation and deactivation times of insulin calculated at euglycemia over a wide range of plasma hormone levels (as high as 1000 mU/mL).⁵⁴ With the reservations inherent in the analysis of non-steady-state tracer data, the results shown in Fig. 10-19 provide evidence that activation and deactivation are inversely related to one another. Thus at higher plasma insulin concentrations, the hormone’s effect is more rapid in onset and takes longer to wane. From the physiologic standpoint, it also is noteworthy that the relationship between onset and offset time is different for the liver (suppression of glucose release) and for peripheral tissues (stimulation of glucose uptake). At any insulin dose, the liver is activated more rapidly, and the effect persists for a longer duration. The more rapid onset of action in the liver may be related to the shorter diffusion time of bloodborne substances into highly perfused organs (1 mL/min per gram of tissue in the liver versus a corresponding value of 0.04 mL/min per gram in resting skeletal muscle; see Table 10-1) and to anatomic differences between liver capillaries (which are fenestrated) and muscle capillaries (which are not fenestrated).

Intracellular Pathways of Glucose Disposal

Free Fatty Acid–Amino Acid–Glucose Interactions

A major part of insulin’s stimulatory action on glucose metabolism is indirect and is mediated via changes in substrate metabolism. In contrast to the hormone’s stimulatory effect on glucose utilization, insulin is a powerful inhibitor of lipolysis and lipid oxidation.⁵⁰ As shown in Fig. 10-27, plasma FFA concentrations decline steeply in response to small increments in circulating insulin levels under conditions of euglycemia. This decrease is the result of a drastic reduction in the rate of FFA appearance into the circulation. The concomitant decline in plasma glycerol concentration is consistent with in vitro studies and indicates that lipolysis is inhibited. The consequence of the reduced availability of FFA is a parallel reduction in both FFA oxidation and nonoxidative FFA disposal, that is, reesterification (Fig. 10-28). The inverse patterns of change in glucose disposal and oxidation on the one hand, and lipid utilization on the other, introduce the important concept of substrate competition. Glucose and long-chain FFAs are the first and best-known example of substrate competition in insulin-dependent tissues.⁸⁵ Physiologically, the increases in plasma glucose (by mass action) and insulin (stimulation of glucose transport) concentrations increase the rate of glucose uptake into fat cells. The resultant increase in intracellular α-glycerol phosphate generated during the stimulation of glycolysis supplies the substrate for augmented reesterification of tissue FFAs, while at the same time insulin stimulates α-glycerol phosphate acyltransferase, the rate-limiting enzyme for triglyceride synthesis.³⁰ These combined effects limit the release of FFAs into the bloodstream. In addition, the glucose-induced increase in plasma insulin concentration quickly inhibits lipolysis by stimulating hormone-sensitive lipase in the adipocyte, and this further reduces the supply of lipid substrates to the oxidative machinery in muscle and liver. A decrease in FFA oxidation by these tissues causes a reciprocal stimulation of glucose oxidation and glycogen synthesis by reversal of the Randle cycle in muscle (see later) and inhibition of gluconeogenesis in the liver.

The glucose-mediated inhibition of FFA metabolism is counterbalanced by an FFA-mediated inhibition of glucose metabolism, creating an FFA–glucose substrate interaction known as the Randle cycle.⁸⁵ When the plasma FFA concentration is elevated, by mass action, FFAs are transported into muscle and liver cells by simple diffusion across the membrane. The intracellular free FFA concentration is maintained low by a specific FFA-binding protein and this ensures a favorable transport gradient. Once inside the cell, FFAs are transported into the mitochondria, where they undergo beta oxidation (Fig. 10-29). Before entering the mitochondria, the long-chain fatty acids (oleic, palmitic, stearic, linoleic, and palmitoleic) are first activated to their acyl-CoA derivative by the appropriate acyl-CoA synthetase. Because the inner mitochondrial membrane is not permeable to the fatty-acyl-CoA, a specific transport system is necessary to transport the fatty-acyl derivative into the mitochondria (see Fig. 10-29).⁸⁶ The enzyme carnitine acyltransferase-I in the outer membrane transfers the cytosolic fatty acyl-CoA to carnitine, and the resulting fatty-acyl carnitine derivative is transported through the mitochondrial membrane. At the inner mitochondrial membrane, carnitine acyltransferase II, the rate-limiting enzyme for beta oxidation, catalyzes the transfer of the fatty-acyl unit from fatty-acyl carnitine back to CoA, and the fatty acyl-CoA then undergoes beta oxidation with the resultant generation of acetyl-CoA.

