Clinical Note continued

and an "overproduction" of glucose by liver. Gluconeogenesis is increased at the expense of muscle protein, which is the chief source of the amino acid substrate. Consequently, there is marked wasting of muscle along with depletion of body fat stores. Devastating cardiovascular complications—atherosclerosis, coronary artery disease, and stroke—often result from high concentrations of blood lipids. Other less obviously related complications include lesions in the microvasculature of the retina and kidneys and in peripheral nerves, and they result from prolonged hyperglycemia and complete the clinical picture. The net effect of insulin lack is a severe reduction in the ability to store glycogen, fat, and protein.

cells. Even within a single cell it produces multiple effects that are both complementary and reinforcing. Insulin acts on adipose tissue, skeletal muscle, and liver to defend and expand reserves of triglyceride, glycogen, and protein. Within a few minutes after intravenous injection of insulin, there is a striking decrease in the plasma concentrations of glucose, amino acids, FFA, ketone bodies, and potassium. If the dose of insulin is large enough, blood glucose may fall too low to meet the needs of the central nervous system, and hypoglycemic coma may occur. Insulin lowers blood glucose in two ways: It increases uptake by muscle and adipose tissue and decreases output by liver. It lowers the concentration of amino acids by stimulating their uptake by muscle and reducing their release. Insulin lowers the concentration of FFA by blocking their release from

hours after oral glucose (100 g)

FIGURE 10 Idealized glucose tolerance tests in normal and diabetic subjects.

hours after oral glucose (100 g)

FIGURE 10 Idealized glucose tolerance tests in normal and diabetic subjects.

adipocytes, and this action in turn lowers the blood ketone level. The decrease in potassium results from stimulation of the sodium/potassium ATPase (sodium pump) in the plasma membranes of muscle, liver, and fat cells. The physiologic significance of this response to insulin is not understood.

Effects on Adipose Tissue

Storage of fat in adipose tissue depends on multiple insulin-sensitive reactions, including (1) synthesis of long-chain fatty acids from glucose; (2) synthesis of triglycerides from fatty acids and glycerol (esterifica-tion); (3) breakdown of triglycerides to release glycerol and long-chain fatty acids (lipolysis); and (4) uptake of fatty acids from the lipoproteins of blood. The relevant biochemical pathways are shown in Fig. 11.

Lipolysis and esterification are central events in the physiology of the adipocyte. The rate of lipolysis depends on the activity of triglyceride lipase. Lipolysis proceeds at a basal rate in the absence of hormonal stimulation but increases dramatically when cyclic AMP is increased. Hormone-sensitive lipase catalyzes the breakdown of triglycerides into fatty acids and glycerol. Fatty acids can either escape from the adipo-cyte and become the FFA of blood or be re-esterified to triglyceride. Fatty acid esterification requires a source of glycerol that is phosphorylated in its a carbon; free glycerol cannot be used. Because adipose tissue lacks the enzyme a-glycerol kinase, all of the free glycerol that is produced by lipolysis escapes into the blood. The only source of a-glycerol phosphate available for esterifica-tion of fatty acids is derived from phosphorylated 3-carbon intermediates formed from oxidation of glucose.

As its name implies, hormone-sensitive lipase is activated by lipolytic hormones, which stimulate the formation of cyclic AMP and thereby promote its phosphorylation by protein kinase A. Insulin accelerates the degradation of cyclic AMP by activating the enzyme glucose r^j

-glucose glycogen

ad'tpoQfte glucose-6-P

glucose-6-P

he,xose, glycolytic monophosphate pathway he,xose, glycolytic monophosphate pathway

triglyceride i

glycerol

CO2 CO2 lipoproteins

FIGURE 11 Carbohydrate and lipid metabolism in adipose tissue. Reactions enhanced by insulin (blue arrows) are as follows: (1) transport of glucose into adipose cell; (2) conversion of excess glucose to glycogen; (3) decarboxylation of pyruvate; (4) initiation of fatty acid synthesis; and (5) uptake of fatty acids from circulating lipoproteins. Breakdown of triglycerides is inhibited by insulin (broken arrow). Esterification of fatty acids to triglycerides follows from availability of a-glycerol phosphate.

cyclic AMP phosphodiesterase and thus interferes with activation of hormone-sensitive lipase. Simultaneously, insulin increases the rate of fatty acid esterification by increasing the availability of a-glycerol phosphate. The net result of these actions is preservation of triglyceride stores at the expense of plasma FFA, whose concentration in blood plasma promptly falls. Decreases in FFA concentrations are seen with doses of insulin that are too low to affect blood glucose and appear to be the most sensitive response to insulin.

