The Mechanisms of Insulin Action

c h a p t e r

33

The Mechanisms of Insulin Action Morris F. White and Kyle D. Copps

CHAPTER OUTLINE INSULIN, IGF, AND THEIR RECEPTORS,  557 STRUCTURE AND REGULATION OF THE IR KINASE, 561 THE INSULIN-SIGNALING CASCADE,  561 IRS-Protein Model of Insulin Action,  562 The PI3K→AKT Cascade,  564 mTOR Cascade,  565 AKT→mTORC1 Cascade,  565 mTORC2⇆AKT Cascade,  565 Downstream of mTOR,  565 AKT→FOXO Cascade,  566 GENETIC VALIDATION OF PROXIMAL INSULIN SIGNALING,  567 Systemic Deletion of the Insulin or IGF1 Receptor Gene, 567 Tissue-Specific Inactivation of the Insulin-Receptor Gene,  567 Inactivation of IRS Genes,  568 Inactivation of the PI3K→PDK1→AKT Cascade, 570

HETEROLOGOUS REGULATION/DYSREGULATION OF THE PROXIMAL INSULIN-SIGNALING CASCADE,  572 REGULATION OF PROTEIN METABOLISM BY INSULIN, 577 INSULIN-REGULATED GLUCOSE TRANSPORT,  578 Insulin-Regulated GLUT4,  578 GLUT4 is Essential for Glucose Homeostasis,  580 MECHANISMS OF INSULIN RESISTANCE,  580 Nutrient Excess and Insulin Resistance,  580 Inflammation and Insulin Resistance,  581 Proinflammatory Cytokines,  581 INSULIN/IGF SIGNALING AND NEURODEGENERATIVE DISEASE,  582 Insulin/IGF1→IRS2 Integrates Longevity and Metabolism, 583 The Protective Effects of Reduced Insulin/IGF1→IRS2 Signaling in Mouse Models of AD,  584 Reduced Insulin/IGF1→IRS2 Signaling in Huntington’s Disease,  584 SUMMARY AND PERSPECTIVES,  585

KEY POINTS • S tructure and function of the insulin receptor tyrosine kinase. • The specificity of insulin- and IGF-receptor hybrids. • Regulation of the insulin-receptor kinase and phosphorylation of the insulin-receptor substrates. • Downstream components of the insulin-signaling cascade. • The relation of the insulin-/IGF-signaling cascade to mouse life span. • The integrative role of IRS2 signaling in pancreatic β-cell function. • Regulation of insulin signaling by feedback and heterologous Ser/Thr phosphorylation of the IRS-proteins. • Mechanisms of cytokine-induced insulin resistance. • Mechanism of insulin-regulated glucose transport. • The relationship between insulin/IGF signaling and neurodegenerative disease.   

Insulin and insulin-like growth factor (IGF) signaling integrates the storage and release of nutrients with animal growth during development and throughout adult life. This signaling is essential for all metazoans, showing a common mechanism used by animals to integrate 556

metabolism, growth, and life span with environmental signals.1,2 Lower animals have a wide array of insulinlike peptides—7 in fruit flies and 38 in C. elegans—that bind to a single insulin-like receptor tyrosine kinase to control metabolism, growth, reproduction, and

33  THE MECHANISMS OF INSULIN ACTION

longevity.3 In honey bees, insulin-like signaling coordinates the division of labor.4 The human genome encodes a superfamily of structurally related insulin-like peptides—including insulin, insulin-like growth factor-1 (IGF1), and insulin-like growth factor-2 (IGF2), which activate five similar receptor tyrosine kinases assembled from two genes. The “insulin” family also includes seven structurally similar relaxin-like peptides that are functionally distinct and activate a family of G-protein coupled receptors.5-7 This chapter focuses on mammalian insulin-signaling mechanisms. Circulating glucose enters pancreatic islet β-cells where it promotes insulin gene expression and insulin secretion.8 By contrast, endocrine IGF1 is secreted largely from hepatocytes stimulated by nutrients and growth hormone; IGF1 and IGF2 are also produced locally in many tissues and cells, including the central nervous system and many tumors.9,10 Dysregulated IGF1 and IGF2 signaling appears to contribute to many but not all cancers, whereas dysregulated insulin signaling is associated mainly with the “metabolic syndrome” and diabetes. However, IGF1 and IGF2 can coordinate with insulin to regulate nutrient homeostasis, insulin sensitivity, pancreatic β-cell function, and cell growth and survival.9,11 Moreover, type 2 diabetes associated with variable insulin sensitivity, nutrient excess, chronic inflammation, and compensatory hyperinsulinemia can promote metabolic stress and cellular damage that might promote cancer progression during chronic cycles of damage and repair.10 Diabetes is a complex disorder that arises from various causes, including impaired glucose sensing or insulin secretion (MODY), autoimmune-mediated β-cell destruction (type 1 diabetes), or insufficient β-cell insulin secretory capacity to compensate for peripheral insulin resistance (type 2 diabetes).12 MODY is caused by mutations in genes associated closely with β-cell function, including HNF4α (hepatocyte nuclear factor-4α, MODY1), GCK (glucokinase, MODY2), HNF1α (hepatocyte nuclear factor-1α, MODY3), PDX1 (pancreatic and duodenal homeobox 1, MODY4), HNF1β (hepatocyte nuclear factor-1β, MODY 5) or NEUROD1 (neurogenic differentiation-1, MODY6), KLF11 (kruppel-like factor 11; MODY 7), CEL (carboxyl-ester lipase, MODY 8), PAX4 (paired box gene 4, MODY 9), INS (insulin, MODY 10), and BLK (tyrosine kinase, B-lymphocyte specific, MODY 11).13-15 By comparison, the autoimmunity of type 1 diabetes is genetically complex, and it is marked by circulating autoantibodies against a variety of islet antigens. Insulin is thought to be one of the principle autoantigens in the pathogenesis of type 1 diabetes, but other antigens deserve attention.16,17 Because new β-cell formation has been found to occur slowly while type 1 diabetes progresses, it might be possible to treat the disease by finding ways to accelerate β-cell regeneration while attenuating the autoimmune response.18 Type 2 diabetes is the most prevalent form of diabetes. Although it typically manifests at middle age, type 2 diabetes in the developed world is becoming more common in children and adolescents.19 Physiologic stress—the response to trauma, inflammation, or excess nutrients— promotes type 2 diabetes by activating pathways that

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impair the postreceptor response to insulin in various tissues.7,20,21 Studies suggest a possible role for gut phyla—the Bacteroidetes and the Firmicutes—in these inflammatory processes.22,23 Moreover, variation in the human genome might promote metabolic disorders that progress to type 2 diabetes during physiologic stress induced by environmental and nutritional factors. In a few informative cases, loss of function mutations in the insulin receptor or its downstream signaling components (AKT2) can explain severe forms of insulin resistance and hyperglycemia.24 By comparison, loss of function mutations in ZNT8 (SLC30A8)—a zinc transporter related to insulin storage and secretion—appears to reduce greatly the risk of diabetes in people25; however, the role of ZNT8 is complex as certain alleles are associated with a risk for developing diabetes, and inactivating mutants in mice can promote glucose intolerance owing to altered insulin crystallization and secretion, or to accelerated hepatic insulin degradation.26,27 Since the mid-2000s, genome-wide association studies (GWAS) revealed more than 60 genetic loci displaying modest or weak—but significant—effects on the risk for type 2 diabetes.25 Many, but not all, of these loci are near genes that are associated functionally with insulin resistance, insulin secretion, or diabetes (Table 33-1). Although the effect of each gene is small, these discoveries provide important clues toward the pathogenesis of type 2 diabetes. Whether the current set of genes implicated in type 2 diabetes will converge on a short list of pathways and signaling networks remains to be established.25 Regardless of the underlying etiology, dysregulated insulin signaling, exacerbated by chronic hyperglycemia, promotes a cohort of acute and chronic sequela.28,29 Untreated diabetes progresses to ketoacidosis (most frequent in type 1 diabetes) or hyperglycemic osmotic stress (most frequent in type 2 diabetes), which are immediate causes of morbidity and mortality.30 In the long term, diabetes is associated with chronic life-threatening complications, including hepatic steatosis, cardiovascular disease, and systemic oxidative stress that damages vascular endothelial cells and mesangial cells of the renal glomerulus.31,32 Diabetes is also associated with age-related degeneration in the central nervous system.33 Humans beyond 85 to 90 years of age display less insulin resistance than expected—and centenarians are surprisingly insulin sensitive.34 Most centenarians escape age-related diseases associated with insulin resistance, including diabetes, and cardiovascular and cerebrovascular events.35,36 The best means to coordinate nutrient homeostasis and insulin signaling with strategies that consistently promote health and longevity across all metazoans, especially people, remains to be established.37

INSULIN, IGF, AND THEIR RECEPTORS Insulin is synthesized in pancreatic β-cells as a single polypeptide (proinsulin) that is processed by PCSK1 (prohormone convertase-1/3) into the bioactive disulfide linked A- and B-chains, and the excised “C-peptide.” Human IGF1 and IGF2 display high-sequence similarity with both the A and B chains of insulin, but they retain the homologous connecting peptide rather than excising it as

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TABLE 33-1  Gene Loci Related to the Susceptibility of Type 2 Diabetes

✓ ✓ ✓ ✓ ✓ ✓

✓ ✓

✓ ✓ ✓

Entrez Gene Name

ABCB10 ABCC8 ACSL1 ADAMTS9 ADCY5 AGPAT2 AKT2 BCL2L11 CDKAL1 CEL DGKB FTO GCK GCKR GIPR GRB14 HHEX HNF1A HNF1B HNF4A IGF2BP2 INS IR IRS1 JAZF1 KCNJ11 KCNQ1 KLF11 LEPR LMNA MC4R MTNR1B NEUROD1 NOTCH2 PARD3B PDX1 PPARG PROX1 RBMS1 SLC30A8 TCF7L2 THADA TSPAN8 WFS1 ZFAND6

ATP-binding cassette, sub-family B (MDR/TAP), member 10 ATP-binding cassette, sub-family C (CFTR/MRP), member 8 Acyl-CoA synthetase long-chain family member 1 ADAM metallopeptidase with thrombospondin type 1 motif, 9 Adenylate cyclase 5 1-acylglycerol-3-phosphate O-acyltransferase 2 v-akt murine thymoma viral oncogene homolog 2 BCL2-like 11 (apoptosis facilitator) CDK5 regulatory subunit associated protein 1-like 1 Carboxyl ester lipase Ciacylglycerol kinase, beta 90kDa Fat mass and obesity associated Glucokinase (hexokinase 4) Glucokinase (hexokinase 4) regulator Gastric inhibitory polypeptide receptor Growth factor receptor-bound protein 14 Hematopoietically expressed homeobox HNF1 homeobox A HNF1 homeobox B Hepatocyte nuclear factor 4, alpha Insulin-like growth factor 2 mRNA-binding protein 2 Insulin Insulin receptor Insulin receptor substrate 1 JAZF zinc finger 1 Potassium inwardly rectifying channel, subfamily J, member 11 Potassium voltage-gated channel, KQT-like subfamily, member 1 Kruppel-like factor 11 Leptin receptor Lamin A/C Melanocortin 4 receptor Melatonin receptor 1B Neuronal differentiation 1 Notch 2 Par-3 family cell polarity regulator beta Pancreatic and duodenal homeobox 1 Peroxisome proliferator-activated receptor gamma Prospero homeobox 1 RNA-binding motif, single-stranded interacting protein 1 Solute carrier family 30 (zinc transporter), member 8 Transcription factor 7-like 2 (T-cell specific, HMG-box) Thyroid adenoma associated Tetraspanin 8 Wolfram syndrome 1 (wolframin) Zinc finger, AN1-type domain 6

Insulin Type 2 Resistance Diabetes X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

MODY

X

Neonatal Hyperglycemia Diabetes X

Glucose Beta Cell Intolerance Hyperplasia Hyperinsulinism

X

X

X X X X X X X X X X X X X X X X X X X X X X

X X X X X X X X X X X X X X

X X

X

X

X X

X

X

X

X

X

X

X X X

X

X

X

X

X

X X

X X X X

X

X X

X

X

X X X

X

X

X

X

X X X

X

X

X

X

X

X

X

X X

X X

The predominant phenotypes associated with each loci “X” are determined by Ingenuity Pathway Assist. These established loci were determined by a meta-analysis of genetic variants on the Metabochip of 34,840 cases and 114,981 controls of European descent.25 The ✓ indicates genes that are previously known to be involved in insulin-signaling cascade and are discussed throughout the text.

X

PART 5  DIABETES MELLITUS

✓

Symbol

33  THE MECHANISMS OF INSULIN ACTION

559

GA1 AB30

L2

L2

Fn31 CR S2

Novel site (orange)

L1

+12 L1

CT

Classical site S1 (red)

Fn32

Fn32 FB1

NA21

S1

+12

Fn33

S S

S-S SS S-S

Fn32

CT

S2

CR

Ins

Fn32 S S

Fn33

A

β-subunit

α-subunit Furin site L1

CR

L2

Fn31

Fn32

Fn32 Fn33 CTIRB

Tyr972

Lys1030

Lys1030

pY1158 pY1162 pY1163

pY1158 pY1162 pY1163

pY1316

pY1316

pY1322

pY

TM ATP binding, lys1030 Tyrosine kinase

NPEpY CTIRA

Tyr972

pY pYpY pY

Substrate Activation binding loop

pY CT

1322 B C Figure 33-1  Insulin and insulin receptor. A, Insulin structure showing the position of some critical amino acids that compose the two binding sur-

faces (S1 and S2) that interact with the L1•CR•CT and Fn31•Fn32 regions of the insulin receptor, respectively. The A-chain is shown in green and the B-chain is shown in blue, and some amino acids composing each binding site are shown as space filling residues in red (S1) or orange (S2). The amino and carboxyl terminal residues of each chain are labeled in black. B, Linear diagram of the insulin-receptor (IR) precursor protein showing the position of important modules in the α- and β-subunits—including leucine-rich regions (L1 and L2), a cysteine-rich region (CR), disulfide bonds ( ), the alternative IRA/IRB splice site that generates CTIRA or CTIRB, a transmembrane region (TM), the furin cleavage site, the IRS-binding motif (NPXpY), the activation loop autophosphorylation sites, and carboxyl terminal tyrosine phosphorylation sites (CT). C, Mature insulin receptor composed of two extracellular α-subunits and two intracellular β-subunits. Contiguous modules of the two α subunits are indicated by black or white labels and dashed tracings. The holoreceptor is stabilized extracellularly by disulfide bonds between cysteine residues (S-S) in the α and β subunits, as well as by noncovalent interactions. Two regions within the α-subunit contribute to insulin binding—including L1•CR (and the extra 12 amino acids encoded by exon-11 in the B form of the insulin receptor) that binds S1 of insulin, and the junction between Fn31 and Fn32 that binds S2 of insulin. The β subunit contains the tyrosine kinase catalytic domain with an ATP-binding site (Lys1030) and a number of tyrosine phosphorylation sites, including those in the juxtamembrane region (pY972), activation loop (pY1158, 1162, 1163), and carboxyl terminal regions.

a “C-peptide.” IGFs also have an extension at the C terminus known as the D domain.38 Insulin possesses two asymmetric receptor-binding surfaces designated “S1” (the classic site) and “S2” (the novel site) (Fig. 33-1, A).39 Natural mutations of insulin together with alanine scanning mutagenesis show that “S1” is composed of residues from the A- and B-chain— including GlyA1, IleA2, ValA3, GluA5, ThrA8, TyrA19, AsnA21, ValB12, TyrB16, GlyB23, PheB24, and PheB25 (see Fig. 33-1, A).40 By comparison, “S2” is composed of SerA12, LeuA13, GluA17, His B10, GluB13, and LeuB17 (see Fig. 33-1, A).40 Together both sites generate high-affinity insulin binding that activates the receptor tyrosine kinase. A 150-kb gene on human chromosome 19p13.3-p13.2 that contains 22 exons encodes the insulin receptor (IR).

Exon-11 is alternatively spiced depending on the tissue and the developmental stage to produce two IR isoforms: IRA lacks the residues encoded by exon-11, and IRB includes the 12 amino acid residues encoded by exon-1139,41,42 (Fig. 33-2). IRB binds only insulin with high affinity, but IRA binds both insulin and IGF2 with about equal affinity, as the absence of the exon-11 encoded C-terminal extension relaxes the specificity of the insulin binding site.43 The homologous IGF1R is assembled without alternative splicing from its 19 exon gene located on human chromosome 15. The IGF1R binds IGF1 and IGF2 with high affinity, but binds insulin weakly (see Fig. 33-2). A third gene encodes the insulin receptor-related receptor (IRR).44 A high-affinity ligand for the IRR is unknown. Regardless, together with the IR and IGF1R, the IRR promotes

560

PART 5  DIABETES MELLITUS

IGF2

CT

IRA

CT

IGF1r

M 5n 0. 70 nM

0.3nM 0.3nM

M 7n 0.