As beta oxidation proceeds, acetyl-CoA accumulates within the cell and becomes a powerful inhibitor of the PDH enzyme complex (Fig. 10-30).^85,86 In addition, the accelerated rate of FFA oxidation consumes nicotinic adenine dinucleotide (NAD) and generates NADH. This shift in redox potential further inhibits PDH and impairs the Krebs cycle. Fatty acyl-CoA derivatives also have been shown to inhibit glycogen synthase in muscle and liver^87,88 (see Fig. 10-30). In healthy humans, a physiologic elevation in the plasma FFA concentration (created by Intralipid/heparin infusion) stimulates FFA oxidation and inhibits both glucose oxidation and glucose storage (glycogen synthesis),⁸⁹ thus providing experimental validation of the Randle cycle. In nondiabetic subjects, a physiologic increment in the plasma insulin level (+100 µU/mL) causes a 50% to 60% decline in plasma FFA concentration and a parallel decline in lipid oxidation (Fig. 10-31). Infusion of Intralipid during the insulin clamp, to maintain or increase the plasma FFA level (see Fig. 10-31), inhibits insulin-mediated stimulation of both glucose oxidation and glucose storage (glycogen synthesis) (Fig. 10-32). These data demonstrate that the Randle cycle operates in vivo in humans in response to physiologic changes in the plasma FFA concentration. The inhibitory effect of elevated plasma FFA levels is observed at all plasma insulin concentrations, spawning the physiologic and pharmacologic range.⁸⁹ The inhibitory effect of an acute increase in plasma FFA concentration on muscle glucose metabolism is time dependent. Thus the earliest (within 2 hours) observed abnormality is a defect in glucose oxidation, as would be predicted by operation of the Randle cycle.⁸⁵ This is followed (between 2 and 3 hours) by defects in glucose transport and phosphorylation and eventually (after 3 to 4 hours) by impaired glycogen synthesis.^3,90

According to the FFA-glucose cycle originally proposed 4 decades ago by Randle and colleagues,⁸⁵ the increase in FFA oxidation restrains glucose oxidation in muscle by altering the redox potential of the cell and inhibiting key glycolytic enzymes. The excessive FFA oxidation, in addition to causing the intracellular accumulation of acetyl-CoA (a potent inhibitor of PDH) and increasing the NADH/NAD ratio (causing a slowing of the Krebs cycle), results in the accumulation of citrate, a powerful inhibitor of PFK. Randle and colleagues proposed that inhibition of PFK caused product inhibition of the early steps involved in glucose metabolism, leading to the accumulation of G6P, which in turn inhibited HK-II. The block in glucose phosphorylation caused a buildup of intracellular free glucose, which restrained glucose transport into the cell via GLUT-4. The resultant decrease in glucose transport, in turn, resulted in impaired glycogen synthesis. This sequence of events by which accelerated plasma FFA oxidation inhibits muscle glucose transport, glucose oxidation, and glycogen synthesis is now referred to as the Randle cycle.⁸⁵ It should be noted that the same scenario would ensue if the FFA were derived from triglycerides stored in muscle⁴² or from plasma.⁸⁹

The original description of the Randle cycle was formulated based on experiments performed in rat diaphragm and heart muscle.⁸⁵ More recent studies performed in human skeletal muscle implicate mechanisms in addition to those originally proposed by Randle and colleagues, in the FFA-induced insulin resistance. Several groups⁹¹ have failed to observe an increase in G6P, or in muscle citrate levels or an inhibition of PFK, when insulin-stimulated glucose metabolism was inhibited by a lipid infusion to increase the plasma FFA concentration. Thus, although increased FFA/lipid and decreased glucose oxidation are closely coupled, as originally demonstrated by Randle and colleagues, mechanisms other than product (i.e., elevated intracellular G6P and free glucose concentrations) inhibition of the early steps of glucose metabolism must be invoked to explain the defects in glucose transport, glucose phosphorylation, and glycogen synthesis (see Fig. 10-30).