Because glucose does not readily diffuse across the plasma membrane, its entry into adipocytes and most other cells depends on carrier-mediated transport. Insulin increases cellular uptake and metabolism of glucose by accelerating transmembrane transport of glucose and structurally related sugars. This action depends on the availability of glucose transporters in the plasma membrane. Glucose transporters (abbreviated GLUT) are large proteins that weave in and out of the membrane 12 times to form stereospecific channels through which glucose can diffuse down its concentration gradient. At least five isoforms of GLUT are expressed in various cell types. In addition to GLUT 1, which is present in the plasma membrane of most cells, insulin-sensitive cells such as adipocytes contain pools of intracellular membranous vesicles that are rich in GLUT 4. Insulin increases the number of glucose transporters on the adipocyte surface by stimulating the translocation of GLUT 4-containing vesicles toward the cell surface and fusion of their membranes with the adipocyte plasma membrane (Fig. 12).

Insulin may accelerate synthesis of fatty acids by increasing the uptake of glucose and by activating at least two enzymes that direct the flow of glucose carbons into fatty acids. Insulin increases conversion of pyruvate to acetyl CoA, which provides the building blocks for long-chain fatty acid synthesis, and stimulates carboxylation of acetyl CoA to malonyl CoA, which is the initial and rate-determining reaction in fatty acid synthesis. In humans, adipose tissue is not an important site of fatty acid synthesis, particularly in Western cultures where the diet is rich in fat. Fat stored in adipose tissue is derived mainly from dietary fat and triglycerides synthesized in the liver. Fat destined for storage reaches adipose tissue

intracellular pool plasma membrane

FIGURE 12 Hypothetical model of insulin's action on glucose transport. Upon associating with its receptors (R) in the cell membrane, insulin (I) signals the translocation of glucose transport systems to the plasma membrane. The stepwise sequence of events is indicated by the blue circled numbers. (From Karnieli E, Zarnowski MJ, Hissin RJ, Salans LB, Cushman SW, Insulin-stimulated translocation of glucose transport systems in the isolated rat adipose cell. Time course, reversal, insulin concentration dependency, and relationship to glucose transport activity. J Biol Chem 1981;256:4772-4777).

intracellular pool

I association plasma membrane

FIGURE 12 Hypothetical model of insulin's action on glucose transport. Upon associating with its receptors (R) in the cell membrane, insulin (I) signals the translocation of glucose transport systems to the plasma membrane. The stepwise sequence of events is indicated by the blue circled numbers. (From Karnieli E, Zarnowski MJ, Hissin RJ, Salans LB, Cushman SW, Insulin-stimulated translocation of glucose transport systems in the isolated rat adipose cell. Time course, reversal, insulin concentration dependency, and relationship to glucose transport activity. J Biol Chem 1981;256:4772-4777).

in the form of low-density lipoproteins and chylomicrons. Uptake of fat from lipoproteins depends on cleavage of ester bonds in triglycerides by the enzyme lipoprotein lipase to release fatty acids. Lipoprotein lipase is synthesized and secreted by adipocytes and adheres to the endothelium of adjacent capillaries. Insulin promotes synthesis of lipoprotein lipase and thus facilitates the transfer of fatty acids from lipoproteins to triglyceride storage droplets in adipocytes.

Effects on Muscle

Insulin increases uptake of glucose by muscle and directs its intracellular metabolism toward the formation of glycogen (Fig. 13). Because muscle comprises nearly 50% of body mass, uptake by muscle accounts for the majority of the glucose that disappears from blood after injection of insulin. As in adipocytes, glucose utilization in muscle is limited by permeability of the plasma membrane. Insulin accelerates entry of glucose into muscle by mobilizing GLUT 4-containing vesicles by the same mechanism that is operative in adipocytes.