0.5nM

0. 25 nM

IRA

M

2n

nM 70

IRB

M

9n

IRB IGF1r

nM 0.5 nM

90nM

IGF1 0 .5

10nM

INS

IGF1r

CT

Figure 33-2  The

insulin and insulin-like growth factor family. The ­insulin/IGF family consists of three peptide hormones: insulin, insulin-like growth factor-1 (IGF1), and insulin-like growth factor-2 (IGF2). These bind as indicated to five distinct homo- or heterodimeric receptor isoforms that generate cytoplasmic signals: two insulin-receptor isoforms, IRA and IRB; the insulin-like growth factor receptor, IGF1R; and two hybrid receptors, IRA•IGF1R and IRB•IGF1R. The insulin receptor is the primary target for insulin throughout development and life. The IGF1 receptor is the primary target for IGF1 and IGF2, whereas IGF1 and IGF2 also binds to IRA because of the absence of the alternatively spliced CT. The activated IR or IGF1R tyrosine kinases phosphorylate the cytoplasmic insulin-receptor substrate proteins IRS1 and IRS2, which mediate somatic growth and metabolism. The approximate binding affinities (Kd, nM) are shown.

male sexual development in mice.45 Moreover, the IRR might sense alkali conditions in the kidney to modulate systemic bicarbonate concentrations.46,47 The insulin and IGF1 receptor precursors have similar structures, which are processed to form holoreceptors composed of two α-subunits and two β-subunits. The process is best characterized for the IR, which is synthesized as a single protein with a classic amino-terminal signal sequence followed by well-defined extracellular modules—including two leucine-rich motifs (L1 and L2) flanking a cysteine-rich (CR) region, which is followed by three fibronectin-III motifs (Fn31, Fn32, and Fn33) ending with the intracellular tyrosine kinase (see Fig. 33-1, B and C). Fn32 is interrupted by a 120-amino acid insert containing a furin cleavage site that generates on cleavage the α- and β-subunits of the holoreceptor (see Fig. 33-1, B). During translation, the proreceptors for insulin and IGF1 can assemble as homodimers, which are linked by disulfide bonds to produce the IR (αβIR•αβIR) or the IGF1R (αβIGF1R•αβIGF1R). However, when expressed together, the αβ dimer of each receptor can associate to form hybrid receptors (αβIR• αβIGF1R).48 Because the insulin receptor occurs in two isoforms, a total of 5 receptors types can be produced from the two receptor genes (see Fig. 33-2).49 The α-subunits are entirely extracellular and create the ligand-binding sites. Each β-subunit contains a transmembrane-spanning segment that separates the extracellular Fn32-Fn33 regions from the intracellular tyrosine kinase (see Fig. 33-1, C).50,51 The holoreceptor (αβ•αβ) has an approximate molecular mass of 350,000 by SDSPAGE—larger than expected owing to glycosylation of

the α- and β-subunits.52,53 On reduction of the disulfide bonds, SDS-PAGE resolves the IR and IGF1R into the αand β-subunits that migrate near 135-kDa and 95-kDa, respectively.54,55 Hybrid receptors can be detected by specific immunoblotting strategies.48 Insulin binds with high affinity (Kd < 0.5 nM) to the homodimeric IRB, which predominates in the classic insulin target tissues—adult liver, muscle, and adipose tissues (see Fig. 33-2). IRB is selective for insulin as its Kd for IGF1 and IGF2 is at least 50-fold to 100-fold higher. Adult liver and adipose are purely insulin-responsive tissues, as they express IRB without detectable IGF1R.9 By comparison, IRA predominates in fetal tissues, the adult central nervous system, and hematopoietic cells.56-59 Most cancers express IRA, IRB, and IGF1R and usually display the hybrid species (see Fig. 33-2).10 IRA binds insulin almost as well and IRB, but IRA also binds IGF2 with moderate affinity (see Fig. 33-2). IGF1 and IGF2 bind with high affinity (Kd < 1 nM) to the homodimeric IGF1R and to the hybrid receptors (αβIRA•αβIGF1R and αβIRB•αβIGF1R), whereas insulin binds poorly to the hybrids (see Fig. 33-2).48,60 Thus, under ordinary conditions insulin never activates the IGF1R tyrosine kinase, whereas IGF1 and IGF2 can activate the IR tyrosine kinase when it forms a hybrid with the IGF1R.61 IRA enhances the effects of IGF2 during embryogenesis and fetal development and in the adult brain.43 Site-directed mutagenesis shows the location of two insulin-binding sites in the α-subunit of the insulin receptor.62,63 Chimeric receptors between the α-subunits of the insulin and IGF1 receptors confirm that one of the sites is located within the first leucine-rich (L1) region of the insulin receptor.64

33  THE MECHANISMS OF INSULIN ACTION

Photoaffinity labeling of mutant insulin receptors shows the second insulin-binding site near the Fn31→Fn32 interface.65 Insulin binding apparently begins through interactions between “S2” on insulin with the Fn31•Fn32 interface in the α-subunit.40 Sixteen amino acid residues at the COOHterminus (CT) of Fn32 interact with the L1•CR-region to create a composite insulin-binding site—L1→CR→CT—that interacts with “S1” on insulin (see Fig. 33-1, C). Although the α-subunits are arranged symmetrically in the dimer, there is a sharp bend between the L2 and Fn31→Fn32 regions that juxtaposes the L1→CR→CT domain antiparallel to Fn31→Fn32 (see Fig. 33-1, C).66-70 Insulin binds to the L1→CR→CT domain of one α-subunit and to the Fn31→Fn32 region of the adjacent α-subunit to create the cross-link that activates the kinase.69 Owing to space constraints, only one insulin molecule can bind with high affinity.66,68,71 Inclusion of exon-11 in IRB lengthens the CT-region by 12 amino acids, which modifies the L1•CR•CT domain to exclude IGF2 binding, and reduces strongly the IGF1 affinity while retaining high insulin-binding affinity (see Fig. 33-2). These details have been investigated.70,72

STRUCTURE AND REGULATION OF THE IR KINASE The tyrosine kinase activity of the insulin receptor was originally discovered by biochemical experiments using [32P]-labeled hepatoma cells or partially purified insulin receptors incubated with insulin and [γ32P]ATP.73-75 However, until the insulin-receptor cDNA was isolated and sequenced,50,51 other mechanisms for signal transduction were proposed—and some still persist.76-79 However, the discovery that rare cases of severe insulin resistance in humans are associated with insulin-receptor mutations that inactivate the tyrosine kinase—without altering insulin binding—supports definitively the central hypothesis of tyrosyl phosphorylation in the mechanism of insulin action.80 The intracellular portion of the insulin-receptor β-subunit is composed of three distinct regions that contain tyrosyl phosphorylation sites (numbered as in IRB): Y965 and Y972 in the juxtamembrane region between the transmembrane helix and the cytoplasmic tyrosine kinase domain; Y1158, Y1162, and Y1163 in the activation loop (A-loop) of the catalytic core; and Y1328 and Y1334 in the COOH-terminus41,81,82 (see Fig. 33-1, B and C). Most receptor tyrosine kinases are activated by ligandinduced dimerization, which brings two intracellular catalytic domains together to mediate tyrosine phosphorylation of the A-loop and the other sites that recruit cellular substrates.83 Because the homologous IR and IGF1R reside in the plasma membrane as inactive covalent dimers, high-affinity insulin or IGF binding adds a transient cross-link to induce and stabilize structural transitions within the receptor that activate the intracellular catalytic site.69 Before insulin stimulation, the unphosphorylated Tyr1162—the second of the three A-loop tyrosine residues—is positioned near the catalytic site, whereas the amino-terminal end of the A-loop (D1150FG-motif) folds into the ATP-binding site elevating the apparent Km for ATP.69,84,85 Apparently, the closed A-loop is in

561

equilibrium with an alternate conformation that allows occasional access by ATP to mediate basal autophosphorylation.86 Infrequent oscillation from the “closed” to an “open” conformation in the basal state might be coupled to complementary changes in the α-subunits, which can be stabilized definitively on insulin binding to accelerate ATP entry and to stimulate autophosphorylation of Tyr1162 (Fig. 33-3, A and B). After initiation, the autophosphorylation cascade progresses to Tyr1158, resulting in a bis-phosphorylated and active kinase.81 Although autophosphorylation of Tyr1163 is relatively slow, it appears to stabilize the open conformation to allow unrestricted access by Mg-ATP and protein substrates (see Fig. 33-3, B and C).69 The final autophosphorylation probably occurs through the interaction between the covalently linked β-subunits.69,75 The substitution of Asp1161 in the middle of the A-loop with alanine shifts the steady-state conformation of the unphosphorylated A-loop toward the open configuration.86 Substitution of Tyr1162 with phenylalanine also increases basal autophosphorylation, consistent with its role in stabilizing the closed conformation or in competing with ATP and protein substrates for binding at the kinase-active site.87,88 Whether other kinases can activate the insulin receptor by phosphorylation of A-loop tyrosine residues independently of insulin is an open question that deserves attention.

THE INSULIN-SIGNALING CASCADE Following the discovery of the insulin-receptor tyrosine kinase, many groups searched for cellular proteins that might mediate downstream signals.89,90 The “substrate” hypothesis was an attractive mechanism from the beginning, but it was difficult to establish because only proteins with unlikely signaling potentials were found to be phosphorylated by the insulin-receptor kinase. The first evidence for a physiologic substrate of any receptor tyrosine kinase came from anti-phosphotyrosine antibody immunoprecipitates that showed a 185-kDa phosphoprotein (pp185) in insulin-stimulated hepatoma cells.91 This protein displayed many features expected for a biologically important insulinreceptor substrate—including immediate phosphorylation on insulin stimulation and no phosphorylation by catalytically inactive or biologically inactive insulin receptors.92 Together these data provided the first clue that receptor autophosphorylation, followed closely by substrate phosphorylation, could be the initial step in signal transduction. Purification and molecular cloning of pp185 showed the first of a large family of signaling scaffolds and the first insulin-receptor substrate called IRS1.93 Four IRSprotein genes exist in rodents, but only three (IRS1, IRS2, and IRS4) are expressed in humans.94 IRS1 and IRS2 are broadly expressed in mammalian tissues, whereas IRS4 is largely restricted to the hypothalamus.95,96 The IRS-proteins are arguably the most important adapter molecules linking the IR and IGF1R to downstream signaling cascades and the heterologous regulatory components used by many signaling systems (see Fig. 33-3, D).

562

PART 5  DIABETES MELLITUS Inactive

Active

Active + YMPMIRS1 peptide NH2

NH2

Y1158

NH2

ANP-PNP

ANP-PNP

pY1158

Y1162

YMPM

pY1158 OPEN A-loop

CLOSED A-loop

A

pY1162

pY1162

Y1163

B

C

*

Y621NPY624PEDY628GDI

*

*

YGSS

IRS2

PH

PTB

IRS1

PH

PTB

*

*

Y538MSM

Y649MPM Y734MRM YTLM

*

Y460ICM

YTEM

*

Y814VLM

Y911INI

Y970MNL

Y1303ASI

YSEM

*

Y671MPM

YMPM

YGSS

Y1242IAI

Y758NLK

Y594MSM

*

Y690GKP

YMPM

Y727MNM

Y935MKM

Y891VNI

*

Y1220ASI

YVDT

Y983MTM

*

Y1173IDL

YPEE YMMM YADM D Figure 33-3  The role of insulin-receptor autophosphorylation. A, Structure of the insulin-receptor activation loop is shown as a series of ribbon

diagrams of the insulin-receptor kinase domain. The activation loop (A-loop) is shown in red as space-filling tyrosine residues (Y1158, Y1162, and Y1163). In the inactive, unphosphorylated state (A), the activation loop blocks access by potential substrates. B, Following autophosphorylation, the activation loop moves out of the catalytic site to allow entry of ATP (yellow space filling) to enter the active site. C, Peptide substrates such as YMXM-peptides of the IRS-proteins enter the catalytic sites when the regulatory loop is removed. D, Comparison of IRS1 and IRS2 phosphorylation sites relative to the amino-terminal pleckstrin homology (PH) and phosphotyrosine-binding (PTB) domains. The amino acid sequences surrounding tyrosine sites are shown, and motifs conserved between Irs1 and Irs2 are color coded. Phosphorylation sites shown by MS/MS are indicated (*). The kinase regulatory loop-binding (KRLB) domain in IRS2 is centered about Y624, and is not conserved in IRS1.

Moreover, work with transgenic mice shows that all insulin responses—especially those that are associated with somatic growth; carbohydrate, protein, and lipid metabolism; hepatic, adipose, skeletal muscle, and cardiovascular physiology; pancreatic β-cell function; and central nutrient homeostasis—are mediated through IRS1, IRS2, or both.97

IRS-Protein Model of Insulin Action IRS-proteins are composed of tandem, structurally similar pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains followed by a long unstructured tail of tyrosine phosphorylation sites that coordinate the insulin/ IGF signal. During insulin and IGF1 stimulation, some tyrosine residues in the tail are phosphorylated and bind to the SH2-domains of various signaling proteins, especially the 85 kDa regulatory subunit (p85) of the PI3K

(class 1A phosphatidylinositol 3-kinase) (Fig. 33-4).98 The interaction between IRS1 and PI3K was the first insulinsignaling cascade to be reconstituted successfully in vivo and in vitro.99 Specific insulin-stimulated tyrosine phosphorylation of the IRS-protein is accomplished through at least two mechanisms—including specific recruitment of IRS to the juxtamembrane region of the IR followed by recognition by the activated catalytic domain of preferred phosphorylation motifs in the unstructured tail. Based on phosphopeptide mapping and phosphorylation of synthetic peptides, several motifs have been identified as optimal insulin-receptor mediated phosphorylation sites in IRS1—including several YMXM-motifs, and the YVNI-, YIDL-, and a YASI-motif.100-103 These motifs are targeted by the IR catalytic domain as antiparallel β-strands relative to the COOH-terminal end

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33  THE MECHANISMS OF INSULIN ACTION

pY

SHC

pY

pY pY

pT

GRB10

RHEBGDP

Lysosome

S6 NFκB

MAPKAPK1

PP-1 pS

MYT1

eIF4A

AKT1,2,3

eIF4G

pS

S6K

SREBP1

FASN GPAT ACLY ACC SCD1 GK

S6K

pS eIF4E eIF4A

Translation Initiation

IMP1

RNA translation

FOXO1/3

TSC1/2

mTORC1

pS

Mnk

pS

pS TSC1/2

eIF4E

eIF4G

CUL7 UbUbUb

RHEBGTP

pS

4E-BP1

FBW8

pS

4E-BP1

Mnk

Transcription

pS

pY

pS

pS

pY

pY

pSY

fos

mTORC2

Liver

P

ERK

elk1

PDK1

pY

APPL1

MEK

pS

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Figure 33-4  A canonical insulin/IGF-signaling cascade. Activation of the receptors for insulin and IGF1 results in tyrosine phosphorylation of

the IRS-proteins, which bind PI3K and Grb2/SOS; tyrosine phosphorylation of SHC also binds GRB2•SOS. The Grb2•SOS complex promotes GDP/GTP exchange on p21ras, which activates the RAS→ RAF→ MEK→ERK1/2 cascade. Activated ERK stimulates transcriptional activity by direct phosphorylation of ELK1 (ETS domain-containing protein) and by indirect phosphorylation of cFOS through MAPKAPK1 (MAPKactivated protein kinase-1). MAPKAPK1 also phosphorylates other proteins including S6 (ribosomal protein S6), NFκB, PP1, and MYT1. The activation of PI3K by recruitment to IRS2 produces PI3,4P2 and PI3,4,5P3 (antagonized by the action of PTEN or SHIP2), which recruits PDK1 and AKT to the plasma membrane. AKT is activated on phosphorylation at T308 by PDK1 and at S473 by mTORC2. mTORC1 is activated by RhebGTP, which accumulates on inhibition of the GAP activity of the TSC1•TSC2 complex following AKT-mediated phosphorylation of TSC2. The S6K is primed through mTORC1-mediated phosphorylation. AKT phosphorylates many cellular proteins—inactivating PGC1α, p21kip, GSK3β, BAD, and AS160, and activating PDE3b and eNOS. AKT-mediated phosphorylation of forkhead proteins, including FOXO1, results in their sequestration in the cytoplasm, which inhibits their influence on transcriptional activity. Insulin stimulates protein synthesis by altering the intrinsic activity or binding properties of key translation initiation and elongation factors (eIFs and eEFs, respectively) as well as critical ribosomal proteins. Components of the translational machinery that are targets of insulin regulation include eIF2B, eIF4E, eEF1, eEF2, and the S6 ribosomal protein.337

of the open A-loop. This orientation positions the hydrophobic side-chain in the Y+1 and Y+3 positions into two hydrophobic pockets on the activated kinase (see Fig. 33-3, C). Tyrosine residues lying NH2-terminal to polar side chains at the Y+1 and Y+3 positions fit poorly in this site, which excludes them from phosphorylation.103 The selective and regulated recruitment of IRS to the activated IR and IGF1R adds an essential and important level of signaling specificity. The IRS-proteins bind through their PTB domain to the juxtamembrane autophosphorylation site at pY972, which resides in a canonical PTB-domain binding motif (NPEpY972).92,104 The juxtamembrane region is about 35 residues long and connects the transmembrane helix of the IRβ subunit to the kinase domain (see Fig. 33-1, B and C). Unlike other

receptor tyrosine kinases, the insulin-receptor kinase is not regulated by autophosphorylation in the juxtamembrane region—although the NPEY-motif modulates receptor trafficking.105,106 However, phosphorylation of Tyr972 creates a docking site for the PTB domain in the IRS-proteins and another substrate called src homology 2 domain-containing transforming protein (SHC) (see Fig. 33-4).92,107 The NPEpY972-motif fills an L-shaped cleft on the PTB-domain, whereas the N-terminal residues of the bound peptide form an additional strand in the β sandwich.104 The NPEpY972-motif is a low-affinity binding site for the PTB domain of IRS1 (Kd ∼ 87 μM), owing to a destabilizing effect of E971 that facilitates autophosphorylation of Y972 by the insulin receptor.69,108 By comparison, the PTB domain of SHC binds to NPEpY972 with a much higher affinity (Kd ∼ 4 μM).

