20 Insulin Secretion and Action

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From: Endocrinology: Basic and Clinical Principles, Second Edition (S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ

20 Insulin Secretion and Action

Run Yu, MD, PhD, Hongxiang Hui, MD, PhD, and Shlomo Melmed, MD

CONTENTS

INTRODUCTION

DEVELOPMENT OF E^NDOCRINE PANCREAS AND REGULATION OF I^SLETΒ-C^ELL M^ASS INSULIN SYNTHESIS

INSULIN SECRETION

INSULIN SIGNALING

required for normal development. Intact insulin function requires four components: islet β-cell mass; insulin synthesis; glucose-dependent insulin secretion; and, ultimately, insulin signaling at the target cells. Although theoretically any of these components can go awry and cause disease, abnormal insulin signaling is the most common problem, followed by decreased islet cell mass.

Abnormal insulin synthesis or secretion rarely causes diseases. Understanding the physiology of all four components, however, is important to prevent and treat diabetes and related diseases.

2. DEVELOPMENT OF ENDOCRINE PANCREAS AND REGULATION

OF ISLET β-CELL MASS

The islet β-cell mass is dynamic and regulated.

Maintaining an appropriate β-cell mass in response to metabolic demand is critical for maintaining glucose homeostasis. Decreased β-cell mass underlies several types of diabetes. In type 1 diabetes, β-cells are destroyed by an autoimmune mechanism. In late-stage type 2 diabetes, β-cells undergo excessive apoptosis owing to glucose toxicity. Intrinsic genetic defects play a central role in causing decreased β-cell mass in maturity-onset diabetes of the young (MODY), diabe-

1. INTRODUCTION

Because glucose is the primary energy source of most cells in the body, control of constant circulating glucose levels is of utmost importance. Too little serum glucose (hypoglycemia) suppresses central nervous system functions and prolonged hypoglycemia leads to death.

Too much serum glucose (hyperglycemia) as seen in diabetes mellitus, results in grave consequences such as kidney, nerve, eye, muscle, and immune system dam- age. The body has an elaborate system to control circulating glucose levels in a narrow range (72–126 mg/dL) to prevent untoward fluctuations. For populations in Western societies, the predominant problem in glucose metabolism is diabetes mellitus although other derangements are significant but not often encountered. Of all the humoral and neuronal regulatory mechanisms for glucose metabolism, insulin is the hormone that lowers serum glucose whereas most other mechanisms function to increase serum glucose.

Insulin is a peptide hormone secreted by β-cells in the pancreatic islets of Langerhans. The main function of insulin is to lower serum glucose. Insulin is a major anabolic hormone that is critical in lipid and protein synthesis, and insulin is also an essential growth factor

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β-cell mass results from the difference between β-cell proliferation and cell death. β-Cell proliferation is achieved in two ways: (1) neogenesis (formation of β−

cells from precursor pancreatic ductal cells) and (2) replication of differentiated β-cells. β-Cell death can be due to either apoptosis (programmed and controlled) or necrosis (associated with inflammation). It has re- cently been realized that β-cells continue to proliferate throughout adult life. The average islet size is more than 10-fold larger in adult mice than in younger animals. The most robust islet growth occurs in the intrau- terine and neonatal period. β-Cell proliferation is also prominent during pregnancy.

An islet of Langerhans comprises three main cell types: (1) glucagon-secreting α-cells at the periphery (15–20% of islet cells), (2) insulin-secreting β-cells at the inside (60–80%), and (3) peripheral somatostatin-secretingδ-cells (5–10%). Glucagon functions to increase serum glucose levels and somatostatin suppresses insulin secretion. During embryonic pancreatic development, primordial islet cell clumps are derived from nascent pancreatic ducts and detach from the ducts to expand and coalesce with other clumps (Fig. 1). β-Cells are first detected at the wk 13 of ges- tation and begin to secrete insulin at the wk 17. β-Cell proliferation and differentiation are under tight control of a number of transcription factors. Pancreatic duode- nal homeobox gene-1 (PDX-1), hepatocyte nuclear factor-1α (HNF-1α), HNF-1β, HNF-4α, insulin promoter factor-1 (IPF-1), and β-cell E-box transactivator 2 (BETA2) are some that have clinical implications since their respective mutations result in neonatal diabetes or MODY. PDX-1 is required for normal pancreatic development. A patient with deficient PDX-1 expression has failed pancreatic development. In adults, PDX-1 is also important for normal islet function because it regulates insulin gene expression. HNF- 1α gene mutations are found in MODY3 and HNF-4α gene mutations in MODY1; mutations in HNF-1β, IPF-1, and BETA2 cause MODY5, MODY4, and