Studies in humans and animals have shown a strong negative correlation between insulin-stimulated glucose metabolism and increased intramuscular lipid pools, including triglyceride, diacylglycerol, ceramides, and long-chain fatty acyl-CoAs.3,92–94 An acute increase in the plasma FFA concentration leads to an increase in muscle fatty acyl-CoA and diacylglycerol concentrations. Both long-chain fatty acyl-CoAs and diacylglycerol activate protein kinase C-θ, which increases serine phosphorylation with subsequent inhibition of IRS-1 tyrosine phosphorylation. In human muscle, elevated plasma FFA levels inhibit insulin-stimulated tyrosine phosphorylation of IRS-1, the association of the p85 subunit of PI 3-kinase with IRS-1, and activation of PI 3-kinase^91,95 (see Fig. 10-30). Direct effects of long-chain fatty acyl-CoAs to inhibit glucose transport, glucose phosphorylation, and glycogen synthase also have been demonstrated in muscle (see Fig. 10-31). Last, increased muscle ceramide levels (secondary to increased long-chain fatty acyl-CoAs) interfere with glucose transport and inhibit glycogen synthase in muscle via multiple mechanisms involving alterations in a variety of intracellular lipid-signaling molecules that exert their inhibitory effects on multiple steps of glucose metabolism (insulin signal transduction system, glucose transport, glucose phosphorylation, glycogen synthase, PDH, Krebs cycle).

The extent to which insulin action in target tissues is direct rather than mediated by shifts in substrate supply can be appreciated by comparing systemic with local insulin administration. When infused intra-arterially into the human forearm, insulin does not alter the circulating substrate supply, in that neither FFA nor glucose concentrations change in the arterial blood recirculating to the forearm tissues. Under these conditions, insulin stimulates forearm glucose uptake and lactate release in a time-dependent manner (Fig. 10-33) but does not induce any detectable change in the local respiratory quotient (0.76) in the blood draining the forearm tissues.⁹⁶ This observation indicates that the forearm tissues, and the muscle in particular, continue to rely mostly on lipid oxidation for energy production, and that the vast majority of insulin-stimulated glucose uptake is channeled to glycogen. In contrast, when comparable hyperinsulinemia is created by systemic infusion of insulin while maintaining euglycemia, the leg respiratory quotient increases from 0.74 to almost 1.00, that is, glucose oxidation increases, whereas lipid oxidation is markedly reduced. In summary, the direct effects of insulin are to promote glucose transport and phosphorylation, glycolysis, and glycogen synthesis; the stimulatory effect of the hormone on glucose oxidation is both direct and indirect, the latter being mediated by a fall in lipid availability.

The liver plays a central role in the regulation of glucose metabolism.1,2,9,10,97 After ingestion of a carbohydrate meal, it is essential that the liver suppress its basal rate of glucose production. In addition, the liver ultimately takes up approximately one third of the glucose in the ingested carbohydrate meal. Collectively, suppression of hepatic glucose production and augmentation of hepatic glucose uptake account for maintenance of approximately half of the maintenance of plasma glucose homeostasis after ingestion of a carbohydrate meal. The regulation of hepatic glucose production is controlled by a number of factors (see Fig. 10-3), of which insulin (inhibits hepatic glucose production) and glucagon and FFA (stimulate hepatic glucose production) are the most important. In vitro studies have demonstrated that plasma FFA are potent stimulators of hepatic glucose production and do so by increasing the activity of pyruvate carboxylase and phosphoenolpyruvate carboxykinase, the rate-limiting enzymes for gluconeogenesis.⁹⁸ FFA also enhance the activity of G6P, the enzyme that ultimately controls the release of glucose by the liver⁹⁹ (Fig. 10-34).