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Unlike SHC, IRS contains a pleckstrin homology (PH) domain immediately upstream of the PTB domain, which helps recruit the IRSs to IR (see Fig. 33-3, D).109 The PH domain is structurally similar but functionally distinct from the PTB domain.110 Although the PHdomain promotes the interaction between IRS and the IR, its mechanism of action remains poorly understood, as it does not bind phosphotyrosine. PH domains are generally thought to bind phospholipids, but the PH domains in IRSs are poor examples of this binding specificity.111,112 The IRS1/IRS2 PH domain binds to negative patches in various proteins, which might also be important for IR recruitment.113 Regardless, the PH domain in the IRS-protein plays an important and specific role as it can be interchanged among the IRS-proteins without noticeable loss of bioactivity. By contrast, heterologous PH-domains reduce IRS1 function when substituted for the IRS1 PH domain, confirming a specific functional role.114 IRS2 uses an additional mechanism to interact with the insulin receptor, which is absent in IRS1. Amino acid residues 591 and 786—especially Tyr624 and Tyr628—in IRS2 mediate a strong interaction with the activated IR catalytic site.115,116 This binding region in IRS2 was originally called the kinase regulatory-loop binding (KRLB) domain, because tris-phosphorylation of the A-loop was required to observe the interaction.115 Structure analysis shows an essential functional part of the KRLB-domain— residues 620 to 634 in murine IRS2—that fits into the “open” catalytic site of the insulin receptor.117 With the A-loop out of the catalytic site—by autophosphorylation or other means—Tyr621 of IRS2 inserts into the receptor ATP-binding pocket whereas Tyr628 aligns for phosphorylation. This interaction might attenuate signaling by blocking ATP access to the catalytic site, or it might promote signaling by opening the catalytic site before tris-autophosphorylation. Interestingly, the KRLB-motif does not bind to the IGF1R, possibly explaining signaling differences between IR and IGF1R, as well as the receptor hybrids.117

The PI3K→AKT Cascade Downstream insulin signaling is composed of a highly integrated network, which coordinates multiple tissuespecific signals that control cellular growth, survival, and metabolism, and modulate the strength and duration of the signal through diverse feedback cascades.118 The cascade begins when insulin stimulates tyrosyl phosphorylation of YXXM-motifs in the IRS-proteins, which directly recruit and activate the class 1A phosphotidylinositide 3-kinase (PI3K) (see Fig. 33-4). PI3Ks are lipid kinases central to numerous signaling pathways, which are organized into three classes—class I, class II, and class III. The growth factor-regulated class IA PI3Ks are composed of two subunits. The catalytic subunit—p110α (PIK3CA), p110β (PIK3CB), or p110δ (PIK3CD)—is inhibited and stabilized on association with one of several homologous 85 kDa regulatory subunits encoded by PIK3R1 (p85α) or PIK3R2 (p85β). Rarely, alternative splicing of PIK3R1 produces p55α or p50α, or a third gene PIK3R3 encodes p55γ, all of which lack some NH2-terminal regulatory

features of p85 while retaining affinity toward the catalytic subunits.118-120 All of the regulatory subunits contain two SH2 (src homology 2) domains that bind phosphorylated YXXM-motifs to disinhibit the catalytic domain that produces PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate).121,122 Inhibition of the PI3K by chemical or genetic means blocks almost all metabolic responses stimulated by insulin—including glucose influx, glycogen, and lipid synthesis, and adipocyte differentiation—confirming that the PI3K is a critical node coordinating insulin action.118 The PI(3,4,5)P3 produced by PI3K plays a pivotal role to recruit to the plasma membrane and activate various proteins—many of which contain PH (pleckstrin homology) domains that bind to PI(3,4,5)P3. A key cascade involves the recruitment of several Ser/Thr-kinases to the plasma membrane, including PDK1 (3’-phosphoinosotide-dependent protein kinase-1) and AKT (v-akt murine thymoma viral oncogene). AKT is activated by phosphorylation of Thr308 in its activation loop by the juxtaposed membrane-bound PDK1. AKT isoforms play a central role in cell biology, as they regulate by phosphorylation many proteins that control cell survival, growth, proliferation, angiogenesis, metabolism, and migration (see Fig. 33-4).121,123,124 More than 100 AKT substrates are known and several are especially relevant to insulin signaling—including GSK3α/β (blocks inhibition of glycogen synthesis); AS160 (promotes GLUT4 translocation); the BAD•BCL2 heterodimer (inhibits apoptosis); the FOXO transcription factors (regulates gene expression in liver, β-cells, hypothalamus, and other tissues); p21CIP1 and p27KIP1 (blocks cell-cycle inhibition); eNOS (stimulates NO synthesis and vasodilatation); PDE3b (hydrolyzes cAMP); and TSC2 (tuberous sclerosis 2 tumor suppressor) that inhibits mTORC1 (mechanistic target of rapamycin complex 1) (see Fig. 33-4). An unbiased MS/MS approach implicates many more AKT substrates in insulin action, suggesting that the majority of PI3Kmediated growth factor (insulin) signaling is coordinated through AKT-dependent mechanisms (see Fig. 33-4).124 A subset of proteins with PH domains that bind selectively to PI(3,4,5)P3 coordinate other tissue-specific signals to control growth, proliferation, survival, vesicular transport, differentiation, and migration, some of which might contribute to an insulin response. In adipocytes and muscle, the aPKC (atypical protein kinase C) is recruited by PI(3,4,5)P3, where, like AKT, it can be activated by PDK1 and can promote insulin-stimulated glucose uptake.118 The localization and/or activity of other effectors can link PI(3,4,5)P3 to cytoskeleton functions. GAB1 and GAB2 (GRB2-associated binder1/2) binds PI(3,4,5) P3, localizing them to the plasma membrane in the vicinity of cell-cell contacts125; PLEKHA1 (pleckstrin homology domain containing, family A phosphoinositide-binding specific member 1) is a PH domain-containing adapter protein specific for PI(3,4)P2, which may be important for remodeling the actin cytoskeleton126; DAPP1 (dual adapter for phosphotyrosine and phosphoinositide) exhibits high affinity for PI(3,4,5)P3 and PI(3,4)P2 to integrate PI3K and SRC kinase signaling to promote B cell adhesion.127 ARAP3 (ARFGAP with RHOGAP

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domain, ANK repeat, and PH domain-containing protein 3) is a specific PI(3,4,5)P3/PI(3,4)P2-binding Arf6-GAP that mediates rearrangements in the cell cytoskeleton and cell shape.128

mTOR Cascade mTOR (mechanistic target of rapamycin) is a Ser/Thr kinase that is regulated through multiple mechanisms. It belongs to the PI3K-related kinase family and forms two large functionally distinct protein complexes—mTORC1 and mTORC2—composed of common and unique subunits. Both complexes are controlled by growth factors and insulin through the PI3K→AKT cascade, but they are recruited to different compartments and respond distinctly to nutrients, stress, hypoxia/energy status, and other stimuli to coordinate a diverse array of biological processes—including protein and lipid synthesis, liposome biogenesis, autophagy, and cell migration, growth, and proliferation.129 In addition to the common catalytic subunit, mTORC1 and mTORC2 share mLST8 (mammalian lethal with sec-13 protein 8), DEPTOR (Disheveled, Egl10, and Pleckstrin domain containing mTOR-­interacting protein), and Tti1 (Telomere maintenance 2 interacting protein 1). However, mTORC1 is distinguished by two specific components, including RAPTOR (RPTOR, regulatory associated protein of mTOR, complex 1) and AKT1S1 (PRAS40, AKT1 substrate 1 proline-rich). mTORC2 lacks the mTORC1-specific components, but includes RICTOR (RAPTOR-independent companion of mTOR, complex 2), mSIN1 (SAPK interacting protein 1, or MEKK2 interacting protein 1), and PRR5 (protor1/2, protein observed with Rictor 1 and 2).129 mTORC1 is strongly regulated by nutrient concentration and inhibited by rapamycin, whereas mTORC2 is inhibited variably by rapamycin and is insensitive to nutrient levels.

AKT→mTORC1 Cascade mTORC1 coordinates many growth factor (insulin) responses owing to its regulation by AKT, nutrient/amino acid concentrations, and subcellular/lysosomal targeting.129 In addition to the stable complex of mTORC1 components, several additional proteins regulate mTOR activity. AKT-dependent activation begins with the phosphorylation of at least five sites (Ser939, Ser981, Ser1130, Ser1132, and Thr1462) on TSC2 (tuberin), which in complex with TSC1 (hamartin) functions as a GTPase-activating protein for the small G protein RHEB (Ras homolog enriched in brain).129 AKT-mediated phosphorylation inhibits TCS1/2, allowing RHEB to accumulate in its GTP-bound form to activate mTORC1 (see Fig. 33-4). In one possible mechanism FKBP38 (FK506-binding protein 8) inhibits mTOR until RHEB-GTP promotes its dissociation from mTORC1.130 Regulation by TSC1/2→RHEB is also augmented by AKT-mediated phosphorylation of AKT1S1 (PRAS40), which promotes its dissociation from RAPTOR to activate mTOR.123 Proinflammatory cytokines can activate mTORC1 through a similar mechanism in which IKKβ (IκB kinase) phosphorylates TSC1/2, leading to the accumulation of RHEB-GTP.131 The second important level of mTORC1 regulation by growth factors (insulin) depends on localization to the

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surface of lysosomes.129 Lysosomal targeting of mTORC1 is coordinated by amino acid-dependent GTP loading of the RAGA•RAGB (Ras-related GTP binding) complex, which interacts with both RAPTOR and a multisubunit complex called RAGULATOR that is located on the lysosome surface.132 RHEB resides on endomembranes, including lysosomes, where it can interact with mTORC1 only if sufficient amino acids are available to promote RAGA•RAGB-mediated recruitment (see Fig. 33-4).129

mTORC2⇆AKT Cascade Compared against the rich detail of mTORC1, the regulation of mTORC2 by growth factors and insulin has many tissue-specific features that complicate our understanding of its emerging mechanisms.133 Like most insulin responses, mTORC2 activation requires PI3K, but its role in insulin action has been difficult to understand owing to variable effects observed on deletion of specific components. However, a central role for mTORC2 growth factor (insulin) signaling emerged when it was found to control several members of the AGC subfamily of kinases, including AKT and SGK1.134 On activation of AKT by PDK1-mediated phosphorylation at T308AKT, some but not all AKT substrates are phosphorylated. For example, TSC2, GSK3, and SIN1 do not require phosphorylation of S473AKT.129 Judging from temporal dependence on insulin stimulation, the SIN1 component of mTORC2 might be recruited to the plasma membrane by PI(3,4,5) P3 where it is phosphorylated at T86 by pT308AKT. Phosphorylated SIN1 appears to promote S473AKT phosphorylation by mTORC2. On bis-phosphorylation, AKT can phosphorylate a wider array of substrates, including FOXO transcription factors (see Fig. 33-4).124 This model fills a conspicuous gap in our understanding of PI3K sensitive mTORC2 activation during insulin stimulation and its role in AKT regulation. However, this pathway is complicated by the finding that SIN1 is phosphorylated by S6K1 at both T86 and T398, which leads to the inhibition of mTORC2.133,135 The integration of mTORC2 activity through multisite SIN1 phosphorylation shows how mTOR signaling can be coordinated through feedforward and feedback mechanisms.133

Downstream of mTOR Although mTORC1 and mTORC2 have several common components, they display unique signaling functions owing to substrate selectivity. mTORC1 regulates various cellular anabolic and synthetic processes needed for growth and proliferation—including the stimulation of glycolytic flux and mitochondrial function, protein and lipid synthesis, and the inhibition of autophagy and lysosomal biogenesis.133 Protein synthesis is one of the best understood mTORC1-regulated processes, which is controlled, at least in part, through the phosphorylation/activation of S6K1 and S6K2 (the ribosomal protein S6 kinases) and phosphorylation/inhibition of 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) (see Fig. 33-4). The dependence on amino acids and energy for full mTORC1 activity ensures that the cellular environment is sufficient to support growth factor (insulin) stimulation. At the whole animal levels, the mTORC1→S6K cascade increases

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cell and animal size, including pancreatic beta-cell growth that is needed for insulin action.136 Although disruption of the S6k1 gene in mice enhances peripheral insulin sensitivity, the reduced size of pancreatic islet β-cells leads to glucose intolerance. It is important to resolve whether the increased systemic insulin sensitivity is a consequence of reduced insulin secretion. mTORC1 also promotes lipid synthesis required for membrane biogenesis in proliferating cells, and for energy storage and lipid secretion from the liver. mTORC1 promotes the expression of SREBP1/SREBP2 and their proteolytic cleavage to mediate translocation of the active transcription factors to the nucleus where they promote the expression of genes involved in fatty acid or cholesterol synthesis (see Fig. 33-4). mTORC1 also inhibits autophagy, which ordinarily degrades damaged proteins and organelles to provide nutrients required to maintain critical cellular functions. Thus mTORC1 controls many key cellular processes that balance cellular integrity and long-term survival. Much less is understood about downstream signaling mediated by mTORC2. As described earlier, mTORC2 phosphorylates the C-terminal hydrophobic motif of AKT and SGK1, which leads to the full activation of the PI3K→AKT cascade. mTORC2 also plays a role in mRNA processing when it phosphorylates IMP1 at Ser181, which strongly enhances insulin-like growth factor 2 mRNAbinding protein 1 (IMPI) binding to enable IGF2-leader 3’-mRNA translational initiation by internal ribosomal entry (see Fig. 33-4).137 Thus, mTORC2-catalyzed cotranslational IMP1 phosphorylation can promote organismal growth by regulating IGF2 production that can activate IRA in the mouse embryo. mTORC2 also regulates protein ubiquitinylation by phosphorylating FBW8 (F-Box and WD Repeat Domain Containing 8), a Cullin 7 E3 ubiquitin ligase-recognition subunit (see Fig. 33-4). The phosphorylation of FBW8 stabilizes and promotes ubiquitinylation of targeted substrates, including IRS1 that contributes to insulin resistance in certain tissues and cells.138 Thus, important mTORC2 signaling might be mediated through its direct effect on the activity of the PI3K→AKT cascade, and its indirect control of important regulatory points, to integrate fully the insulin/IGF-signaling cascade.137

AKT→FOXO Cascade Forkhead box O (FOXO) subfamily of transcription factors (FOXO1, FOXO3a, FOXO4, and FOXO6) regulate expression of target genes involved in DNA damage repair response, apoptosis, metabolism, cellular proliferation, stress tolerance, and longevity.139,140 FOXOs contain four highly conserved domains, including the N-terminal region containing several AKT phosphorylation sites, a highly conserved forkhead DNA-binding domain (DBD), a nuclear localization signal (NLS) located just downstream of the DBD, a nuclear export sequence (NES), and a C-terminal transactivation domain.141 The DBD domain contains three α-helices, three β-sheets, and two loops that are referred to as the wings. All the regulation mechanisms involve the retention of FOXO in the nucleus where it can promote or suppress the transcription of target genes containing a consensus DNA-binding sequence (TTGTTTAC).