decreasedβ_cell mass observed in this syndrome. Dia- betes is found in a number of knockout mice deficient in various genes. In many cases, the experimentally dis- rupted gene is expressed in many cell types, but the β-cells seem to be particularly vulnerable to the disruption. Securin (also called PTTG), a regulatory protein critical for progression of mitosis, is expressed in all proliferative cells. Disruption of murine securin results in defective proliferation of β-cells, a cell type not known for rapid division, while sparing more proliferative cells such as hemopoietic and spermatogenic cells.

Another intriguing feature of diabetes owing to genetic defects is that in most cases, they do not immediately manifest themselves but occur after a considerable latent period in childhood or young adulthood, suggesting that other insults during postnatal growth must cooperate with the genetic defects to result in the clinical phenotype.

Besides genetic determinants, β-cell mass is regulated by nutrients, growth factors, and hormones. Glu- cose is a major stimulator of β-cell proliferation, and in rodents, infusion of glucose for 24 h results in a rapid increase in β-cell mass, mostly owing to neogenesis.

Ironically, glucose also induces β-cell apoptosis, as seen in prolonged type 2 diabetes. Amino acids and free fatty acids are also potent stimulators of β-cell proliferation.

Several growth factors such as epidermal growth factor, fibroblast growth factor, and vascular endothelial growth factor stimulate β-cell proliferation. Many hormones including insulin, insulin-like growth factor-1 (IGF-1), IGF-2, glucagon, gastroinhibitory peptide, gastrin, cholecystokinin, growth hormone (GH), pro- lactin (PRL), placental lactogen, leptin, and glucagon- like peptide-1 (GLP-1) stimulate β-cell proliferation.

Most of the hormones have significant systemic effects and therefore are not good candidates for potential therapeutic agents, whereas GLP-1 is rather specific in increasingβ-cell mass. GLP-1, a gastrointestinal peptide hormone secreted by enteroendocrine L-cells, stimulatesβ-cell mass growth in animal models and in

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human islet cultures through three potential pathways:

(1) promotion of β-cell proliferation, (2) stimulation of β-cell neogenesis from ductal epithelium, and (3) inhi- bition of β-cell apoptosis. In rodents, GLP-1 treatment results in an increase in β-cell proliferation. Multiple signaling pathways including phosphatidylinositol 3- kinase (PI3K), Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) mediate the proliferative effects. Islet neogenesis may also be stimulated, because the number of small islets increases after chronic administration of GLP-1 analogs. PDX-1 appears important in mediating the GLP-1 stimulation of islet neogenesis. GLP-1 prevents apoptosis in immor- talized rodent β-cell lines treated with apoptosis-pro- moting agents including peroxide, streptozotocin, fatty acids, and cytokines. GLP-1 also prevents apoptosis in human islet cultures. PI3K, Akt, and MAPK are the likely mediators for the antiapoptotic effects.

3. INSULIN SYNTHESIS

Insulin synthesis appears to be very error proof because defects are extremely rare. The only known defects are rare mutations in the insulin gene producing defective proinsulin. Understanding insulin synthesis, however, is important, because pharmacologic interven- tions can be designed to manipulate insulin synthesis.

The human insulin gene is located on chromosome 11p15.5 and contains three exons and two introns. The final spliced mRNA transcript is 446 bp long and encodes preproinsulin peptide (with a B-chain, a C- chain, and an A-chain). The structure of the insulin gene has been remarkably conserved throughout evolution.