In normal subjects, an acute physiologic increase in plasma FFA concentration stimulates gluconeogenesis, whereas a decrease in plasma FFA concentration reduces gluconeogenesis.¹⁰⁰ A significant portion (as much as 25%) of the suppressive effect of insulin on hepatic glucose production is mediated via inhibition of lipolysis and a reduction in circulating plasma FFA concentration.^10,23 The relationship between increased plasma FFA concentration, FFA oxidation, and hepatic glucose production can be explained as follows: (1) Increased plasma FFA levels, by mass action, augment FFA uptake by hepatocytes, leading to accelerated lipid oxidation and accumulation of acetyl-CoA. The increased concentration of acetyl-CoA stimulates pyruvate carboxylase, the rate-limiting enzyme in gluconeogenesis, as well as G6Pase, the rate-controlling enzyme for glucose release from the hepatocyte (see Fig. 10-34). (2) The increased rate of FFA oxidation provides a continuing source of energy (in the form of ATP) and reduced nucleotides (NADH) to drive gluconeogenesis. The net result is an enhanced flux of three carbon precursors from pyruvate to oxaloacetate and thus into the gluconeogenic pathway. Finally, it should be noted that an increase in plasma FFA concentration need not be associated with an increase in hepatic glucose production, even though gluconeogenesis is enhanced. This is especially true in nondiabetic subjects in whom, except under very unique experimental conditions, elevation of the plasma FFA level rarely leads to an accelerated rate of hepatic glucose output, because the stimulation of hepatic gluconeogenesis by FFA is precisely offset by an inhibition of glycogenolysis, thereby yielding no net change in hepatic glucose production. This hepatic autoregulation in response to FFA infusion is similar to that observed during the infusion of gluconeogenic precursors.¹⁰¹ Thus, in dogs and nondiabetic humans, intravenous administration of alanine, lactate, and glycerol augment gluconeogenesis but fail to increase total hepatic glucose production because of a reciprocal decline in glycogenolysis. The situation in insulin-resistant states, such as obesity and diabetes, appears to be different. Here, FFA infusion impairs the suppression of hepatic glucose production by insulin and in some instances may actually elevate the basal rate of hepatic glucose production.^102,103

Amino acids also can enter into a substrate competition cycle with glucose, although somewhat less effectively than FFAs.¹⁰⁴ Increased amino acid provision enhances glucose production under conditions of insulin deficiency or resistance and limits glucose utilization in the insulinized state. Furthermore, an increase in the plasma FFA concentration exerts a hypoaminoacidemic effect in humans.¹⁰⁵ In summary, each of the three major substrates (i.e., glucose, FFAs, amino acids), if present in excessive amounts (whether by endogenous production or exogenous administration), can lower the level of the other two by stimulating insulin release. In this situation, glucose metabolism obviously is favored because it is a much more potent insulin secretagogue than fat or amino acids. In addition, multiple substrate effects (not mediated by insulin) participate in the regulation of the substrates themselves: high FFA and amino acid concentrations increase glucose, whereas a high FFA concentration lowers the amino acid levels. The net result is the creation of a glucose–FFA–amino acid cycle in which each member of the triad influences its fellow members both directly and indirectly through the stimulation of insulin secretion (Fig. 10-35).

Lipid Synthesis

In addition to its potent restraining effect on lipolysis and inhibition of FFA oxidation, the increase in plasma insulin concentration that occurs in the fed state stimulates fatty acid synthesis and storage as triacylglycerol in adipose depots throughout the body. As discussed previously, these fat stores are mobilized during conditions of fasting and serve as an important metabolic fuel for skeletal muscle, heart, kidney, liver, and other organs. In addition to its important role in lipid synthesis, insulin also enhances cholesterol formation and cholesterol ester storage and promotes phospholipid metabolism.