FOXOs are regulated by several posttranslational modifications, including AKT-mediated phosphorylation, or by acetylation, methylation, glycosylation, and ubiquinylation.141-143 The modifications affect protein–protein and protein–DNA interactions that eventually alter the DNAbinding characteristics that regulate transcriptional activity.141,143-147 AKT-mediated phosphorylation of FOXO1, FOXO3a, and FOXO4 causes their nuclear exclusion leading to ubiquitinylation and degradation in the cytoplasm. Moreover, FOXO transcriptional activity can be inhibited by IkB (inhibitor of NF-kB) kinase (IKK) and the serum/ glucocorticoid-inducible protein kinase (SGK) that also causes phosphorylation and nuclear exclusion.141,148 By comparison, arginine methylation of FOXO blocks AKTinduced phosphorylation and inactivation,143 whereas the addition of O-linked beta-N-acetyl-glucosamine to FOXO promotes the expression of its target genes involved in stress resistance during hyperglycemia and cell stress.149 Acetylation of FOXO has variable effects, as lysyl acetylation can promote FOXO phosphorylation to attenuate its ability to bind cognate DNA sequences,150 whereas deacetylation by SirT1 can suppress transcriptional activity directly.151 The evidence from genetically modified mouse models suggests that the FOXOs have distinct functions during development, but functional redundancy in adults.152-154 Through the IR→IRS→PI3K→AKT signal cascade, FOXO integrates insulin action with the systemic nutrient, energy homeostasis, and organism growth. FOXOs can regulate genes controlling hepatic glucose production; insulin secretion in β-cells and β-cell growth and differentiation; survival and function; and fat and muscle mass (see Fig. 33-4).155 Thus, FOXO could be a therapeutic target for metabolic disorders of insulin resistance, including obesity, diabetes, and nonalcoholic fatty liver diseases. In mammalian liver, decreased circulating insulin during fasting promotes the nuclear localization of FOXO, where it interacts with PGC1α (Ppargc1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and CREB•CRTC2 (CREB, cAMP response element binding protein; CRTC2, CREB regulated transcription coactivator 2) to increase the expression of the key gluconeogenic enzymes G6PC (glucose-6-phosphatase) and PCK1 (phosphoenolpyruvate carboxykinase 1),156-161 which ensures the production of sufficient glucose to prevent life-threatening hypoglycemia.150 However, these processes can proceed without the FOXOs, which emphasizes the integration of alternative mechanisms for this important process.155 FOXO1 also coordinates decreased nutrient availability with reduced somatic growth by increasing the hepatic expression of IGFBP1 (insulin-like growth factor-binding protein 1)—a liver-secreted protein that binds to circulating IGF1 to limit its systemic bioavailability.160 Skeletal muscle growth and metabolism are regulated by insulin and IGF1, at least in part, by the switch of FOXO1 activity—nuclear during fasting and cytoplasmic during feeding—which modulates nutrient and energy homeostasis in the skeletal muscle by promoting the use of lipid or carbohydrate, respectively. Severe and persistent starvation can trigger FOXO1-mediated autophagy and atrophy that breaks down muscle protein for use by the liver

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to make glucose. Reduced insulin and IGF1 signaling in skeletal muscle can underlie the loss of muscle mass and glycemic control during prolonged insulin resistance especially during traumatic injury.162

GENETIC VALIDATION OF PROXIMAL INSULIN SIGNALING Alterations in glucose homeostasis are important hallmarks of insulin resistance, which are investigated extensively in humans and in experimental animals. However, other aspects of cellular and systems physiology are disrupted by insulin resistance, which are difficult to assess in people. The purification and genetic cloning of insulin-­ signaling components and their analysis in cell-based assays have played a key role in the discovery of the insulin-signaling framework. Moreover, our synthesis of the molecular physiology of insulin signaling and its failure has been achieved largely with the use of genetically modified mice.163 Although there are important physiologic differences between humans and mice, genetically modified mice provide an essential integrative experimental model that guide us in the discovery of novel treatments for insulin resistance and its progression to diabetes.164

Systemic Deletion of the Insulin or IGF1 Receptor Gene Insulin and IGF1 receptors have many overlapping functions during development and adult life, even though some details are different between mice and humans. In mice, the complete deletion of the IR or the IGF1R includes serious physiologic consequences that cause death shortly after birth.165 The IGF1/2→IGF1R signaling is the principle growth regulatory pathway in fetal mice, which is not influenced by growth hormone or augmented by the insulin receptor until after birth.166 IGF1R-deficient mice are born 50% smaller than normal littermates, and they die after a few days owing to developmental defects.167 By contrast, mice lacking the insulin receptor are nearly normal in size at birth, except for a reduced adipose tissue mass. Regardless, insulin receptor-deficient mice also die a few days after birth owing to severe hyperglycemia.166 Mice retaining at least 20% of the normal insulin-receptor expression throughout the body can survive with severe postnatal growth retardation and hyperglycemia that resembles human leprechaunism.165 This growth defect might arise, at least in part, from elevated hepatic IGFBP1 (FOXO-mediated) that reduces IGF1 bioavailability. Thus, small mice with severely reduced insulin or IGF-1 signaling have short life spans owing to developmental and metabolic defects. Although highly informative, the mouse knockout experiments do not reflect the critical role of human insulin receptors during gestation. Rodents are born at a developmental stage corresponding to about 26 weeks of human gestation, so the IR-dependent phase of mouse embryonic growth is minimal.163 By contrast, the occasional humans lacking insulin receptors display a syndrome originally called leprechaunism, which is now known to arise from missense mutations in the IR that might retain some activity. Without a functional IR, intrauterine growth is reduced severely and the fetus is

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usually not viable; however in rare cases, IR-deficient human neonates might survive owing to extreme hyperinsulinemia that can activate residual IR activity or the homologous IGF1Rs.168

Tissue-Specific Inactivation of the Insulin-Receptor Gene Owing to metabolic interactions between various tissues and the lethal consequences of whole-body insulinreceptor knockout, it is difficult to establish the tissuespecific behavior of insulin signaling in whole-body knockout mice. The best approach is to use Cre-loxP technology to delete the insulin receptor in single tissues or organs.169 A tissue-specific knockout of the insulin receptor is produced by introducing two short bacterial DNA sequences called lox (locus of crossing-over) sites around exon 4 in the insulin receptor to produce a “floxed” allele. In the absence of the bacterial Cre recombinase, this floxed allele usually behaves normally in all tissues studied; however, in the presence of Cre recombinase, these lox sites facilitate the excision of exon 4 of the insulin receptor and produce a nonsense mRNA with a premature stop codon, which fails to encode a receptor that binds insulin or fails to generate an insulin signal. Tissue-specific knockouts have limitations, as the complete absence of insulin signaling is never involved in common metabolic disease. Regardless, conditional insulin-receptor knockout mouse models generated with the Cre/lox system are remarkably informative regarding the role of insulin receptors in classic insulin target tissues, and reveal other unexpectedly important sites of insulin action.163,170 Liver. The liver is an important site of insulin action that plays a role in systemic glucose and lipid homeostasis. LIRKO (liver-specific insulin-receptor knockout) mice display a moderately elevated fasting glucose concentration; however, the liver does not respond to insulin, and the LIRKO mice develop severe postprandial hyperglycemia and glucose intolerance.171 This metabolic disorder is related, at least in part, to constitutive hepatic glucose release owing to dysregulated hepatic gene expression— including elevated PCK1 and G6PC, and decreased GCK (glucokinase, hexokinase 4) and PK1 (pyruvate kinase).164,171 Moreover, LIRKO mice exhibit marked hyperinsulinemia owing to a combination of decreased insulin clearance and increased insulin secretion associated with β-cell mass expansion. The chronic hyperinsulinemia might promote systemic insulin resistance, which could also contribute to the postprandial hyperglycemia.172 However, it is unlikely that the mechanisms of systemic insulin resistance are similar between LIRKO and more conventional models such as leptin-deficient ob/ob mice or diet-induced obesity. Although genetic hepatic insulin resistance of LIRKO mice is exacerbated by secondary systemic insulin resistance, LIRKO mice display reduced levels of circulating free fatty acids and triglycerides.171 However, on an atherogenic diet, LIRKO mice develop dyslipidemia by 12 weeks of age—including decreased circulating HDL cholesterol and increased nonHDL cholesterol—which progresses to atherosclerosis.173

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Unexpectedly, glucose intolerance of the LIRKO mice resolves with age, which appears to be associated with hepatic failure and mitochondrial dysfunction.163 Thus, insulin signaling is essential for normal hepatic function beyond the anticipated regulation of glucose metabolism. Skeletal Muscle. Insulin resistance in muscle, liver, and fat has been viewed as central to the pathogenesis of the insulin resistance syndromes, particularly type 2 diabetes. So one of the most informative and unexpected discoveries was obtained with the skeletal muscle-specific IR knockout (MIRKO) mice.169 Although muscle insulinstimulated IRS→PI3K→AKT signaling is lost in MIRKO mice, systemic glucose tolerance is normal, at least in part, because insulin-independent glucose uptake remains intact, and even increases owing to the activation of AMPK (AMP-dependent kinase)-mediated glucose influx resulting from reduced glucose oxidation.174 Moreover, fat mass increases in MIRKO mice because of a shift of nutrients into the adipose tissues.169 Whereas muscle insulin resistance promotes some aspects of metabolic disease—including mild obesity and elevated circulating free fatty acids and triglycerides—elevated glucose and insulin never develop.164 Adipocytes. White adipose tissue displays several important physiologic functions, including the storage of postprandial glucose as triglyceride, and the secretion of signaling factors that regulate appetite and energy homeostasis. Genetic insulin resistance of adipose tissue caused by the deletion of the insulin receptor (FIRKO mice) dysregulates insulin action on glucose influx, triglyceride synthesis, and antilipolysis.175 FIRKO-mice consume the same amount of food and accumulate less brown and white adipose tissue, but they display increased systemic insulin sensitivity that persists during aging. Remarkably, FIRKO mice experience a longer life span, suggesting that leanness and insulin sensitivity can be associated with longevity even in the absence of reduced calorie intake.176 These beneficial effects might arise from reduced adipocyte-related inflammation. Pancreatic β-Cells. The deletion of insulin receptors from pancreatic β-cells (βIRKO mice) shows a role for insulin

signaling in the control of glucose-stimulated insulin secretion.177 The βIRKO mice develop progressive glucose intolerance, which is associated with reduced insulin content and β-cell mass, and diminished first-phase glucosestimulated insulin secretion. These results are especially intriguing, as they reflect some of the pathophysiology that develops in humans with type II diabetes. Despite numerous reviews on the subject, whether or not insulin itself is the physiologically relevant ligand regulating βcell function remains controversial.178 However, normal glucose-stimulated insulin secretion also depends on IGF1R in the β-cells, suggesting that Igf1 and Igf2 produced in pancreatic islets might play important roles.179

Inactivation of IRS Genes As with the insulin receptor, genetic deletion of IRS1 and/ or IRS2 in mice reveals their required role in metabolic regulation and growth. IRSs couple the receptors for insulin and IGF1 to the PI3K→AKT cascade and other downstream signals. In many cell-based assays, IRS1 and IRS2 exhibit similar roles in insulin signaling; however, they display unique signaling potential in certain tissues owing to different regulation, function, or expression. Systemic deletion of IRS1 produces small insulin-resistant mice with nearly normal glucose homeostasis owing to β-cell expansion and lifelong compensatory hyperinsulinemia.180 These results suggest that IRS1 mediates most of the IGF1 signal for somatic growth, but is not essential for β-cell growth during insulin resistance. By contrast, mice lacking IRS2 display nearly normal body growth and even gain excess weight, but they develop life-threatening diabetes between 8 and 15 weeks of age for male mice (later for females) owing to reduced β-cell mass and insufficient compensatory insulin secretion.181 Whereas the complete deletion of IRS1 and IRS2 is embryonically lethal, littermates retaining one allele of IRS1 (IRS1+/-•IRS2-/-) or one allele of IRS2 (IRS1-/-•IRS2+/-) can be born alive.182 IRS1+/-•IRS2-/- mice develop severe fasting hyperglycemia and die by 4 weeks of age because IRS2 is required for pancreatic β-cell survival and growth (Fig. 33-5). By contrast, Irs1-/-•Irs2+/- mice reach only 30% of normal size, but they display nearly normal glucose tolerance and circulating insulin concentrations at 6 months of age182 (see Fig. 33-5). Regardless, the small

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Figure 33-5  The life span of genetically modified mice, indicated in days or as a percentage of the life span of C57BL/6 mice. The targeted genes indicated on the left of the figure are shown against life span (days) or the C57BL/6 life span (%). Except where indicated, mice are either wild-type [+/+], heterozygous [+/-] or homozygous [-/-] knockout, or Tg+ transgene positive.

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IRS1-/-•IRS2+/- mice are fragile and require extraordinary care to live beyond this age. Thus, IRS1 and IRS2 are essential for development and nutrient homeostasis. Liver. Hyperglycemia and dyslipidemia owing to hepatic insulin resistance are key pathologic features of type 2 ­diabetes.183,184 In mice, near total hepatic insulin resistance can be introduced via the systemic or liver-specific knockout of key insulin-signaling genes.171,185-188 Among these approaches, the compound suppression or deletion of the insulin-receptor substrates, IRS1 and IRS2, is the least complicated by defective insulin clearance.188,189 The central role of IRS-proteins in the PI3K→AKTsignaling cascade is validated by a wide array of cell-based and mouse-based experiments. The simplest experiments use an intraperitoneal injection of insulin into ordinary mice, or mice lacking hepatic IRS1, IRS2, or both. Insulin rapidly stimulates AKT phosphorylation and the phosphorylation of its downstream substrates FOXO1 and GSK3α/β in wild-type mice. However, both IRS1 and IRS2 must be deleted before insulin receptors are completely uncoupled from the PI3K→PDK1→AKT cascade in hepatocytes.190 These results show the shared but absolute requirement for IRS1 or IRS2 for the hepatic insulin response in mice. In general, IRS1 plays a dominant role in liver because most nutrient-sensitive transcripts—including gluconeogenic and lipogenic genes—are expressed nearly normally in liver lacking both IRS2 alleles (LKO2 mice), whereas these transcripts are dysregulated significantly in liver lacking IRS1, or IRS1 and IRS2 together.191 Even IRS1 reduced by 50% in the absence of IRS2 is sufficient to maintain nearly normal gene expression, fasting glucose concentrations, and postprandial glucose tolerance.191 Thus, IRS1 appears more important than IRS2 for glucose tolerance during nutrient excess. This distinction appears related to the stability of IRS1 compared against IRS2 during metabolic stress. Other aspects are also involved, including differential transcriptional regulation. We conclude that hepatic IRS1 is a principal mediator of the transition between fasting and postprandial glucose homeostasis, especially during chronic nutrient excess, whereas IRS2 modulates the signal daily during ordinary metabolic challenges.191 Skeletal Muscle. Like the liver, IRS1 and IRS2 display similar but not identical signaling functions in skeletal muscle. Insulin-like signaling in skeletal muscle is initiated by the activation of the insulin and/or IGF1 r­ eceptor tyrosine kinases,192 which can exist as hybrids linked to the downstream pathways through the IRS1 and IRS2 branches of the cascade. Based on insulin- or IGF1-stimulated AKT→mTORC1 signaling, IRS1 has a slightly stronger role than IRS2 in insulin-like signaling in muscle. Consistent with this result, mice without muscle IRS1 display a small reduction in mass and protein content, whereas no reduction is detected without IRS2.193 These results are consistent with previous work showing that conventional systemic IRS1 knockout mice are smaller than control mice.180,194 Regardless, when IRS1 is inactivated in muscle, IRS2 can promote AKT→mTORC1 signaling and muscle growth—including maintenance

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of cardiac muscle.193 Because the deletion of IRS1 and IRS2 further reduces muscle growth—and either IRS1 or IRS2 can prevent sudden cardiac death in these mice— we conclude that both IRS-proteins contribute to i nsulin-like signaling in skeletal and cardiac muscle.193 Insulin-like signals promote muscle growth through the AKT→mTOR pathway.195,196 But without IRS1, AKT phosphorylation at T308AKT and S473AKT is mildly impaired, whereas AKT phosphorylation is normal without muscle IRS2: Without IRS1 and IRS2, insulin-stimulated phosphorylation of T308AKT and S473AKT is even lower.193 These results are consistent with the conclusion that both IRS1 and IRS2 can promote AKT activity, but IRS1 has the strongest effect. On deletion of IRS1—or the deletion of both IRS1 and IRS2—insulin-stimulated T308AKT phosphorylation is more strongly reduced than S473AKT phosphorylation. This difference might arise because T308AKT is a direct target of PDK1, which is immediately downstream of the PI3K, whereas S473AKT phosphorylation is mediated by mTORC2.197 The role of mTORC2 in skeletal muscle S473AKT phosphorylation is confirmed in mice by loss of this phosphorylation on muscle-specific deletion of Rictor, a functional component of the mTORC2 complex.198 Pancreatic β-Cells. The insulin signal transduction pathway in pancreatic β-cells is similar to that in most other cell types, except that it can be regulated by glucose through an indirect mechanism that depends largely on IRS2 expression. Mice lacking the IRS1 or IRS2 genes are insulin resistant, with impaired use of peripheral glucose.181,199,200 Both types of knockout mice display metabolic dysregulation, but only IRS2-/- mice (males) develop diabetes between 10 and 15 weeks of age owing to the a rapid and progressive loss of pancreatic β-cells.181 These results position the insulin-like signaling cascade through IRS2 at the center of β-cell function (Fig. 33-6).181,199,201,202 Before these experiments, the predominant thought was that insulin resistance was the main cause of type 2 diabetes, but it was not widely acknowledged until the 1990s that the onset of type 2 diabetes is marked by a failure of the functional β-cell mass to meet the increased systemic requirements.203-205 On realizing that IRS2 signaling in β-cells is important, many studies established a role for various upstream and downstream elements that regulate or mediate IRS2 signals in β-cells, which link mechanistically peripheral target tissues to pancreatic β-cell function (see Fig. 33-6).203,206 Although β-cell loss in IRS2-/- mice progresses steadily after weaning, at 4 weeks the β-cells are still sufficient to maintain glucose tolerance. By comparison, β-cell mass decreases by 50% in 4-week-old IGF1R+/-•IRS2+/- mice, and it is nearly undetectable in IGF1R+/-•IRS2-/- mice.201 By contrast, the targeted deletion of the IGF1R in β-cells has insignificant effects on β-cell growth and survival,179,207 whereas the β-cell-specific deletion of both IR and IGF1R in β-cells causes loss of β-cell mass by 2 weeks of age. Together these experiments suggest that both IR and IGF1R—and possibly receptor hybrids—promote the IRS2 signaling needed for β-cell growth and survival (see Fig. 33-6).208 Many factors are required for proper β-cell function, including the homeodomain transcription factor

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Wnt

INS Glc PI(3,4,5)P3

Frizzled Dishevelled

GSK3β

mTORC2 PDK1

PI3K

pY

pY

pS

Glc6P PTB1B

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kip

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o

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S6K

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β-cell mass expansion Figure 33-6  The integrative role of IRS2 signaling in pancreatic β-cell function. The diagram shows the relationship between the IRS2-branch of

the insulin-signaling pathway and upstream and downstream mechanisms regulating β-cell growth and function. The production of PI3,4,5P3 by the PI3K recruits the Ser/Thr-kinases PDK1 and AKT to the plasma membrane, where AKT is activated by PDK1 and mTORC2-mediated phosphorylation. AKT phosphorylates many proteins that play important physiologic roles—GSK3β (glycogen synthesis), the BAD•BCL2 heterodimer (apoptosis inhibition), TSC1•TSC2 (protein synthesis and nutrient sensing), and FOXO (transcriptional regulation). Activation of GLP1→cAMP→PKA→CREB, glucose→Ca2+→CRTC2, and calcinurin→NFAT induce IRS2 expression in β-cells, showing a mechanism that places β-cell growth, function, and survival under the control of glucose and incretins. Because insulin and IGF1R are constitutively active, IRS2 expression can act as the regulatory gateway for these signaling cascades: IRS2→PI3K→AKt signaling promotes mTOR signaling and inhibits Foxo1 and p27kip; Irs2─┤Gsk3β might couple into the β-catenin cascade regulated by Wnt. PTEN and PTP1B modulate IRS2 signaling. Together this integrated pathway shows how signals known to promote β-cell/islet growth and function can be integrated by the IRS2-signaling cascade into common pathways regulated by glucose and other nutrients.