Most animals have a single copy of the insulin gene, with the exception of rat and mouse, which carry a duplicate. The mammalian insulin gene is exclusively

expressed in β-cells of the islet of Langerhans. Intensive physiologic and biochemical studies have led to iden- tification of regulatory sequence motifs along the insulin promoter and the binding proteins, such as islet-restricted proteins (BETA2, PDX-1, RIP3b1-Act/

C1) and ubiquitous proteins (E2A, HEB). Their DNA- binding activity and transactivating potency can be modified in response to nutrients (glucose, free fatty acids) or hormonal stimuli (insulin, leptin, GLP-1, GH, PRL) through kinase-dependent signaling pathways (PI3K, p38MAPK, PKA, calmodulin kinase). It is nota- ble that transcriptional regulation results not only from specific combinations of these activators through DNA- protein and protein-protein interactions, but also from their relative nuclear concentrations, generating cooperativity and transcriptional synergism unique to the insulin gene.

As in the case of many other peptide hormones, insulin mRNA encodes preproinsulin peptide. A signal sequence is removed from preproinsulin while preproinsulin is inserted into the endoplasmic reticulum, which results in proinsulin. Proinsulin is further processed to form insulin and a byproduct, C peptide, both of which are packaged into secretory vesicles. The mature insulin molecule comprises two peptide chains (A and B) linked together by two disulfide bonds, and an additional disulfide bond is formed within the A chain (Fig. 2). In most species, the A chain consists of 21 amino acids and the B chain, 30 amino acids. Although the amino acid sequence of insulin varies among species, key structural features (including the positions of the three disulfide bonds, both ends of the A chain, and the carboxyl-terminal residues of the B chain) are highly conserved, and the three-dimensional conformation is quite similar.

Insulin from one species is usually readily active in Fig. 2. Insulin synthesis and processing. SS = signal sequence.

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another one. Insulin exists primarily as a monomer at low concentrations (~10^–6 M) and forms a dimer at higher concentrations at neutral pH. At high concentrations and in the presence of zinc ions, insulin aggre- gates further to form hexameric complexes. Monomers and dimers readily diffuse into blood, whereas hexamers diffuse poorly. Hence, absorption of insulin preparations containing a high proportion of hexamers is delayed and slow. By preparing different insulin analogs bearing amino acid substitutions and deletions, and comparing their ability to activate the insulin receptor, several regions of insulin have been determined to be linked to receptor binding. The three conserved regions in insulin—amino terminal A-chain (GlyA1-IleA2- ValA3-GluA4 or AspA4), carboxyl terminal A-chain (TyrA19-CysA20-AsnA21), and carboxyl terminal B- chain (GlyB23-PheB24-PheB25-TyrB26)—are located at or near the surface of insulin and therefore may interact with the insulin receptor. Understanding insulin structure has stimulated development of a number of insulin analogs, and recombinant DNA technology allows their production.

4. INSULIN SECRETION

β-cells are excitable endocrine cells that secrete insulin. The essential secretory machinery is analogous to that for other peptide hormones such as GH and PRL, as described elsewhere in this book. Although abnor- malities in insulin secretion are not common, insulin secretion is of pharmacologic interest because oral hypoglycemic agents act by increasing insulin secretion. Multiple mechanisms mediate insulin secretion, the most important being glucose-stimulated, K_ATP channel dependent. Other pathways either augment or complement that pathway.

Glucose-stimulated, K_ATPchannel-dependent pathway starts with intracellular transport of extracellular glucose by a glucose transporter (GLUT2) (Fig. 3).

Intracellular glucose undergoes cytosolic glycolysis catalyzed by glucokinase (glucokinase gene mutations

resultant biphasic increase in intracellular calcium trig- gers fusion of secretory vesicles with the plasma membrane and insulin is released.

The K_ATPchannel plays the crucial role of converting metabolic to electric signals. All components of glucose-stimulated, K_ATPchannel-dependent insulin secretion are found in several other cell types, except for the K_ATP channel, which is unique for β_cells (a similar channel is also found in muscle and brain). Each β-cell posseses thousands of K_ATPchannels, which is a high density considering the small size of β-cells. This channel has eight subunits; four identical smaller subunits, called Kir6.2 (“ir” stands for “inward rectifier”), which form the inner pore for K⁺passage; and four identical larger subunits, termed the sulfonylurea receptors (SUR1), which form the outer regulatory structure. ATP regulates K_ATPchannel by binding to Kir6.2, and oral sulfonylurea hypoglycemic medications bind to SUR1 and close the K_ATP channel, thus stimulating insulin secretion. Not surprisingly, mutations in the K_ATPchan- nel cause either hyperglycemia or hypoglycemia. Muta- tions in SUR1 and Kir6.2 that render the K_ATPchannel less open cause insulin hypersecretion with neonatal hypoglycemia (nesidioblastosis). Conversely, other mutations in Kir6.2 that maintain the K_ATPchannel in an open state result in neonatal diabetes. K_ATP channel mutations are also found in some patients with type 2 diabetes.