In humans, adipose tissue and the liver represent the primary sites of fatty acid synthesis. Insulin augments fatty acid synthesis in these tissues primarily by activating acetyl-CoA carboxylase.³⁰ This enzyme converts acetyl-CoA in the cytosol to malonyl-CoA (see Fig. 10-29). Insulin directly activates acetyl-CoA carboxylase by increasing its phosphorylation state and enhancing its synthesis. Insulin also indirectly activates the enzyme by increasing the supply of citrate, which activates acetyl-CoA carboxylase, and by stimulating the pentose phosphate cycle, thus providing the necessary reducing equivalents (NADH) for fat biosynthesis. Insulin also phosphorylates, thereby activating, ATP-citrate lyase, the step immediately preceding that catalyzed by acetyl-CoA carboxylase (see Fig. 10-29).³⁰ The increase in cytosolic malonyl-CoA concentration simultaneously activates fatty acid synthase and binds to carnitine palmitoyl transferase-I, inactivating the enzyme and inhibiting fatty acid oxidation.⁸⁶ The net result is an enhanced availability of fatty acyl-CoA for triglyceride synthesis. Insulin appears to have little effect on any of the enzymes involved in triacylglycerol synthesis. This is in marked contrast to insulin’s powerful inhibitory action on triacylglycerol lipase, the rate-limiting enzyme in the regulation of lipolysis.⁸⁶ In addition to the stimulatory effect of insulin on triglyceride synthesis, the hormone also appears to enhance cholesterol formation through an effect exerted on hydroxymethylglutaryl-CoA reductase.

Ketone Metabolism

Ketones are formed in the liver from the oxidation of FFA, and their synthesis is tightly regulated by the circulating levels of insulin and glucagon as well as malonyl-CoA. In the fed state, insulin levels rise, lipolysis is inhibited, and intracellular malonyl-CoA levels rise⁸⁶ (see Chapter 20). This latter key intermediate inhibits carnitine acyltransferase-I, thus favoring triglyceride synthesis and retarding fatty acid oxidation and ketone body formation. Conversely, in the starved or diabetic state, plasma insulin levels decline and glucagon increases. The increase in glucagon-to-insulin ratio is associated with a decrease in malonyl-CoA concentration, and fatty acid oxidation and ketogenesis are favored (see Fig. 10-30). With the exception of the liver, most tissues can oxidize ketone bodies. Their utilization by peripheral tissues, including muscle, is primarily regulated by their plasma concentration. Ketone bodies represent a major fuel for muscle during prolonged fasting, once their circulating blood levels increase to 1 to 3 mmol/L. Reversal of the elevated glucagon-to-insulin ratio after feeding rapidly inhibits ketogenesis, and the associated decline in plasma ketone levels abolishes ketone utilization by peripheral tissues.

Oral Glucose

At any given point in time, the glycemic response to an exogenous glucose load represents the balance between the rate of glucose appearance in the systemic circulation from all sources (i.e., ingested glucose entering via the gastrointestinal tract and endogenous glucose production) and the rate at which glucose is disposed of by all tissues in the body.97,106 Oral glucose appearance in the systemic circulation depends on (1) the rate at which the gastric contents are passed on to the small intestine; (2) the rate of intestinal glucose absorption; (3) the extent of gut glucose utilization; (4) the degree of hepatic glucose trapping; and (5) the dynamics of glucose transfer through the gut, liver, and posthepatic circulation to the right heart. The contribution of endogenous glucose production to the glycemic response to feeding depends on the extent and rate of change of hepatic glucose release. Initially, disposition of an ingested glucose load depends on changes in the pattern of hormonal stimuli and substrate availability. Because it represents a summation phenomenon, the response to oral glucose explores the whole of glucose tolerance, not the individual contribution of the various components. As discussed previously in this chapter, the rate-limiting step in the transfer of ingested glucose from the stomach to the liver is the rate of gastric emptying. This depends on the volume, temperature, osmolarity, and sodium content of the glucose solution when glucose alone is ingested. Glucose absorption through the intestinal epithelial cells is rapid, efficient, and well in excess of ordinary needs. Glucose utilization by intestinal tissues is small when glucose is presented from the vascular side, that is, when there is no oral glucose (see Table 10-2). The major fuels used by the gastrointestinal tissues in the postabsorptive state are FFA and glutamine. In the presence of glucose at high concentrations on the luminal side, it appears that gut glucose metabolism increases in response to the increased energy needs for absorption; the quantitative aspects of this stimulation of gut glucose utilization remain undetermined in the human. The possibility also exists that systemic hyperglycemia and/or hyperinsulinemia may impede intestinal glucose absorption.