PDX1. PDX1 regulates downstream genes needed for β-cell growth and function, and mutations in PDX1 cause autosomal forms of early-onset diabetes in people (MODY4)209,210 (see Fig. 33-6). PDX1 is reduced in IRS2-/islets, and PDX1 haploinsufficiency further diminishes the function of β-cells lacking IRS2. By comparison, transgenic PDX1 expressed in IRS2-/- mice can maintain sufficient β-cell function and normalizes glucose tolerance, linking functional IRS2 signaling to the network of β-cell transcription factors.211,212 Transgenic expression of IRS2 or suppression of FOXO1 increases PDX1 concentrations in IRS2-/- mice, supporting the hypothesis that PDX1 can be modulated by the IRS2→FOXO1 cascade in β-cells (see Fig. 33-6).212,213 Haploinsufficiency for PTEN also prevents β-cell failure in IRS2-/- mice, owing at least in part to the simulation of the PI3K→PKB/AKT cascade that inhibits FOXO1 (see Fig. 33-6). IRS2 is critical because it is a highly regulated “gatekeeper” of islet β-cell homeostasis. IRS2 expression is increased by glucose, incretins such as GLP1 (glucagonlike peptide 1), and other factors that increase cytosolic

[Ca2+]i and [cAMP]i in β-cells.214-217 By contrast, IRS2 can be downregulated by proinflammatory cytokines, physiologic stress, and feedback inhibition of normal IRS2-signaling.206,218-220 It is conceivable that the relatively high expression of IRS2 and its quick turnover in β-cells217 might offset any need for an insulin-stimulated receptor kinase—as in the liver.221 With a controlled upregulation of IRS2 when β-cell compensation is needed to maintain glucose homeostasis, and downregulation of IRS2 when β-cell compensation is not needed, the responsibility for insulin/IGF1 itself to trigger downstream signaling in β-cells seems to be removed and placed on glucose, incretins, and neuronal connections—which are known to be physiologically relevant regulators of pancreatic β-cell function (see Fig. 33-6).

Inactivation of the PI3K→PDK1→AKT Cascade PI3K. The IRS→PI3K→AKT cascade is robust because of the expression of multiple isoforms of the proximal components. As outlined earlier, the type 1A PI3K is a dimer composed of a catalytic subunit (p110α, p110β, or p110δ)

33  THE MECHANISMS OF INSULIN ACTION

and one of five regulatory subunit isoforms encoded by three different genes (Pik3r1, Pik3r2, and Pik3r3).123 In most cells, including the liver, products of Pik3r1— p85α, p55α, and p50α—and Pik3r2 (p85β) stabilize and inhibit the catalytic subunits.222,223 The complete disruption of hepatic Pik3r1 and Pik3r2 markedly reduces insulin-stimulated PI3K activity and PIP3 accumulation—at least in part by destabilizing the catalytic monomers— which dysregulates glucose and lipid homeostasis, hepatic size, and function.224 Unexpectedly, partial genetic deletion of the regulatory subunits increases insulin sensitivity. Because the concentration of regulatory monomers ordinarily exceeds that of the catalytic subunit, it is possible that activation of the p85•p110 complex provides a competitive advantage when less regulatory subunits are available to compete for binding to IRS.222,225 Physiologically, this regulation might be accomplished by sequestering p85 in the nucleus through its interaction with other proteins, including XBP1 (X-box binding protein 1).226 p85 also appears to modulate JNK (c-Jun terminal kinase) activity, which can promote insulin resistance.227 Thus, genetic manipulations show that p85 can regulate the insulin-signaling cascade through unanticipated mechanisms, but how these mechanism are accessed physiologically remains to be established. The complete systemic deletion of either p110α or p110β is embryonically lethal,228,229 whereas deletion of p110δ causes immune-system defects.230 By comparison, mice heterozygous for deletion of either p110α or p110β show no phenotypic abnormalities, whereas combined heterozygosity for p110α and p110β causes mild glucose intolerance and fasting hyperinsulinemia.231 The inactivating mutation of p110α (D933p110α→A933p110α) abolishes its lipid kinase activity, and homozygous A933p110α knock-in mice die during embryogenesis.232 The negative consequences of the heterozygous systemic D933p110α knock-in are more pronounced on insulin signaling in muscle and adipose than in liver.232 Heterozygous A933p110α mice are viable and fertile, but they develop insulin resistance, glucose intolerance, and hyperphagia with increased adiposity. Because the function of p110α is not compensated by p110β, the unique functions of p110α might arise from the highly selective recruitment of p110α to IRS-signaling complexes following insulin stimulation. It is of interest to note that beneficial metabolic effects are observed as heterozygous A933p110α mice age, which are probably derived in part from attenuated but not absent insulin signaling that mimics the effects of calorie restriction to improve insulin action.233 AKT. The mammalian genome contains three genes that separately encode AKT isoform 1, 2, and 3. The analysis of knockout mice shows that each isoform regulates important biological functions, including cell proliferation, cell growth, cell survival, cell differentiation, and glucose metabolism in vivo; however, the AKT isoforms are not redundant components of the insulin-like signaling cascade.234,235 AKT1 plays a major role in embryonic development, growth, and survival, but includes only minor effects on metabolism.236 By comparison, systemic AKT2-deficient mice display metabolic defects, whereas AKT3-deficient

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mice display neural defects.123 AKT2 is important for metabolic regulation largely because it promotes insulinstimulated GLUT4 translocation and regulates liver glucose metabolism.237 Two human subjects with a dominant negative mutation in AKT2 display many features of type 2 diabetes—including hyperglycemia, increased lipogenesis, elevated liver-fat content, TG-enriched VLDL, hypertriglyceridemia, and low HDL cholesterol levels.24,238 Targeted disruption of AKT2 impairs insulin-stimulated glucose uptake in murine muscle and adipocytes, and it prevents the suppression of hepatic glucose output by insulin. Systemic AKT2 deletion causes glucose intolerance and insulin resistance that progress to diabetes and β-cell failure.185 Moreover, the related SGK3 (glucocorticoid-regulated kinase 3) synergizes with AKT2 in pancreatic β-cells to stimulate proliferation and insulin release.239 Work with IRS1/2 and AKT1/2 hepatic-specific knockout mice shows the important role of the inactivation of FOXO1. As expected, mice without hepatic AKT1 and AKT2—or without IRS1 and IRS2—are glucose intolerant, insulin resistant, hyperinsulinemic, and defective in their transcriptional response to feeding in the liver.190,240 Remarkably, in both cases these defects are normalized on concomitant liver-specific deletion of FOXO1. In the absence of both AKT1/2 and FOXO1—or without IRS1/2 and FOXO1—mice are no longer hyperinsulinemic and they adapt appropriately to both the fasted and fed states, even though insulin fails to promote an hepatic response.190,240 Gene expression analysis shows close concordance for dysregulation of FOXO1-dependent gene expression on deletion of AKT1/2 or IRS1/2, whereas deletion of FOXO1 restores a nearly normal metabolic response to nutrient intake. These results show that a major role of hepatic IRS→AKT signaling in the liver is to restrain the activity of FOXO1. Remarkably, in the absence of FOXO1, IRS→AKT signaling is largely dispensable for insulin- and nutrient-mediated hepatic metabolic regulation in vivo.241 It is unclear how liver metabolism can be normalized without direct insulin signaling, unless the indirect signals generated in the brain (or other peripheral tissues) are sufficient. PDK1. PDK1 controls the activation of numerous kinases including AKT and SGK.235 Unlike most kinases in the insulin-signaling cascade, PDK1 is constitutively active and is encoded by a single gene. Phosphorylation of its substrates is regulated by mechanisms that control interactions with PDK1.242 PDK1 is required for normal development, as mouse embryos lacking it die at day E9.5; however, PDK1-hypomorphic mice—retaining 10% of normal PDK1 expression in all tissues—are viable, fertile, and small.243 Regardless, activation of AKT1 and S6K1 by insulin is largely normal in PDK1-hypomorphic mice. Tissue-specific deletion of PDK1 confirms the important role of PDK1 for metabolic regulation.187 Liverspecific PDK1-/- mice display normal blood glucose and insulin concentrations under ordinary fasting and postprandial conditions; however, these mice develop marked hyperglycemia during a glucose tolerance test.187 Thus, basal activity of AKT2 appears sufficient under ordinary conditions, whereas insulin-stimulated phosphorylation

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of pS308AKT2 is especially important during acute metabolic challenge when definitive activation of AKT is required for tight metabolic control. Glucose intolerance of liver-specific PDK1−/− mice appears to arise from an inability of insulin to promote glycogen storage or to suppress gluconeogenesis. Moreover, these mice die between 4 to 16 weeks of age with severe liver failure. Thus hepatic PDK1 signaling is required for systemic glucose homoeostasis and normal liver function.187

HETEROLOGOUS REGULATION/DYSREGULATION OF THE PROXIMAL INSULIN-SIGNALING CASCADE Insulin resistance—reduced responsiveness of tissues to normal insulin concentrations—is a principle feature of type 2 diabetes that leads to compensatory hyperinsulinaemia.244 It also underlies risk factors—including hyperglycaemia, dyslipidaemia, and hypertension—for the clustering of type 2 diabetes with cardiovascular disease, nonalcoholic fatty liver disease, and related maladies (metabolic syndrome).164 Although numerous genetic and physiologic factors interact to produce and aggravate insulin resistance, rodent and human studies implicate dysregulated signaling by the insulin-receptor substrate proteins IRS1 and IRS2 as a common underlying mechanism.245,246 In this section, we describe several mechanisms—including transcriptional regulation, translational control, posttranslational modification, and IRS degradation—which can conspire to dysregulate the proximal steps of the insulin-signaling cascade and contribute to metabolic disease. Concerted Regulation of Proximal Insulin Signals More than a decade of genetic experiments in mice establishes that changes in the relative function of a broad array of insulin-signaling components, nutrient sensors, and their downstream metabolic effectors can have profound effects on insulin sensitivity and nutrient homeostasis.164 Although this work is remarkably informative, the complexity of heterologous regulation complicates the identification and design of new strategies for the treatment of insulin resistance and its pathologic sequela. Although the list of insulin-signaling components and their interactions continues to grow by functional and genetic approaches, the IRSs retain a special position as the integrating node that coordinates insulin responses in all tissues and cells. Indeed, a 50% reduction in the concentration of the IR, IRS1, and IRS2 achieved by genetic methods causes growth deficits and diabetes in mice.247 Thus, reduced IR→IRS signaling throughout life causes metabolic disease. We are now aware of many heterologous pathways that regulate the concentration and function of these proximal insulin-signaling components, but how the dysregulation of these mechanisms contributes to the progression of insulin resistance, metabolic disease, and type 2 diabetes in people is not understood. Studies show several mechanisms of concerted transcriptional regulation of proximal components of the insulin-signaling cascade. In skeletal muscle, YY1 (Yin Yang 1) regulates transcription of IGF1 and IGF2, IRS1 and IRS2, and AKT1, AKT2, and AKT3.248 YY1 is a

ubiquitous homeobox transcription factor related to the Polycomb family that can activate or repress these genes and many others. YY1 includes many functions, including interactions with histone acetyltransferase and histone deacetylase complexes, which alter chromatin structure and function. In its active state, YY1 recruits PRC (Polycomb repressive complex), PC2 (Polycomb protein 2), and EZH2 (enhancer of zeste homolog 2) to the promoters of the proximal insulin-signaling genes to promote histone acetylation that prevents the binding of the “transcription activator complex.” However, YY1 can also interact with other proteins including mTORC1 and S6K2.248,249 mTORC1 phosphorylates YY1, which disrupts the acetylation complex leading to increased transcription of its target genes.248 This regulatory mechanism was recognized because the inhibition of mTORC1 with rapamycin suppresses the expression of the proximal insulin-signaling genes, whereas hyperactivation of mTORC1 can promote expression of these YY1-regulated genes.248 This unexpected “feed forward” mechanism can explain, at least in part, why patients treated with mTOR inhibitors might develop glucose intolerance, insulin resistance, and dyslipidemia—which prevents the long-term use of these drugs for the treatment of metabolic disease.248 Transcriptional Control of IRS1 Although the concerted repression of multiple signaling components can produce strong effects, reduced expression of individual signaling molecules can also lead to insulin resistance. Decreased expression of IRS1 in patients and rodents is associated with diabetes, but few investigators have studied whether dysregulated transcription of IRS1 might contribute to metabolic disease. Few studies have included descriptions of transcription factors that promote IRS1 expression, leading to the view that IRS1 is a constitutive mediator of long-term insulin action. Regardless, recent work suggests that IRS1 expression might be regulated by transcriptional repressors, including AP2β (transcription factor AP-2-beta), or the p160 family of nuclear receptor coactivators p/CIP and SRC1.250,251 AP2β is expressed in adipose tissue where it promotes adipocyte hypertrophy, inhibits adiponectin expression, and enhances the expression of inflammatory adipokines such as IL-6 and MCP-1.250 AP2β binds directly to the IRS1 promoter and decreases IRS1 mRNA and protein concentration in adipocyte cell lines.250 Interestingly, GWAS shows AP2β as a candidate gene for the risk of obesity and type 2 diabetes, which might involve negative regulation of IRS1 expression.252 p/CIP and SRC1 serve as transcriptional coactivators for nuclear hormone receptors and certain other transcription factors.253 Compound knockout of p/CIP and SRC1 in mice prevents obesity and increases energy expenditure, consistent with a role of nuclear hormone receptor target genes in these processes. Without p/CIP and SRC1, mice display increased glucose uptake and enhanced insulin sensitivity in white adipose tissue and skeletal muscle. Interestingly, IRS1 expression increases significantly in p/CIP and SRC1 knockout mice, suggesting that steroidregulated nuclear receptors can regulate IRS1 transcription through the action of p160 coactivators.251

33  THE MECHANISMS OF INSULIN ACTION

Multiple Factors Regulate IRS2 Transcription Unlike IRS1, IRS2 transcription is regulated by multiple metabolically important factors, including CREB (cAMP response element-binding protein) and its coactivator CRTC2 (CREB-regulated transcription coactivator 2), FOXO1/3, NFAT (nuclear factor of activated T cells), TFE3 (transcription factor E3), HIF2α (hypoxia-inducible factor-2α encoded by Epas1), and SREBP1 (sterol regulatory element-binding protein 1).215,254 Under fasting conditions, the cAMP-responsive CREB coactivator CRTC2 promotes glucose homeostasis by stimulating gluconeogenesis in liver on assembly of CREB•CRTC2 on relevant CRE promoter sites, including a half-CRE on IRS2.254 The induction of hepatic IRS2 during fasting appears critical for glucose homeostasis, as it triggers a feedback response that limits glucose output from the liver even when insulin concentration are low. In addition to CRE sequences responsive to cAMP→CREB•CRTC2, the promoter region of the IRS2 gene includes insulin response elements that bind FOXO family members. FOXO links the PI3K→AKT cascade to the expression of genes important for cell growth, survival, and metabolism (see Fig. 33-4). On deletion of AKT1/2 in liver, FOXO promotes IRS2 transcription, whereas the deletion of FOXO1/3 strongly reduces IRS2 transcription even when AKT is deleted.240 Deletion of hepatic FOXO1 attenuates CREB•CRTC2-mediated gene expression— including the IRS2 gene—which inhibits gluconeogenesis and promotes glycogen storage while augmenting other postprandial responses.150 Thus, regulation of IRS2 transcription by AKT→FOXO establishes a direct feedback loop in the liver to promote insulin signaling during fasting and to inhibit it during prolonged intervals of insulin stimulation. FOXO action on IRS2 transcription during fasting ensures that the liver can respond strongly to insulin immediately on feeding, but with a diminishing response during chronic nutrient intake when the insulin signal is sustained by IRS1. TFE3 is a basic helix-loop-helix protein that binds to E-box consensus cis-elements in many genes, including some related to glycolysis, lipogenesis, and insulin signaling.255 Because the E-box within the IRS2 gene overlaps with an IRE that binds FOXO1/3, TFE3 converges with FOXO to promote IRS2 expression. However, these elements also overlap with an SRE (sterol regulatory element DNA sequence, TCACNCCAC) that binds the SREBP1c. SREBP-1c is an important transcriptional regulator of lipid synthesis.256 Active SREBP-1c concentrations increase during nutrient excess and chronic insulin stimulation.257,258 Elevated hepatic SREBP-1c is associated with a decrease in IRS2 mRNA, owing to competition between overlapping SRE and IRE.258 Upregulation of IRS2 expression by TFE3/FOXO and downregulation by SREBP-1c parallels the switch from starvation-induced glycogenolysis and gluconeogenesis to postprandial lipogenesis. An imbalance in this reciprocal regulation might ultimately contribute to pathophysiologic effects of overnutrition leading to the development of the metabolic syndrome and diabetes. More work is needed to establish the details of this complex regulatory mechanism in human metabolic disease.