Mitochondria produce ATP, the signal for the K_ATP channel, and they are implicated in a subtype of diabetes termed mitochondrial diabetes (1% of all diabetes). The most common is a mutation in the mitochondrial gene encoding transfer RNA for leucine, which decreases production of some mitochondrial proteins. Interest- ingly, this mutation causes two distinct syndromes: (1) maternally inherited mitochondrial diabetes and deaf- ness, and (2) mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS syndrome), the latter often associated with diabetes. Uncoupling protein 2 (UCP2) is an anion carrier located on the

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mitochondrial inner membrane that uncouples proton gradient and ATP synthesis, thus decreasing ATP production and increasing heat generation. Mice deficient in β-cell UCP2 have increased ATP production and insulin hypersecretion. UCP2 is upregulated in obesity and may contribute to diabetes associated with obesity.

Certain UCP2 gene polymorphisms are weakly associated with body mass index (measure of obesity) and type 2 diabetes.

Three other pathways are also known to stimulate insulin secretion. The augmentation pathway may facilitate the K_ATPchannel–dependent pathway but is controversial. The second pathway is also calcium dependent and works through Gq-coupled receptors that produce inositol triphosphate (IP₃), which releases calcium from the endoplasmic reticulum through IP₃ receptors. Acetylcholine releases insulin through this mechanism. GLP-1 and vasoactive intestinal peptide act through binding to Gs-coupled receptors that produce cyclic adenosine monophosphate, which activates PKA. PKA stimulates insulin secretion through a calcium-independent mechanism. GLP-1 is an ideal hormone candidate for diabetic treatment. Besides the stimulatory effects on β-cell mass growth, GLP-1- stimulated insulin release is regulated by serum glucose and is suppressed by hypoglycemia. In clinical trials, GLP-1 normalizes serum glucose in patients with type 2 diabetes without development of hypoglycemia.

5. INSULIN SIGNALING 5.1. Insulin Receptor

Insulin is released from the islet into the bloodstream and its actions are mediated by the insulin receptor (IR) on the surface of target cells. The most common etiol- ogy in diabetes, insulin resistance, results from defects in insulin signaling. Native IR is a large cylindrical tetrameric protein with a relative mobility (Mr) of 3,000,000, consisting of two α- and two β-polypeptide chains linked by disulfide bonds. IR is expressed in most mammalian tissues; adipose and liver have the highest IR density (>300,000 receptors/cell). High IR density is necessary for more rapid binding kinetics since normal plasma insulin levels range from 10^–10to 10^–9M, which are lower than the insulin-binding affinity. IR gene is located on the short arm of chromosome 19 and is approx 150 kb long with 22 exons and 21 introns. Most cell types have multiple RNA transcripts ranging from 5.7 to 9.5 kb in size, and all appear to code for the complete proreceptor. Exons 1–12 encode the α-subunit, and exons 13–22 encode the β-subunit. Two IR isoforms exist: IR-A is more abundant in fetal tissues, whereas IR-B is more abundant in adult tissues except brain. The difference between the two isoforms is that the IR-A

transcript does not include exon 11, whereas the IR-B transcript does, and, consequently, IR-A misses 12 amino acids near the carboxyl terminus of the α-subunit. Both isoforms have similar affinities for insulin.

IR as a whole is an allosteric enzyme. The β-subunit has tyrosine kinase activity, and the α-subunit regulates the β-subunit in an insulin-binding-dependent manner. The extracellular IR α-subunit is responsible for ligand binding. It has two insulin-binding pockets that interact with corresponding regions of insulin molecule. The carboxyl-terminal domain of α-subunit interacs with the β-subunit and transmits signal from insulin binding to activation of the IR kinase. The β- subunit has a short extracellular domain interacting with the α-subunit, a transmembrane domain, and a large intracellular domain with an intrinsic tyrosine (Tyr) kinase activity that undergoes tyrosine autophosphorylation on insulin binding to α-subunit and starts the insulin signaling network. Insulin-induced IR β-subunit autophosphorylation further increases the tyrosine kinase activity, and β-subunit autophosphorylation also forms recruitment sites for bringing substrate proteins to the IR. Tyrosine residues 1146, 1150, and 1151 are essential for tyrosine kinase activity. NPEY motif (Asn-Pro-Glu-Tyr) at the juxtamem- brane region is important for substrate recruitment.