Glucose uptake by the liver is stimulated by portal hyperglycemia (see following),9,10,107 and this has a major impact on hepatic glucose release. The traversal of glucose through the hepatic space is relatively quick, and this is unlikely to introduce a significant delay in the systemic appearance of oral glucose. In summary, the dynamics of oral glucose appearance are essentially determined by gastric emptying, whereas intestinal transport, transit across the gastrointestinal mucosa into the portal blood, and transhepatic passage together introduce only a small time delay. Stated otherwise, if neither gastrointestinal nor liver tissues used glucose, the time course of oral glucose appearance in the systemic circulation would follow that of gastric emptying, with a time shift of a few minutes. For this reason, the glucose absorption step represents a major component of the shape of the glycemic response to glucose. Fig. 10-36 shows the pattern of appearance of ingested glucose in healthy individuals, as reconstructed by a double tracer technique.⁹⁷ Glucose appearance in the systemic circulation peaks within 30 to 45 minutes, declines slowly thereafter, but remains significantly above zero at 210 minutes after glucose ingestion. At least 4 to 5 hours are necessary for the complete absorption of an oral glucose load, and this is further prolonged by the presence of fat and protein in the meal. Fig. 10-36 also shows the time course of suppression of endogenous glucose release by oral glucose.^97,107 A sustained nadir is reached between 60 and 120 minutes, and this is followed by a slow return to the fasting rate; however, hepatic glucose production is still significantly inhibited 210 minutes after the glucose challenge. Overall suppression of hepatic glucose production during the 3- to 4-hour period after glucose ingestion averages 50%. This is surprisingly less than what would be expected based on the combined portal hyperglycemia and hyperinsulinemia (see Fig. 10-5). Because circulating levels of insulin antagonistic hormones (except norepinephrine) do not change after oral glucose, it is likely that activation of the sympathetic nervous system, and specifically those nerves innervating the liver, keeps liver glucose outflow open.¹⁸ In Fig. 10-37, the observed arterial plasma glucose concentration is broken down into the component contributed by the appearance of oral glucose and that derived from hepatic glucose production.^97,107 The resemblance of oral glucose appearance rate to the plasma glucose curve is readily evident, especially during the first 60 to 90 minutes. Less appreciated is the fact that absorption is still incomplete 3 to 4 hours after ingestion. After a lag of approximately 30 minutes, tissue glucose uptake is stimulated by 50% to 100% throughout the period of observation. Hyperglycemia (mass action effect of glucose to promote its own uptake) contributes more to whole-body glucose disposal during the first half hour; thereafter, hyperinsulinemia provides the predominant stimulus. Oral glucose also elicits a marked vasodilation of the splanchnic vascular bed, and this regional increase in splanchnic blood flow persists for at least 4 hours. Thus both the metabolic and the hemodynamic perturbations induced by oral glucose extend beyond the time that it takes for the plasma glucose concentration to return to its pre-ingestion level.