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Studies show that HIF2α induces transcription of mouse or human IRS2 in liver.259 Under normal oxygen tension, HIF1α and HIF2α are destabilized on hydroxylation of critical proline residues by prolyl hydroxylase domain– containing proteins (PHD1, 2, and PHD3). Interestingly, acute inhibition of hepatic PHD3 improves insulin sensitivity and resolves diabetes by specifically stabilizing HIF2α, which increases IRS2 transcription that promotes insulin-stimulated AKT activation.259 Physiologically, the HIF2α-mediated mechanism of IRS2 transcriptional regulation might be important in the perivenous zone of the liver, which displays more hypoxia and less gluconeogenesis as compared to other zones in the liver.260 Moreover, vascular endothelial growth factor (VEGF) inhibition can improve glucose tolerance by inducing hepatic hypoxia through HIF2α stabilization and induction of IRS2.261 These results show an interesting and unexpected intersection between HIF2α-mediated hypoxic signaling and IRS2-mediated hepatic insulin action. Complex and multifactor transcriptional regulation of IRS2 is also found in pancreatic β-cells, which can depend on IRS2 expression for growth, function, and survival. IRS2 transcription increases through the action of FOXO1/3, NFAT, and the CREB•CRTC2 (see Fig. 33-6).214,215,218,219 Beta-cells express both FOXO1 and FOXO3a,32 and both factors can bind to the insulin response element in the promoter, but in primary rat β-cells only FOXO3 appears to drive the basal IRS2 gene promoter activity that maintains IRS2 expression.219 In β-cells, FOXO3 can account for as much as 80% of the basal IRS2 expression.219 Regardless, deletion of FOXO1 can rescue β-cell function in IRS2-/- mice.262 Because the IRS2→PI3K→AKT cascade p ­hosphorylates and inhibits FOXO, insulin or IGF1 have inhibitory effects on FOXO-mediated transcription of IRS2, which can coordinate a feedback control loop to attenuate IRS2 expression. Regardless, in the postprandial state or during overnutrition when FOXO is strongly inhibited, IRS2 expression can be maintained and enhanced by alternative mechanisms. Elevated glucose produces ATP—which depolarizes β-cells to promote both Ca2+ influx and cAMP production—and induces IRS2 expression under conditions of overnutrition to help sustain compensatory β-cell function. In addition to its immediate role in insulin secretion, Ca2+ activates calcineurin, which dephosphorylates NFAT to facilitate its entry into the nucleus where it upregulates IRS2 and other genes (Fig. 33-6).214 Glucose or glucagon-like peptide-1 also increases the cAMP concentration in β-cells, which provides many important effects, including the activation of CREB•CRTC2 that also promotes IRS2 transcription (see Fig. 33-6).202,216 Thus glucose sensing is coupled directly to IRS2 expression in β-cells, which can stimulate β-cell growth and compensatory insulin secretion. miRNA-Mediated Translational Regulation miRNAs are short (∼20 nucleotides), noncoding RNA molecules that act as posttranscriptional regulators of gene expression, which bind to target sites in the 3’-untranslated regions (3’UTR) to form a complex that inhibits translation and renders the target mRNA molecule unstable. Several proximal components of the IR→PI3K→mTOR-signaling

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cascade are regulated by RNA interference mediated by the LIN28a/b─┤LET7 axis.263 LET7 was originally discovered in C. elegans where it controls the timing of stem-cell division and differentiation.264 LET7 and its family members are highly conserved across species in sequence and function, and it was the first known human miRNA whose dysregulation leads to less differentiated cellular states and cancer. LET7 interferes with many targets, including the translation of several proximal insulin-signaling proteins, including IGF1R, INSR, IRS2, PIK3IP1, AKT2, TSC1, and RICTOR.263 LIN7 interference is inhibited by the RNAbinding proteins Lin28a and Lin28b, which block production of mature LET7 leading to the increased translation of the insulin-signaling components. LIN28 overexpression in mice can cause gigantism and a delay in puberty onset, consistent with human genomewide association studies suggesting that polymorphisms in the human LIN28B gene are associated with human height and puberty timing.265 Moreover, LIN28a/b can promote glucose homeostasis in mammals by increasing insulin→PI3K→mTOR signaling and insulin sensitivity, which promotes resistance to type 2 diabetes and obesity induced by a high-fat diet (HFD).263 Several other miRNAs have been shown to suppress the translation of IRS1, including miRNA-96, miRNA-128a, miRNA-126, miRNA-143, miRNA-144, miRNA-145, miRNA-487b, and miRNA489. In one example, chronic angiotensin-II induced hypertension can increase the expression of miRNA-487b in the rat aorta.266 In this case, downregulation of IRS1 might contribute to hypertensioninduced cardiovascular disease, including aortic aneurysm formation owing to the loss of medial smooth muscle. Degradation of the IRS-Proteins Proteasome-mediated degradation regulates many biological processes including signal transduction, gene transcription, and cell-cycle progression.267 Proteins targeted for destruction by the 26S proteasome are polyubiquitinylated by a complex containing a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3). It is the concerted interactions between the bound substrate, the E3, and the ubiquitin-charged E2 that yield a polyubiquitinylated target protein. IRS1 and IRS2 can be polyubiquitinylated during sustained insulin or IGF1 stimulation or chronic inflammatory states, and these states correlate closely with feedback or heterologous inhibition of insulin signaling.268 The first pathway identified to mediate IRS1 degradation/polyubiquitinylation was associated with proinflammatory cytokine-mediated upregulation of suppressors of cytokine signaling (SOCS) proteins, which include eight isoforms that contain an NH2-terminal SH2 domain and a COOH-terminal SOCS box. The SH2 domains in SOCS bind to phosphotyrosine residues in various cytokine receptors or associated Janus kinases.269 SOCS1 and SOCS3 also bind to the insulin receptor to inhibit phosphorylation of IRS1 and IRS2.270,271 Moreover, SOCS1/3 also bind to elongin BC-containing E3 ubiquitin ligase complexes via the conserved SOCS box, which can promote ubiquitinylation/degradation of IRS1 and IRS2 in multiple cell types.272 In mouse liver, adenoviral-mediated

expression of SOCS1 dramatically reduces hepatic IRS1 and IRS2 protein levels, which leads to systemic glucose intolerance. Mutations in the conserved SOCS box prevent SOCS-mediated degradation of IRS1 or IRS2. Moreover, inhibition of SOCS by antisense oligonucleotides improves insulin sensitivity and normalizes SREBP-1c expression in obese and diabetic mice.269 Thus, SOCS1 and SOCS3-mediated polyubiquitinylation appears to be an important pathway linking infection, inflammation, or metabolic stress to insulin resistance and glucose intolerance (Fig. 33-7) 273-277. It is interesting to note that the core protein of hepatitis C virus upregulates SOCS3, which may help explain why infected patients have increased fasting insulin levels compared to patients with other chronic liver diseases.278 The cullin-RING E3 ubiquitin ligase 7 (CRL7) can mediate IRS1 degradation downstream of a feedback phosphorylation signal generated by the PI3K→AKT→mTORC1 cascade.279 CRL7 is a member of the Cullin-RING finger E3 ligases, which comprise the largest E3 family responsible for directing the polyubiquitinylation of substrate proteins by the 26 S proteasome.280 The CRL7 complex contains CUL7 (cullin 7)—a molecular scaffold that assembles Fbw8 (F-box/WD repeat-containing protein and 8), Skp1 (S-phase kinase-associated protein 1), and ROC1 (RING-box protein 1) that recruits an E2 conjugating enzyme. FBW8 binds to IRS1 through phospho-S/T residues generated by the mTORC1→S6K cascade—including human pS307IRS1, pS312IRS1, and pS527IRS1, but possibly others—which then leads to polyubiquitinylation of IRS1 that progresses to degradation (Fig. 33-8).279,280 However, ordinary mTORC1→S6K activity appears insufficient to engage the CRL7 pathways against IRS1, as unusually high levels of mTORC1→SK6 activity are required to drive IRS1 degradation.280 A molecular understanding of insulin resistance induced by nutrient excess has been difficult to establish and many mechanisms have been proposed.162 Reports suggest that mechanisms involving polyubiquitinylation of IRS1 can contribute to the disorder. Chronic consumption of highcalorie diets upregulates Cbl Proto-Oncogene B (CBLB), which is a RING-type E3 ubiquitin ligase that belongs to the Casitas B-lineage lymphoma family of proteins. CBL proteins share a conserved NH2-terminal region containing a tyrosine kinase-binding domain and a RING-finger domain to facilitate E3 ubiquitin ligase activity. Calorie excess induces carbohydrate-responsive element-binding protein (ChREBP) and SREBP1c, which upregulates in murine muscle and liver Myostatin (MSTN) that induces CBLB expression to drive insulin resistance through the polyubiquitinylation and degradation of IRS1.281 Yet another ubiquitin ligase—MG53 (Mitsugumin 53), a TRIM (tripartite motif-containing) family musclespecific E3 ubiquitin ligase, also promotes IRS1 and insulin-receptor degradation in muscle during calorie excess. MG53 is found mainly in skeletal muscle and the heart where it was originally shown to promote repair by acting as a scaffold for assembly of repair complexes.282 However, MG53 also targets the IR and IRS1 to promote their polyubiquitinylation and degradation.283,284 Although it is unclear how high-calorie diets upregulate MG53,

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some. Many proinflammatory cytokines that cause insulin resistance also induce the expression of SOCS family members, which contain an NH2terminal SH2 domain and a COOH-terminal SOCS box.276,277 SOCS1 or SOCS3 can target phosphotyrosine-containing proteins such as IRS1 or IRS2 for ubiquitinylation and degradation, because the SOCS-box associates with elongin BC-containing ubiquitin ligase E3.278,280

mouse studies show that MG53–null-mice that are fed a high-calorie diet are protected from insulin resistance and degradation of IR and IRS1.284 Degradation of IRS-proteins is stimulated in various cell-based systems by multiple factors, including tumor necrosis factor-α (TNFα), interferon-γ, insulin and IGF1, platelet derived growth factor (PDFGF), free fatty acids (FFA), 12-myristate 13-acetate (PMA), inhibitors of Ser/ Thr phosphatases, and inhibitors of calcineurin.285 Activation of mTORC1 is at least partially responsible for initiating IRS1 degradation in some cell backgrounds, especially during extreme conditions of mTORC1 activation.286-289 Hyperactivation of the mTORC1 complex in cells lacking TSC1 or TSC2 increases serine phosphorylation of IRS1;288-289 however, there is some evidence that IRS-protein degradation in these cells is a result of coincident activation of endoplasmic reticulum (ER) stress-signaling pathways and is partially separable from the effects of IRS1 phospho-S/T.290 Although the mechanism of mTORC1-mediated IRS1 degradation is not completely clear, CUL7/Fbw8 might play an important role.279 However, CUL7/Fbw8 does not target IRS2, so other mechanisms must also be involved for this branch of insulin-signaling cascade.291 Multisite Ser/Thr-Phosphorylation of IRS-Proteins IRS1 and IRS2 can be regulated through a complex mechanism involving phosphorylation of more than 50 serine/threonine residues (phospho-S/Ts) within their unstructured tail regions.292 Understanding how phospho-S/Ts regulate signaling is a difficult problem because so many sites and mechanisms appear to be involved. Heterologous signaling cascades initiated by proinflammatory cytokines or metabolic excess—including tumor

necrosis factor-α (TNFα), endothelin-1, angiotensin II, excess nutrients (free fatty acids, amino acids, and glucose) or endoplasmic reticulum stress—are implicated in IRS1 phospho-S/T.293,294 Many biochemical and genetic experiments in cell-based systems suggest that individual phospho-S/T sites throughout the structure of IRS1 are associated with a reduction of insulin-stimulated tyrosine phosphorylation by up to 50%.295 This level of inhibition is sufficient to cause glucose intolerance that could progress to diabetes, especially if pancreatic β-cells fail to provide adequate compensatory hyperinsulinemia.247 Numerous cell-based studies show IRS1 phospho-S/T to be a physiologically integrative mechanism modulating insulin sensitivity.292 Insulin is clearly an important input to IRS1 phospho-S/T, as the vast majority of sites detected by specific monoclonal antibodies are stimulated by insulin, and they are diminished by the inhibition of the insulin-stimulated PI3K→Akt→mTOR cascade.296 Moreover, the IRS1 phospho-S/T patterns produced during druginduced “metabolic stress” correlate significantly with that stimulated by insulin. These results suggest that IRS1 phospho-S/T is first and foremost a feedback mechanism that develops during insulin stimulation, but that this mechanism can be co-opted by metabolic stress—such as ER stress or inflammation—to inhibit insulin signaling and to promote metabolic disease.292,297-302 An implicit corollary is that hyperinsulinemia may be an important physiologic mediator of insulin resistance in animals, and there is some experimental evidence to corroborate this implication.303 In cultured cells, insulin-stimulated kinases—including aPKC (atypical protein kinase C), AKT, SIK2 (salt-inducible kinase 2), mTORC1, S6K1, ERL1/2 (extracellular signal-regulated kinase 1/2), ROCK1 (rho-associated

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coiled-coil containing protein kinase 1)—mediate feedback (autologous) IRS1 phospho-S/T with positive or negative effects on insulin sensitivity.292 An emerging view is that the positive/negative regulation of IRS1 by autologous pathways is subverted/co-opted in disease by inappropriate phospho-S/T levels mediated by heterologous kinases—including AMPK (AMP-activated protein kinase), GSK3 (glycogen synthase kinase 3), GRK2 (G protein-coupled receptor kinase 2), novel and conventional PKC isoforms, JNK (c-Jun N-terminal protein kinase), IKKβ (inhibitor of nuclear factor ĸB kinase β), and mPLK (mouse Pelle-like kinase). The use of siRNA shows additional kinases that might be involved, including Pim2 (Proviral Integrations of Moloney virus 2), PDHK (pyruvate dehydrogenase complex kinase), CaMKI-like, DAPK2 (death-associated protein kinase 2), DCLK1 (doublecortin-like kinase 1), STK10 (serine/ threonine kinase 10), STK25 (serine/threonine kinase 25), MKK4 (MAP kinase kinase 4), MKK6 or MKK7, and LIMK2 (LIM domain kinase 2).304 One of the best-studied regulatory phosphorylation sites in IRS1 is Ser307 (S307IRS1) in the rodent protein (human S312IRS1).301,305-309 Phosphorylation of S307IRS1 might be a common mechanism of insulin resistance (see Fig. 33-8). S307IRS1 phosphorylation is associated with reduced tyrosine phosphorylation of IRS1 in cultured cells, which decreases the activation of the PI3K→AKT pathway in response to insulin.310,311 Insulin itself promotes rat/mouse S307Irs1 phosphorylation through activation of the PI 3-kinase, showing feedback regulation that can be mediated by many kinases—PKCξ, IKKβ, JNK, mTOR, and S6K1 (see Fig. 33-8).295,296 Insulinstimulated degradation of IRS1 via the PI3K pathway is in part dependent on the pS307IRS1.312 JNK can bind directly to IRS1, which appears to facilitate rat/mouse

pS307Irs1 during stimulation of cells with proinflammatory cytokines. S307Irs1 is poorly phosphorylated in ob/ ob (obese) mice that lack Jnk1, suggesting that this mechanism of inhibition has physiologic significance.313 Free fatty acids that contribute to insulin resistance promote pS307IRS1, possibly through PKCθ307; however, associated hyperinsulinemia has not been excluded. IRS1 can be phosphorylated by PKCδ on at least 18 sites in BL21 DE3 cells, including S307IRS1, S323IRS1, and S574IRS1, which appear to play an inhibitory role.314 Hyperactivated mTOR also promotes S307IRS1 phosphorylation, which is diminished in mice lacking S6K.288,315,316 IKKβ inhibitors (aspirin and salicylates) block S307IRS1 phosphorylation,306 which improves insulin sensitivity in obese rodents and in type 2 diabetes patients.317-319 Phosphorylation of S307IRS1—located near the PTB domain— inhibits insulin-stimulated IRS1 tyrosine phosphorylation by disrupting the association between the insulin receptor and IRS1,311,320 although other phosphorylation sites might be involved.321 So far, only two studies have included direct investigations of the function of IRS1 S/T phosphorylation in mice using transgenesis or genetic knock-in to augment or replace endogenous (wild-type) IRS1 with a mutant version. Shulman and colleagues created transgenic mice having moderate overexpression in skeletal muscle (twofold vs. littermates) of nonmutant IRS1, or mutant IRS1 with alanine substitution of three serine residues— S302/307/612A (hS307/312/616A)—to block phosphorylation.322 Thus, in the triple-mutant transgenic mice, possibly half the total IRS1 protein is endogenous in origin. Matching of IRS1 expression is a potentially important consideration, given the role of autologous feedback. Regardless, the mutant transgenic mice showed better glucose tolerance than nonmutant transgenic mice when

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Figure 33-8  Heterologous and feedback inhibition of insulin signaling mediated by Ser/Thr-phosphorylation of IRS1. Various kinases in the insulin-

signaling cascade are implicated in this feedback mechanism, including PKB, mTOR, S6K, ERK, AKT, and atypical PKC isoforms. Other kinases activated by heterologous signals, including lipids such as ceramide, are also involved. Adiponectin signaling might promote insulin signaling by driving the conversion of ceramide to nontoxic sphingosine. Serine phosphorylation of IRS1 can recruit CRL7, which can promote ubiquitinylation and degradation of IRS1.