Insulin receptor substrate-1 (IRS-1) and Shc, both IR substrates, interact with the NPEY motif. Mutations of autophosphorylation sites demonstrate that both increased kinase activity and recruitment of target proteins are critical for insulin signaling. Autophos- phorylation is achieved within seconds after insulin binding. Seconds to minutes later, substrate protein phosphorylation occurs.

IR signaling is regulated in a variety of ways. Two protein tyrosine phosphatases (PTPases) dephosphory- late the IR, terminating insulin action without degrad- ing the IR. One of the PTPases, leukocyte common antigen-related phosphatase, is membrane bound; the other, PTPase1B, is intracellular. Cell-surface IR density is regulated by addition from the Golgi apparatus and insulin-stimulated IR endocytosis. IR serine phosphorylation also contributes to the negative regulation of IR signaling.

5.2. Insulin Signal Network

The major substrates of IR tyrosine kinase are adapter protein Src-homology-collagen-like protein (SHC) and multifunctional docking proteins IRS-1 and IRS-2 (Fig. 4). Tyrosine-phosphorylated SHC recruits the small adapter protein growth factor receptor–binding protein 2 (Grb2), which, in turn, recruits and activates the ras-guanosine 5´-diphosphate exchange

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factor mammalian son-of-sevenless protein (m-sos).

The addition of m-sos into the receptor tyrosine kinase–

induced complex at the plasma membrane activates ras small guanosine 5´-triphosphate–binding protein, which leads to eventual activation of the MAPK cascade involved in stimulating mitogenesis. A number of other hormones and growth factors also regulate MAPK cascade, and IR-stimulated MAPK activation mostly does

not take part in insulin regulation of metabolic events.

IRS-1 and IRS-2 are the major mediators for insulin metabolic action. Phosphorylation of specific tyrosine residues on IRS-1 and IRS-2 by activated IR allows recruitment of a number of signaling molecules through their Src homology 2 (SH2) domains. These include Grb2, the small adapter protein Nck, the tyrosine phosphatase Syp, and PI3K.

Fig. 4. (A) Insulin receptor structure; (B) signal transduction for IR.

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PI3K plays a central role in insulin signaling, leading to metabolic consequences. PI3K has two subunits. The p85 subunit acts as an adapter and targets the p110 catalytic subunit to the appropriate signaling complex. p85 contains two SH2 domains, which bind to tyrosine-phosphorylated motifs on IRS-1, IRS-2, and growth factor receptors. An inter-SH2 domain in p85 interacts with the p110 catalytic subunit. The p110 subunit is a kinase that phosphorylates phosphoinositides at the 3-position to form phosphatidylinositol-3-phosphates. Signaling proteins containing pleckstrin homology domains bind to the membrane-bound phosphaotidylinositol-3-phosphate, and their activity or localization is altered by the binding. p110 also has some serine kinase activity that appears to be largely directed toward the p85 subunit and IRS-1. PI3K recruitment increases PI3K activity because p85 interaction with specific phosphotyrosine motifs increases the p110 kinase activity and recruitment brings PI3K closer to its substrate near plasma membranes. PI3K activates phosphatidylinositol-3,4- biphosphate/phosphatidylinositol-3,4,5-triphosphate- dependent kinase 1, which activates PKB/Akt, a serine kinase. PKB in turn deactivates glycogen synthase kinase-3 (GSK-3), leading to glycogen synthase activation and thus glycogen synthesis. PKB activation also results in translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane, where they transport glucose into the cell. Another target of PKB is mTOR-mediated activation of protein synthesis by PHAS/elf4 and p70s6k.

5.3. Physiologic Actions of Insulin

Insulin has profound, diverse, and indispensible physiologic functions in growth and metabolism (Fig. 5).