The tissue destination of absorbed glucose has been the subject of intense investigation. Although the liver was classically reputed to be responsible for the eventual disposal of the majority of an oral glucose load, more recent studies that have employed the hepatic vein catheter technique in combination with radioisotope methodology have demonstrated that peripheral tissues dispose of approximately two thirds of the ingested glucose, whereas the splanchnic tissues account for the remaining one third.108,109 However, it must be remembered that the splanchnic tissues (i.e., liver) also contribute to glucose conservation by reducing their output of glucose. When this amount of glucose is added to that which is taken up by the liver, it can be seen that splanchnic (hepatic) and peripheral tissues contribute approximately equally to the maintenance of normal glucose homeostasis. Obviously, these approximate proportions vary according to the period during which the study is conducted, as well as with the nature of individual responses to glucose ingestion. A robust insulin secretory response directs more posthepatic glucose to the peripheral tissues, whereas a large increase in splanchnic blood flow channels the delivery of incoming sugar to the liver. In humans, for example, a glucose drink sipped slowly over 3.5 hours rather than swallowed in one bolus generates the same overall glucose curve but a 50% smaller endogenous insulin response. Last, the route of glucose administration exerts an important influence on the metabolic fate of glucose in that the portosystemic glucose concentration gradient per se enhances hepatic glucose uptake independently of portal glycemia and total glucose delivery to the liver.¹⁰ In humans, the oral route of glucose administration also has been shown to enhance splanchnic (hepatic) glucose uptake.¹⁰⁷ Recent studies also have shown that the liver takes up glucose in proportion to delivery.¹¹⁰

After glucose ingestion, glucose oxidation in the brain continues unabated during the absorptive period. Approximately 50% of the glucose that is taken up by peripheral tissues (muscle) is oxidized, and the remainder is stored as muscle glycogen or as lactate in the lactate pool. During absorption from the gastrointestinal tract, lactate release by the intestinal tissues into the portal vein is markedly increased. It has been estimated that approximately 5% of the ingested glucose load is converted into three carbon precursors (lactate, pyruvate, and alanine) that are passed on to the liver and synthesized into glycogen via the indirect pathway of gluconeogenesis. Because the lactate concentration in the hepatic vein also increases after glucose ingestion, it follows that the sum of hepatic lactate production and gut lactate formation must exceed hepatic lactate extraction. Liver glycogen formation during absorption of oral glucose occurs both directly from glucose and indirectly via gluconeogenesis.¹¹¹ The relative contributions of the direct (from glucose) versus indirect (from gluconeogenic precursors) pathway to hepatic glycogen synthesis in humans remains uncertain owing to methodological difficulties. However, current data suggest that gluconeogenesis participates in liver glycogen repletion to a much lesser extent in humans than in rats.

Counterregulatory Hormones

A review of the role of the insulin counterregulatory hormones (glucagon, epinephrine, cortisol, growth hormone, and thyroid hormones) and their roles in intermediary metabolism is beyond the scope of this chapter, and the reader is referred to Chapter 21 and Ref. 16. The most important role of the counterregulatory hormones is in the defense against hypoglycemia. Glucagon and epinephrine play an important role in the acute recovery from hypoglycemia by stimulating both glycogenolysis and gluconeogenesis. When hypoglycemia is prolonged, cortisol and growth hormone contribute to the restoration of normoglycemia by impairing the tissue’s ability to respond to insulin. Epinephrine also is a potent peripheral insulin antagonist. Except for glucagon (whose concentration increases during fasting, leading to a stimulation of hepatic glucose production primarily by augmenting gluconeogenesis), it is unclear what role if any these insulin antagonistic hormones play in glucoregulation during the transition from the fed to the fasted state. More likely, these counterregulatory hormones contribute to the mobilization of competitive substrates, especially FFAs, from adipose depots to the liver and muscle, where they serve as an important energy source. Circulating levels of these counterregulatory hormones (except glucagon) do not increase during fasting. Rather, the plasma insulin concentration declines in response to the decrease in blood glucose, and the resultant hypoinsulinemia leaves the lipolytic and ketogenic activities of the counterregulatory hormones unopposed, thus facilitating the shift from glucose to FFA metabolism.

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