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both were fed an HFD. Compared to an HFD with true wild-type littermates, the mutant transgenic mice showed increased total and muscle glucose disposal during clamp, and enhanced muscle IRS1 tyrosine phosphorylation and p85 binding in response to insulin. Although somewhat complicated in design, this experiment is consistent with the notion that S/T phosphorylation of IRS1 in skeletal muscle contributes to the development of insulin resistance in animals/humans. We used genetic knock-in experiments to replace wildtype IRS1 in mice with a mutant (A307IRS1) lacking the ability to be phosphorylated at S307 (equivalent to human S312IRS1).323 To control for potential ancillary effects of the knock-in process on IRS1 expression/function,324 nonmutant control knock-in mice were also generated (S307S, “S”). Surprisingly, given the sensitizing effect of the A307IRS1 mutation in cell-based assays, homozygous A307IRS1 mice show increased fasting insulin versus control mice, as well as very mild glucose intolerance. Furthermore, high-fat-fed mutant homozygous A307IRS1 mice exhibit higher fasting insulin and more severe glucose intolerance than wild-type mice, and the A307IRS1 protein exhibits decreased PI3K binding in insulin-­stimulated primary hepatocytes. Thus, S307 phosphorylation appears permissive, rather than inhibitory, for insulin signaling in mice. But why is this? Among other, less prosaic explanations, it is possible that a serine is structurally required at position 307 of the IRS1 protein for its normal function. Alternatively, S307 phosphorylation could have a partial positive effect on insulin signal transduction that is more important in tissues/primary cells than its desensitizing function in continuous cell lines. By analogy with JNK1 activity, A307IRS1 phosphorylation could also have mixed, tissue-specific effects that are obscured by the standard knock-in approach.325 In any case, the phenotype of A307IRS1 mice confirms that (non)phosphorylation of unique S/T sites on IRS1 can affect whole-body insulin sensitivity. Modulation of Insulin Signaling by Protein and Lipid Phosphatases Many phosphatases can modulate the action of insulin by dephosphorylating key proteins or lipids in the signaling cascade—including PTP1B (PTPN1, tyrosineprotein phosphatase nonreceptor type 1), PTPN2, pTEN (phosphatase and tensin homolog), and PP2A (protein phosphatase 2A). PTP1B and PTPN2 are related phosphotyrosine phosphatases that attenuate insulin signaling by dephosphorylating the bisphosphorylated regulatory loop of the IR326; however, their biological effects are distinct owing to a different time-course of action and differential expression (muscle expresses PTP1B, whereas liver expresses both enzymes).327 Both PTP1B and TCPTP can be inactivated by reactive oxygen species generated during insulin stimulation, which provides an additional level of regulation.79,328 PTP1B-/- mice display increased insulin sensitivity, lower circulating insulin concentrations, and decreased pancreatic β-cell mass.329 Furthermore, PTP1B is a selective inhibitor of leptin signaling (LepRb→JAK2) as it dephosphorylates JAK2, but not JAK1 or 3, whereas TCPTP dephosphorylates JAK1⁄ 3, but not JAK2.326

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Thus, CNS inhibition of PTP1B can protect against obesity, whereas peripheral inhibition of PTP1B promotes glucose tolerance.330 In pancreatic β-cells, PTP1B attenuates IRS2→PI3K→AKT cascade that is important for growth, function, and survival of these cells.331 Thus, the deletion of PTP1B maintains β-cells mass in mice lacking IRS2, which prevents the early onset of diabetes. Regardless, IRS2-/-•PTP1B-/- mice eventually lose sufficient β-cell mass to develop diabetes between 8 to 9 months of age (see Fig. 33-5). PTEN is a potent negative regulator of insulin action and cellular proliferation, and one of the most frequently mutated genes in many forms of human cancer.332,333 PTEN attenuates downstream insulin-like signaling by dephosphorylating PI(3,4)P2 and PI(3,4,5)P3 at the 3-position, which reduces the recruitment and activation of PDK1 and AKT.333 PTEN+/- mice are glucose tolerant even as β-cell mass and circulating insulin levels decrease.334 PTEN heterozygosity also increases peripheral insulin sensitivity in Irs2-/-•PTEN+/- mice and normalizes glucose tolerance, as the small islets in these mice produce sufficient insulin until death from lymphoproliferative disease between 10 to 12 months age (see Fig. 33-5). These experiments highlight the complex relationship between nutrient homeostasis, insulin sensitivity and secretion, and cancer that emerges in rodents and humans. Despite this complexity, mild inhibition of PTEN, especially if it can be accomplished in a tissue-specific or transient way, might provide therapeutic value. The serine–threonine phosphatase PP2A plays an important regulatory role for insulin signaling. It dephosphorylates Ser/Thr residues on IRS1, which can enhance tyrosine phosphorylation to generate the insulin signal. By contrast, the inhibition of PP2A by okadaic acid strongly increases phospho-S/T and degradation of IRS-1, which is associated with reduced tyrosine phosphorylation and decreased insulin signaling.335 Thus, compared against the effects of PTP1B, PP2A generally displays the opposite effect on insulin signaling. The specificity of PP2A is largely coordinated through a scaffolding unit that binds to various substrates. The scaffolding unit is composed of HEAT (huntingtin-elongation-A subunit-TOR) repeats, which are thought to target PP2A to its substrates to confer specificity on the constitutive catalytic domain.

REGULATION OF PROTEIN METABOLISM BY INSULIN Multiple translation initiation factors (eIFs), many of which are multisubunit complexes, facilitate and regulate translation initiation (eIFs) and elongation (eEFs).336 Insulin stimulates protein synthesis by altering the intrinsic activity or binding properties of these factors. Several components of the translational machinery are targets of insulin regulation, including eIF2B, eIF4E, eEF1, eEF2, and the S6 ribosomal protein.337 Met-tRNAiMet (the initiator methionyl-tRNA) is delivered to the 40S subunit as part of a ternary complex with eIF2 and GTP. However, the eIF2B multisubunit guanine nucleotide exchange factor for eIF2 is inactivated by GSK3 (glycogen synthase kinase3)-mediated phosphorylation of the eIF2Bε subunit.338

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Inhibition of GSK3 by AKT-mediated phosphorylation during insulin stimulation leads to dephosphorylation of eIF2Bε, which promotes the formation of eIF2GTP that recruits Met-tRNAiMet to the ribosome.338-340 Thus, the insulin-stimulated IR→IRS→PI3K→AKT─┤GSK3 cascade leads to an overall increase in translation initiation by eIF2B.341 Diabetic rats exhibit significantly lower eIF2B activity in muscle.315 Initiation of translation also involves the interaction of key factors with the 5’-end of an mRNA molecule, the 5’-cap, as well as with the 5’-UTR. The eIF4F complex mediates cap-dependent translation initiation, a multisubunit complex composed of several proteins, including eIF4A, eIF4B, eIF4E, and eIF4G. eIF4E (eukaryotic initiation factor 4 gamma) recognizes and binds to the 5’-cap structure of mRNA, whereas eIF4G binds to the Poly(A)binding protein associated with the 3’-end of mRNA. These interactions form the characteristic loop structure of eukaryotic protein synthesis and stimulate the activity of the polyadenylate polymerase. Initiation of translation is strongly inhibited by 4E-BP1, which blocks the activity of eIF4E (see Fig. 33-4). 4E-BP1 directly interacts with eIF4E, which is a limiting component of the multisubunit complex that recruits 40S ribosomal subunits to the 5’ end of mRNAs. Interaction of 4E-BP1 with eIF4E inhibits complex assembly and represses translation. Insulin dissociates the 4E-BP1•eIF4E complex on mTORC1-mediated phosphorylation of 4E-BP1, facilitating the interaction between eIF4E and eIF4G, which is the scaffold protein for the eIF4F complex.342 Mnk, an insulin-stimulated kinase activated through the Ras→ERK cascade, also resides in the eIF4F complex where it phosphorylates eIF4E at Ser209 (see Fig. 33-4).343,344 Phosphorylation of eIF4E increases the binding affinity for mRNA caps to enhance translation initiation. The regulation of translation elongation in is one of the important steps in protein synthesis, because it consumes a great deal of metabolic energy. Mammals require a set of nonribosomal proteins called eukaryotic elongation factors (eEFs)—including eEF1A and eEF1B that mediate amino acyl-tRNA recruitment, and eEF2 that mediates ribosomal translocation. eEF2 is inactive when phosphorylated at Thr56 by the eEF2 kinase (eEF2K).345,346 Insulin stimulates the dephosphorylation of eEF2 through a rapamycin-sensitive (mTORC1) route, potentially involving the phosphorylation and inactivation of eEF2K by p70S6K.345 In vitro, p70S6K phosphorylates eEF2K at Ser366, a modification that reduces the activity of the kinase.345 Insulin-stimulated phosphorylation of the ribosomal S6 protein by p70S6K can promote elongation of specific mRNAs corresponding to components of the translational machinery.347 The atypical protein kinase C isoforms PKCξ and PKCλ also promote insulin-stimulated protein synthesis.348 The IRS→PI3K appears to represent a bifurcation in the insulin-signaling pathway to protein synthesis: One branch leads through PDK1→PKCλ/ξ to general protein synthesis, and the other branch goes through Akt→mTORC1 to regulate protein synthesis required for cell growth and cell-cycle progression; however, these mechanism are certain to be tissueand cell-type specific.

INSULIN-REGULATED GLUCOSE TRANSPORT Glucose transport is the prototype insulin response.349,350 The sodium-independent, facilitated-diffusion glucose/ hexose transporters (GLUTs) are integral membrane proteins that mediate transport across the plasma membrane. The members of the GLUT family are distinguished by substrate specificity and affinity, tissue distribution, and regulation. Cells express one or more members of the glucose transporter family, including 12 isoforms organized into 3 classes: class I includes the well-characterized GLUT1, 2, 3, and GLUT4 transporters; class II includes GLUT5, GLUT7, GLUT9, and GLUT11; and class III includes GLUT6, GLUT8, GLUT10, and GLUT12.351,352 Insulin stimulates glucose influx into adipose tissue, and cardiac and skeletal muscle.349,350 Insulin-stimulated glucose influx is stimulated by the translocation of GLUT4 from the intracellular to the plasma membrane. The molecular mechanisms linking the insulin signal to increased glucose influx involve complex multistep coupling between the signaling cascade and vesicle trafficking.349 GLUT1 is expressed in most cells and tissues and resides permanently on the plasma membrane where it constitutively transports glucose from the extracellular space into the cell. Although insulin does not stimulate translocation of GLUT1, chronic insulin treatment can increase the expression of cellular GLUT1 via the p21ras→ERK kinase pathway.353,354 GLUT2 is mainly expressed in liver and rodent pancreatic β-cells, where its low affinity and high transport capacity provides a constant flux of glucose into these organs at physiologic plasma glucose concentrations. In the β-cell, the uptake of glucose through GLUT2 is the first step in the detection of circulating glucose concentrations needed to stimulate insulin secretion. GLUT3 (Glucose transporter 3, encoded by SLC2A3) has a relatively high affinity for glucose. Although glucose delivery and use in the mammalian brain is mediated primarily by GLUT1 at the blood-brain barrier, GLUT3 is most abundant in the central nervous system where glucose concentrations are lower than in the bloodstream. GLUT5 is a fructose transporter expressed on the apical border of enterocytes in the small intestine.352 GLUT8 is a high-affinity glucose transporter (Km, 2 mM) that can also transport fructose and galactose. GLUT8 is expressed at high levels in testis, at intermediate levels in the brain—including hippocampal excitatory and inhibitory neurons, the dentate gyrus, amygdala, primary olfactory cortex, some hypothalamic nuclei, and the nucleus of the tractus solitaries—and at lower levels in the heart and other tissues.355 GLUT8 is not required for embryonic development, and GLUT8-deficient mice display normal development, growth, and glucose homeostasis.

Insulin-Regulated GLUT4 GLUT4 is the principle insulin-responsive glucose transporter, which is expressed selectively in adipose tissue, and skeletal and cardiac muscle where it plays a direct role in glucose disposal and homeostasis.349,356 In the unstimulated state, GLUT4 is sequestered mainly in small intracellular storage vesicles (GSVs) until the GSVs translocate and insert into the plasma membrane on insulin

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stimulation or energy depletion (Fig. 33-9).357 The GSVs contain various components that contribute to their assembly and insulin sensitivity—including GLUT4 itself, IRAP (insulin-regulated aminopeptidase), LRP1 (lowdensity lipoprotein receptor-related protein 1), SORL1 (sortilin), GGA (ADP-ribosylation factor-binding protein), ACAP1 (ARFGAP with coil-coil and ankyrin repeats protein 1), and clathrin.358 The components contribute to the formation of GSVs following endocytosis of GLUT4 from the plasma membrane. After formation, the GSVs are retained within unstimulated cells by the action of TUG (tether containing UBX domain or GLUT4-Ubiquitin Like 1), Ubc9 (SUMO-conjugating enzyme 9), and other proteins.356 Insulin acts at several sites to promote GLUT4 translocation—including GSV assembly, release from intracellular retention sites, and transport and fusion at the plasma membrane. The PI3K→AKT cascade plays a key role in this process.349 Important understanding came from the identification of a 160kD AKT substrate originally called AS160 (TBC1D1, TBC1 domain family member 1) and TBC1D4.359 AS160 is a GTPase-activating protein that

maintains its target RAB proteins—including RAB8A, RAB10, RAB14, and others—in an inactive GDP-bound state under basal conditions. AS160 contains AKT phosphorylation sites that inhibit the GAP activity to promote the accumulation of the active RAB-GTP through the action of guanine exchange factor DENND4C (DENN/ MAP-kinase activating death domain containing 4C).349,356 Activation of RAB8A, 10, or RAB14 on GTP binding promotes dissociation of the GSVs from TUG to facilitate its relocation (see Fig. 33-9).356 AKT also phosphorylates and inactivates RALGAP, which leads to the accumulation and activation of RAL-GTP, which binds to exocyst components (Sec5 and Exo70) that tether the incoming GSV to the plasma membrane (see Fig. 33-9). Thus, insulin-stimulated AKT plays an important role in the regulation of translocation, docking, and fusion of GLUT4 vesicles with the plasma membrane.356 Insulin also regulates the assembly of the GSVs during endocytosis of GLUT4 from the plasma membrane. By accelerating the formation of GSVs, all the subsequent steps of GLUT4 translocation can be enhanced.360 GRP1 (general receptor for 3-phosphoinositides 1) is a guanine

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Figure 33-9  A mechanism of insulin-stimulated glucose transport. The PI 3-kinase→PDK1→AKT←→mTORC2 branch of the insulin-signaling path-

way is shown. AKT phosphorylates RALGAP, AS160, and GRP1, which mediates various steps of the assembly and trafficking of GSV (Glut4 storage vesicles). GCVs recycle through the trans-golgi network under the control of GRP1•ARF6 signaling. TUG restrains the GSVs until they are released for translocation to the plasma membrane. AKT-mediated phosphorylation of AS160 and RALGAP promote the translocation of GSVs to the plasma membrane. Activation of these pathways by insulin promotes the accumulation of GLUT4-containing vesicles in the plasma membrane, which increases glucose influx.

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nucleotide exchange factor that acts on ARF6 (ADP-ribosylation factor 6), which promotes GLUT4 vesicle recycling through the trans-Golgi network (see Fig. 33-9). Insulin activates GRP1 through AKT-mediated phosphorylation, which leads to the accumulation of activated ARF6 to promote GSV formation. Thus, activation of GRP1 by insulin allows the coordination of multiple steps of GLUT4 recycling to stimulate glucose influx. The atypical protein kinase C isoforms—PKCξ and PKCλ—are also implicated in the regulation of GLUT4 translocation in adipose tissue and muscle.361-363 PKCξ and PKCλ are downstream of the PI3K→PDK1 cascade, but independent of AKT. Constitutively active PKCλ or PKCξ promotes GLUT4 translocation and glucose uptake in adipocytes and muscles in the absence of insulin.361,363-365 Overexpression of inactive PKCλ inhibits insulin-stimulated activation of endogenous PKCλ, which inhibits GLUT4 translocation and glucose influx.361 Microinjection of PKCλ antibodies inhibits insulininduced GLUT4 translocation, and recombinant PKCξ promotes insulin-stimulated glucose uptake.366 Impaired PKCξ activity is associated with obesity-induced insulin resistance in monkeys, and insulin-stimulated activation of PKCξ is reduced in obese patients with insulin resistance.364,367,368

GLUT4 is Essential for Glucose Homeostasis GLUT4 in muscle and adipose tissue is indispensable for glucose homeostasis.349 Specific deletion of GLUT4 from muscle or adipocytes dysregulates glucose homeostasis and promotes insulin resistance to a much greater extent than deletion of insulin-signaling components from these tissues alone.350 Muscle-specific GLUT4–knockout (MG4KO) mice develop hyperglycemia, glucose intolerance, and insulin resistance by 8 weeks of age. Interestingly, these mice also display dysregulated glucose metabolism in adipose and liver. Hyperglycemia owing to diminished muscle glucose use appears to drive the heterologous insulin resistance, because normalization of circulating glucose by promoting its excretion by the kidney can restore insulin action in adipose and liver.350 Deletion of GLUT4 in adipocytes also causes systemic insulin resistance, which is associated with the release of RBP4 (retinol-binding protein 4) into the serum from GLUT4deficient adipose tissue.349 RBP4 might cause insulin resistance by promoting an inflammatory state in adipose tissue through a novel JNK- and TLR4-dependent activation of proinflammatory cytokines in macrophages.369 By comparison, genetic disruption of insulin signaling in muscle—on deletion of the IR or IRS1 and IRS2— produces minor effects on systemic glucose homeostasis and circulating insulin. Without insulin signaling, basal muscle glucose influx shows an increase, owing at least in part to energy depletion and activation of AMPK, which provides an alternative pathway to AS160 phosphorylation and insulin-independent GLUT4 translocation to the plasma membrane (see Fig. 33-9).193 This unexpected result is consistent with the effect of transgenic overexpression of GLUT4 in muscle or adipose tissue to lower circulating insulin concentration while improving glucose tolerance.350