Insulin can act very rapidly, within minutes (e.g., increased glucose uptake in muscle and fat cells); or in minutes to hours (e.g., regulation of gene expression and protein degradation); or slowly, in days (e.g., cell differentiation and organ development). Insulin is one of the few quintessential growth factors required for cell survival, proliferation, and differentiation. Indeed, most synthetic growth media (serum-free “defined media”) for cell culture must contain insulin. It is not entirely clear how insulin signals as a growth factor but the MAPK pathway contributes at least partially. The metabolic activities of insulin, however, are more important clinically because metabolic derangements are the most common problems encountered in abnormal insulin signaling.

Insulin regulates the metabolism of glucose, fat, and proteins and the general effects are anabolic. The metabolic activities of insulin are mediated through modu- lating the expression and activity of key enzymes in metabolism via the PI3K pathway.

Insulin stimulates glucose uptake, utilization, and storage in various tissues. Glucose cannot diffuse pas- sively into the cells; the only way for a cell to take up glucose is through facilitated diffusion with hexose transporters (GLUTs). Four types of GLUT exist:

GLUT1 is present in most tissues; GLUT2 is in liver and pancreaticβ-cells, and GLUT2 in β-cells is important for regulating insulin secretion, as discussed earlier;

GLUT3 is in brain; GLUT4 is in skeletal muscle, heart, and adipose tissue and is closely regulated by insulin through the PI3K-Akt pathway. In the absence of insulin, GLUT4 is on the membrane of intracellular vesicles, unable to transport glucose. Increased insulin levels, as seen in the postprandial state, result in binding of insulin to IR, which leads rapidly to fusion of those vesicles Fig. 5. Insulin-regulated metabolic activities in muscle, liver, and fat.

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degradation. Before insulin became available for treatment of type 1 diabetes, patients with this disease were invariably thin, reflecting the importance of insulin in lipid metabolism. Insulin increases fatty acid synthesis in the liver. When the liver is saturated with glycogen (roughly 5% of liver mass), glucose is diverted into synthesis of fatty acids and lipoproteins. The lipoproteins are released from the liver and undergo lipolysis in the circulation, providing free fatty acids for use in other tissues including the adipose tissue. Insulin also facili- tates glucose uptake into adipocytes and glucose can be used to synthesize glycerol in these cells. Adipocytes use fatty acids produced in the liver and from food and glycerol produced in adipocytes to synthesize triglyceride. Insulin inhibits triglyceride breakdown in adipose tissue by inhibiting an intracellular lipase that hydro- lyzes triglycerides. Thus insulin promotes lipid anabo- lism and has a fat-sparing effect so that insulin drive most cells to preferentially utilize carbohydrates instead of fatty acids for energy expenditure. Insulin also has profound effects on activity of lipoprotein lipase, fatty acid synthase, and acetylCoA carboxylase.

Insulin stimulates the intracellular uptake of amino acids through the PI3K-Akt-p70^rsk pathway and promotes protein synthesis. Insulin also increases the per- meability of many cells to potassium, magnesium, and phosphate ions.

5.4. Insulin Resistance

The most important and readily measurable physiologic effect of insulin is on serum glucose. In a given person, serum insulin levels correspond to serum glucose levels. The relationship is inverse: the higher the insulin, the lower the glucose level. The concept of insulin resistance developed after it was found that in patients with early type 2 diabetes, both insulin and glucose levels are high, suggesting that glucose is

“resistant” to insulin in these patients. Insulin resistance is defined as a clinical state of decreased insulin biologic responses to a normal or higher insulin level.

bolic syndrome” (central obesity, hypertension, insulin resistance, and dyslipidemia).

Mechanisms for insulin resistance are not clear but are likely multiple. About 50% of insulin resistance can be attributed to genetic factors and another 50% to lifestyle factors. The molecular abnormality underlying insulin resistance may involve any of the multiple steps required for insulin signaling and can be classified as prereceptor or postreceptor defects. Prereceptor insulin resistance is extremely rare in humans and includes insulin mutants defective in binding with IR and anti- bodies to circulating insulin. Most clinical insulin resistance is owing to postreceptor defects.