Metabolism of glucose begins with conversion to glucose-6-phosphate catalyzed by either of the two isoforms of the enzyme hexokinase that are present in muscle. Insulin not only increases the synthesis of hexokinase II, but it also appears to enhance the efficiency of hexokinase II activity by promoting its association with the outer membrane of mitochondria, which optimizes access to ATP. In the basal state, glucose is phosphorylated almost as rapidly as it enters the cell, and hence the intracellular concentration of free glucose is only about one-tenth to one-third that of extracellular fluid.

Glucose-6-P is an allosteric inhibitor of hexokinase and an allosteric activator of glycogen synthase. Stimulation of glycogen synthesis by insulin and glucose-6-phosphate protects hexokinase from the inhibitory effect of glucose-6-phosphate when entry of glucose into the muscle cell is rapid. Glycogen synthase activity is low when the enzyme is phosphorylated and increased when it is dephosphorylated. The degree ofphosphorylation of glycogen synthase is determined by the balance of kinase and phosphatase activities. Insulin shifts the balance in

FIGURE 13 Metabolism of carbohydrate and lipid in muscle. Rate-limiting reactions accelerated by insulin (blue arrows) are as follows: (1) transport of glucose into muscle cells; (2) phosphorylation of glucose by hexokinase; (3) storage of glucose as glycogen; (4) addition of the second phosphate by phosphofructokinase; and (5) inhibition of fatty acid entry into mitochondria by malonyl CoA.

FIGURE 13 Metabolism of carbohydrate and lipid in muscle. Rate-limiting reactions accelerated by insulin (blue arrows) are as follows: (1) transport of glucose into muscle cells; (2) phosphorylation of glucose by hexokinase; (3) storage of glucose as glycogen; (4) addition of the second phosphate by phosphofructokinase; and (5) inhibition of fatty acid entry into mitochondria by malonyl CoA.

favor of dephosphorylation in part by inhibiting the enzyme glycogen synthase kinase 3 (GSK-3) and in part by activating a phosphatase. Dephosphorylation of glycogen synthase not only increases its activity directly, but also increases its responsiveness to stimulation by its substrate, glucose-6-P. Hence the powerful effects of insulin on muscle glycogen synthesis are achieved by the complementary effects of increased glucose transport, increased glucose phosphorylation, and increased gly-cogen synthase activity.

The alternative fate of glucose-6-P, metabolism to pyruvate in the glycolytic pathway, is also increased by insulin. Access to the glycolytic pathway is guarded by phosphofructokinase, whose activity is precisely regulated by a combination of allosteric effectors including ATP, ADP, and fructose-2,6-bisphosphate. This complex enzyme behaves differently in intact cells and in the broken cell preparations typically used by biochemists to study enzyme regulation. Because conflicting findings have been obtained under a variety of experimental circumstances, no general agreement has been reached on how insulin increases phosphofructokinase activity. In contrast to the liver, the isoform of the enzyme that forms fructose-2,6-bisphosphate in muscle is not regulated by cyclic AMP. The effects of insulin are likely to be indirect.

Note that oxidation of fat profoundly affects the metabolism of glucose in muscle and that insulin also increases all aspects of glucose metabolism in muscle as an indirect consequence of its action on adipose tissue to decrease FFA production. When insulin concentrations are low, increased oxidation of fatty acids decreases oxidation of glucose by inhibiting the decarboxylation of pyruvate and the transport of glucose across the muscle cell membrane. In addition, products of fatty acid oxidation appear also to inhibit hexokinase, but recent studies have called into question the relevance of earlier findings that fatty acid oxidation may inhibit phosphofructokinase. Insulin not only limits the availability of fatty acids, but also inhibits their oxidation. Insulin increases the formation of malonyl CoA, which blocks entry of long chain fatty acids into the mitochondria as described for liver (Fig. 7). These effects are discussed in Chapter 42.

Protein synthesis and degradation are ongoing processes in all tissues and in the nongrowing individual are completely balanced so that on average there is no net increase or decrease in body protein (Fig. 14). In the absence of insulin there is net degradation of muscle protein and muscle becomes an exporter of amino acids, which serve as substrate for gluconeogenesis and ureogenesis in the liver. As with its effects on carbohydrate and fat metabolism, insulin intercedes in protein synthesis at several levels, and has both rapidly apparent and delayed effects. Insulin increases uptake of amino

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