Type A severe insulin resistance is an extremely rare clinical syndrome owing to IR mutations. In mice models, Drosophila melanogaster, and Caenorhab- ditis elegans, IR mutations have been identified that alter insulin binding and tyrosine kinase activity. Some mutations decreased mRNA stability with reduced IR expression at the cell surface. Because PI3K is critical for insulin metabolic functions, defects in PI3K could result in insulin resistance. A single mutation (326met- ile) was found in the coding sequence of p85. Mice lacking IRS-1 or IRS-2 are insulin resistant and defective in PI3K stimulation in muscle and adipocytes.

These observations suggest that mutations in the insulin-signaling machinery are rare, which may not be surprising considering the critical importance of insulin signaling for survival.

IR serine/threonine phosphorylation contributes to insulin resistance. In muscle preparations derived from patients with type 2 diabetes, IR phosphorylation on serine or threonine residues is accompanied by decreased IR tyrosine phosphorylation. Serine phosphorylation of IRS molecules is elevated in the tissues of insulin-resistant or diabetic subjects with concomi- tant decrease in IRS tyrosine phosphorylation. Increased serine phosphorylation in insulin resistance of obesity may be due to elevated serum lipids and/or increased tumor necrosis factor-α (TNF-α). Furthermore, down-

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stream enzymes such as PKC isoenzymes are serine/

threonine kinases and phosphorylate both IR and IRS.

IR serine phosphorylation is also elevated in tissues derived from patients with polycystic ovary syndrome, who often have insulin resistance.

Because the skeletal muscles take up the majority of glucose, and muscle glycogen synthesis is about 50%

lower in type 2 diabetic patients than in healthy subjects, impaired insulin-stimulated muscle glycogen synthesis is important in insulin resistance. Glucose transport (by GLUT4), phosphorylation (by hexoki- nase), and storage (by glycogen synthase) are three rate-limiting steps for muscle glycogen synthesis, and their defects have been suggested to be responsible for insulin resistance. In muscle of patients with type 2 diabetes, intracellular glucose and glucose-6-phosphate levels are both decreased, suggesting that glucose transport by GLUT4 is decreased, which causes impaired muscle glycogen synthesis.

Although decreased muscle glucose transport is a common theme in insulin resistance, the underlying mechanism is not clear. An interesting hypothesis based on nuclear magnetic resonance spectroscopy studies of intracellular muscle metabolites suggests that impaired mitochondrial fatty acid oxidation may be a primary lesion for insulin resistance. Compared with matched healthy control subjects, healthy, young lean but insulin-resistant offspring of patients with type 2 diabetes have an 80% increase in intracellular muscle lipid con- tent with a 30% decrease in mitochondrial phosphorylation. The increase in intracellular fatty acids results in increased fatty acid metabolites such as diacylglycerol, fatty acid CoA, and ceramide, which may activate PKC

isoenzymes, leading to serine/threonine phosphorylation of IR and IRS molecules. A decrease in muscle glucose transport ensues.

This hypothesis explains most features of insulin resistance and type 2 diabetes (Fig. 6). A genetic primary defect in mitochondrial fatty acid oxidation leads to an increase in intramyocellular fatty acid levels, which leads to insulin resistance and an increase in triglyceride synthesis. A sedentary lifestyle and high- calorie diet further increase triglyceride synthesis and result in obesity. Central or abdominal obesity wors- ens insulin resistance and eventually diabetes devel- ops. The mechanisms by which obesity promotes clinical manifestation of diabetes are poorly under- stood. Currently, it is thought that molecules released from adipose tissue are involved in insulin resistance, including metabolic products (e.g., free fatty acids), hormones (e.g., leptin, resistin, and adiponectin), and cytokines (e.g. TNF-α, interleukin-6, and acylation- stimulating protein). In animal models, some adipose hormones (e.g., resistin) are necessary and sufficient to cause insulin resistance. In humans, however, none has been shown to be consistently important in linking obesity to insulin resistance. The difference between mice and humans may be that adipose hormones may cause insulin resistance only in a selected genetic back- ground. A class of drugs referred to as thiazolidine- diones, including troglitazone, rosiglitazone, and pioglitazone, are insulin sensitizers and enhance insulin-stimulated glucose disposal in muscle by interacting with peroxisome proliferator–activated receptor γ, a nuclear receptor regulating adipogenesis.

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Fig. 6. A hypothesis on mechanism for insulin resistance.