MECHANISMS OF INSULIN RESISTANCE Whereas the consequences of insulin resistance have been studied in detail with genetically modified mice, other heterologous mechanisms cause insulin resistance associated with metabolic disease. Several pathologic mechanisms associated closely with obesity, inactivity, and overnutrition—including glucotoxicity, lipotoxicity, oxidative stress, endoplasmic reticulum stress, and amyloid deposition—have emerged to explain the relationship between insulin resistance, hyperglycemia, hypertension, increased proinflammatory and cytokine signaling, kidney dysfunction, neurodegeneration, and the progression of β-cell dysfunction to type 2 diabetes.7,370 Regardless, establishing the mechanism that conspires to cause type 2 diabetes and its life-threatening sequela in humans and animals continues to be a complex problem.7 Clinical staging of type 2 diabetes suggests that early insulin resistance in skeletal muscle—the primary site of glucose disposal in humans—is important in disease initiation.162,371,372 Compatible with this hypothesis, healthy human subjects that display skeletal muscle insulin resistance exhibit decreased incorporation of glucose into muscle glycogen, which parallels increased liver triglyceride synthesis.21 However, animal models of pure skeletal muscle insulin resistance—including mice that lack the insulin receptor or IRS1/2 in muscle—show surprisingly mild phenotypes.169,193 By contrast, many but not all features of type 2 diabetes and the associated metabolic syndrome are recapitulated by deletion in hepatocytes of the insulin receptor, IRS1/2 or AKT1/2.171,173,190,240 These and other experiments support the importance of tissue selective insulin resistance in diabetes etiology.183

Nutrient Excess and Insulin Resistance Nutrient excess can promote the development of type 2 diabetes as the organism struggles with sustained insulin secretion and dysregulated metabolic use and storage. The progressive rise in compensatory insulin concentrations during nutrient excess promotes negative feedback to the insulin-signaling cascade (see Fig. 33-8).296 The accumulation of excess triglycerides within insulin sensitive tissues can inhibit signaling through heterologous mechanisms that inhibit IR→IRS1/IRS2 signaling through Ser/Thr phosphorylation or ubiquitin-mediated degradation (see Figs. 33-7 and 33-8).270,282,373,374 Increased adipose tissue mass is also associated with macrophage infiltration into white adipose tissue depots, which promotes the production of inflammatory cytokines that lead to insulin resistance.162 Serum branched-chain amino acids in conjunction with protein overloads can promote insulin resistance in humans and rodents owing at least in part to elevated mTORC1 activity.375 Increased activation of the mTOR pathway because of nutrient excess drives synthetic processes that can promote endoplasmic reticulum (ER) stress-mediated insulin resistance (see Fig. 33-8). UPR activation owing to mTORC1 hyperactivation can also inhibit IR→IRS1 signaling associated with Ser/Thr phosphorylation of IRS and other mechanisms.290 The ER is a luminal network of interconnected membrane-enclosed tubes that are continuous with the outer

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membrane of the nuclear envelope. Rough ER is coated with ribosomes where protein synthesis is especially prominent; smooth ER lacks ribosomes and functions in lipid and cholesterol biosynthesis, carbohydrate metabolism, and detoxification.376,377 Successful protein folding requires a tightly controlled environment of substrates, including glucose, calcium, and redox buffers that maintain the oxidizing environment required for disulfide bond formation.378 The unfolded protein response (UPR) is activated when circumstances disrupt protein folding, including glucose and energy deprivation, viral infection, increased protein trafficking, the accumulation of unfolded proteins, cholesterol accumulation, or exposure of test cells to chemical agents such as tunicamycin and thapsigargin.376,377 Hyperactivation of mTORC1 owing to nutrient excess and hyperinsulinemia also promotes ER stress through the sustained flux of newly synthesized proteins through the ER lumen. Distinct branches of the UPR are initiated by two typeI transmembrane kinases PERK (PKR-like endoplasmic reticulum kinase) and IRE1 (inositol requiring enzyme-1), and by a type-II transmembrane protein ATF6 (activating transcription factor-6).376,377 PERK reduces general translation in cells thereby reducing this branch of the insulin response to suppress protein influx into the ER. PERKmediated phosphorylation of eIF2 contributes to accumulation of lipids in the liver, which can promote insulin resistance.379 Activation of JNK by IRE1 signaling in the liver and adipose tissues might cause insulin resistance resulting from serine phosphorylation of IRS1 or other substrates.380 ATF6 upregulates expression of the XBP1 (X-box binding protein 1) transcription factor whose products increase the protein-folding capacity of the cell to compensate for the consequence of protein overloads. Mice lacking one allele of XBP1 develop higher levels of ER stress, obesity, and insulin resistance.380 Furthermore, when ER stress is attenuated in the obese and diabetic mice through the use of chemical chaperones—agents that have the ability to increase ER-folding capacity—insulin resistance decreases, glucose tolerance improves, and blood glucose concentrations approach the normal range.381 The presence of ER stress in the liver and fat tissues of obese patients strengthens the conclusion that this system plays a role in the development of insulin resistance.382 Although decreased ER stress likely plays an important role to normalize the insulin response, ATF6→XBP1 also interacts with FOXO1 to direct it toward proteasome-mediated degradation, which can contribute to the restoration of the insulin response.383

Inflammation and Insulin Resistance Components of the immune system are altered during obesity, insulin resistance and type 2 diabetes with the most apparent changes occurring in adipose tissue, liver, and muscle, but also in pancreatic islets, cadiovascular system, and circulating leukocytes.7,384 Although adipose tissue is not the primary site of glucose disposal, it is the major site of energy storage that can inform the CNS and other tissues about energy availability through the secretion of various adipokines. Increased triglyceride storage during obesity gives rise to an expanded adipose tissue

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compartment that can be compromised by hypoxia and adipocyte death, which leads to a microenvironment conducive to macrophage infiltration.385-387 Proinflammatory “M1” macrophages accumulate in adipose tissue during obesity, whereas noninflammatory “M2” macrophages accumulate in the adipose tissue of lean animals.388 M2 macrophages mediate lipid trafficking through the lysosomes to recycle excess lipids into the circulation. They also remove dead cells to maintain adipose tissue homeostasis without promoting inflammation.386 Chronic obesity decreases (adiponectin) and increases adipokine (leptin, resistin, RBP4) secretion that can dysregulate systemic metabolism through myriad mechanisms.389 The “M1” macrophages contribute to local and circulating concentrations of proinflammatory cytokines, including monocyte chemoattractant protein (MCP1), interleukin 6 (IL6), and TNFα.390 There can be extensive crosstalk between these mechanisms because adipokines affect inflammation and lipid deposition in tissues.391 Some features of in vivo adipose insulin resistance can be captured by various in vitro models, including treatments with TNFα, hypoxia, dexamethasone, high insulin, or a combination of TNFα and hypoxia in differentiated 3T3L1 adipocytes.373 Insulin resistance in visceral adipocytes can give rise to circulating free fatty acids, which contribute to insulin resistance.387 Excess triglycerides that are not stored in adipose tissues accumulate and dysregulate lipid flux within muscle, liver, and other tissues. A variety of clinical and experimental data supports the hypothesis that intramyocellular triglyceride (IMTG) causes skeletal muscle insulin resistance in humans and rodents.392 The accumulation of DAG and/or ceramide can impair insulin signaling by heterologous pathways involving IRS1 (and possibly IRS2) Ser/Thr-phosphorylation, direct inhibition of AKT, or other signaling pathways (see Fig 33-8).385,390,393 Infusion of triglyceride into humans or rodents—which transiently increases circulating free fatty acid concentrations—inhibits insulin-stimulated glucose disposal into muscle by a mechanism associated with decreased IRS1associated PI3K activity.307,394,395 In mice and rats, this resistance is associated with increased muscle S307IRS1 phosphorylation,307 which—based on studies of knockout mice—occurs via PKCθ- and IKKβ-dependent pathways.317,396 In liver, PKCε functions similarly to PKCθ in muscle,393 and PKCε might contribute to the loss of inhibition of hepatic gluconeogenesis by insulin caused by FFA as evident in human studies.397,398 Elevated cellular diacylglycerols (DAG) owing to decreased fatty acid oxidation or increased triglyceride synthesis activate both PKCθ and PKCε.393 In addition to its glycerol-based counterpart DAG, ceramide correlates with impaired insulin action in muscle.

Proinflammatory Cytokines Obesity and its progression to diabetes are associated with the secretion of the proinflammatory cytokines, including resistin, TNFα, and IL6.20 Epidemiologic data show that elevated circulating IL6 correlates with adiposity in humans.399 IL6 is generally thought to promote systemic insulin resistance, especially during obesity, because it

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is secreted from fat cells of insulin-resistant humans.399 However, in type 2 diabetes patients, the plasma concentrations of IL6 and TNFα might only reflect the level of adiposity rather than the insulin sensitivity.400 INTERLEUKIN 6. Contradictory and multisystemic effects under various physiologic states complicate the understanding of how IL6 ­regulates central and peripheral nutrient homeostasis.401 IL6 is best known as a proinflammatory cytokine that regulates the immune response, the acute phase response (APR), and hematopoiesis.402 In response to physical trauma, burns, or bacterial infection, IL6 is secreted from T cells and macrophages, which mobilizes substrates in muscle and adipose tissues to provide substrates and energy to fight infection and to repair tissue.403-405 Adipose and skeletal muscles are also important sites of IL6 production, suggesting that IL6 includes hormone-like effects to integrate peripheral physiology with nutrient homeostasis.406 In rats, the central effects of IL6 resemble those of leptin, suggesting that IL6 can regulate nutrient homeostasis.401,407 Epidemiologic data suggest that elevated circulating IL6 in humans correlates with adiposity.399,408-410 IL6 is produced in greater quantity by fat cells from insulin-resistant humans, suggesting that it might promote systemic insulin resistance, especially during obesity. Infusion of murine IL6 causes hepatic and muscle insulin resistance in mice that is associated with reduced insulin signaling.411 Regardless, IL6 also displays antiinflammatory characteristics by inhibiting TNFα and IL1, and by activating IL10.412-415 In mice, IL10 can reverse the effects of IL6 and can promote insulin sensitivity.411 Moreover, adipose-derived IL6 can have autocrine effects that increase leptin secretion and fat oxidation, and can reduce the expression and activity of lipoprotein lipase in human adipose tissues—which might attenuate the progression of obesity.416 Infusion of recombinant hIL6 to sustain physiologic concentrations in healthy individuals or patients with type 2 diabetes increases lipolysis in the absence of adverse effects.417,418 Chronic overexpression of hIL6 in mice reduces daily food consumption and promotes energy expenditure.419 Consistent with the reduced adiposity, circulating insulin decreases and glucose tolerance improves, confirming that hIL6 promotes systemic insulin sensitivity especially in animals on an HFD. Moreover, circulating leptin and daily food consumption decrease, suggesting that hIL6 improves central leptin sensitivity or action. Although the IL6-stimulated STAT3 to SOCS3 cascade inhibits IR→IRS1 signaling, it prevents the loss of IR→IRS2 signaling in mice on an HFD, which is consistent with improved systemic glucose tolerance.419 In humans, physical activity promotes the production and release of IL6 from skeletal muscle.401,420 Muscle-derived IL6 appears to promote metabolic homeostasis during periods of altered demand such as muscular exercise or insulin stimulation.421 In humans, IL6 might thus mediate at least in part the effect of exercise to improve insulin sensitivity and glucose tolerance.422 Tumor necrosis factor-α. TNFα activates signaling cascades in insulin-sensitive tissues that result in the activa-

tion of JNK1 (Jun N-terminal Kinase-1) and IKKβ (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta).390 Both of these kinases are implicated in multiple mechanisms of insulin resistance.7 Although increased circulating TNFα is generally accepted as a cause for insulin resistance, there is substantial disagreement about the target tissues and whether the increase contributes to insulin resistance in humans.390 Improved insulin sensitivity reported for mice lacking TNFα is not, in at least one report, shared by mice that lack the two TNFα receptors.423 Similarly, the administration of a highly effective TNFα inhibitor to insulin-resistant humans for 3 months does not improve glucose homeostasis, despite the reduction in some parameters of systemic inflammation.424 Thus, despite some strong experimental evidence, the physiologic relevance of TNFα as a cause of insulin resistance in humans remains unclear. Regardless, TNFα and FFAs activate JNK1 and IKKβ, which are associated with obesity and insulin resistance. JNK1-deficient mice are protected from obesity and insulin resistance caused by HFD feeding, indicating an important role of this kinase in metabolic dysregulation.313 Inhibition of JNK activities in obese mice using kinase inhibitors increases insulin sensitivity and improves glucose homeostasis425; however, the role of JNK1 in metabolic regulation appears to be more complex, as liver-specific knockdown of JNK1 lowers circulating glucose and insulin concentrations while increasing triglyceride levels in mice on an HFD.426 Moreover, tissue-specific JNK1 ablation in many peripheral tissues— including adipose, liver, muscle, and myeloid—does not reduce obesity.325 Unexpectedly, genetic experiments focusing on the CNS suggests that JNK1 promotes obesity owing to the reduced energy expenditure associated with increased DIO2 (iodothyronine 5’-deiodinasegene). DIO2 negatively regulates HPT (hypothalamic-pituitarythyroid) axis-mediated energy expenditure. HFDs cause JNK-mediated DIO2 expression, which decreases HPT axis and promotes obesity.427 Although chronic inflammation is associated with metabolic dysregulation, the exact mechanism involved remains difficult to establish.

INSULIN/IGF SIGNALING AND NEURODEGENERATIVE DISEASE Systemic metabolic dysregulation and insulin resistance might contribute to the progression of adult onset neurodegenerative diseases—including Alzheimer’s (AD), Parkinson’s (PD), Huntington’s (HD), and others.428-430 AD is the most common form of dementia, accounting for 50% to 70% of all cases leading to the loss of cognitive abilities progressing to death.431 AD is characterized by the formation of extracellular β-amyloid plaques and neurofibrillary tangles. The plaques are composed of aggregated amyloid-β (Aβ), a 39 to 42 amino acid peptide generated by the proteolytic cleavage of APP (amyloid polypeptide) by β-secretase and γ-secretase.432,433 Based on current projections, 11 to 16 million Americans might develop Alzheimer’s disease by 2050.434 Inspection of an Alzheimer’s disease patient registry (Mayo Clinic) suggests that 80% of AD patients exhibit glucose intolerance

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or diabetes.435 Epidemiologic studies suggest that diabetes increases by 50% to 100% the risk for progressive AD.415 Normal aging is associated with declining IGF1 levels and rising insulin levels owing to progressive peripheral insulin resistance.34,437 Type 2 diabetes is associated with many pathologic features associated with progressive AD—including vascular lesions, cardiovascular disease, hyperinsulinemia, and glucose intolerance; dysregulated insulin-signaling, glucose metabolism, hypercholesterolemia, and oxidative stress; abnormal protein processing, accumulating advanced glycation end products; elevated brain concentration of Aβ, τ phosphorylation, and oxidative stress; and the activation of inflammatory pathways.434 Whether neurodegeneration is a direct result of central insulin resistance or a consequence of peripheral metabolic disease is difficult to resolve. Whether the restoration of insulin-like signaling in the CNS can slow the progression of neurodegeneration and AD is an important question of intense scientific and clinical interest. Nasal administration of insulin can improve cognitive function in AD patients.438-442 Studies in rats demonstrate that infusion of IGF1 protects from Aβ-mediated toxicity.443 Injection of IGF1 into AD mice reduces the typical behavioral impairments associated with increased concentrations of Aβ. Regardless, the relationship between insulin signaling and neurodegeneration remains an open question, because reduced insulin/IGF1 signaling slows aging, which is the major risk factor for the development of neurodegenerative disorders in lower organisms and rodents.444,445 IGF1R heterozygous mice have a longer life span and are more resistant to oxidative stress.425,426 Reduced insulin-like signaling protects C. elegans from the toxic effects of aggregated amyloid β (Aβ) in daf-16 and HSF-1-dependent manners, and long-lived age-1 mutants show improved thermotaxis and learning with age.448,449 Inactivation of Drosophila CHICO (IRS1 ortholog) attenuates the progression of age-related decline in motor function, suggesting that reduced insulin-like signaling might prevent CNS decline.450,451 Female fruit flies live up to 80% longer when the activity of the Drosophila insulin-like receptor is reduced by 80%.450 Although the long-lived flies show resistance to oxidative stress, they also accumulate excess lipids and carbohydrate.452 Thus, reduced insulinlike signaling can extend the life span in lower metazoans, while causing physiologic alterations that are associated with life-threatening diseases in rodents and humans. Insulin-Receptor Signaling in CNS-Regulated Metabolism Insulin receptors are expressed throughout the CNS where its signaling cascade mediates important metabolic effects.453 Intracerebroventricular infusion of insulin reduces food intake and body weight, and CNS insulin resistance is associated with overweight phenotypes in rodents and primates.454,455 The role of insulin signaling in the brain is not uniform, because insulin can produce distinct physiologic effects in various regions. To establish the function of brain insulin signaling, the insulin receptor has been deleted throughout the CNS using the nestin-regulated Cre transgene (NIRKO mice), or in specific neuronal populations using various targeted Cre transgenes.164 NIRKO mice display a normal life span

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while developing many features of the insulin-resistance syndrome—including mild obesity, hypertriglyceridemia, hyperinsulinemia, and infertility. Male and female NIRKO mice have a larger fat pad mass; however, only females consume more chow and display greater body mass.456 Moreover, CNS insulin signaling is essential for fertility, as NIRKO mice of both sexes show low concentrations of circulating follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and female NIRKO mice display few antral follicles and corpora lutea.456 Nutrient homeostasis and fertility are controlled by hypothalamic signals, so the role of insulin receptors in the arcuate nucleus has been investigated by targeted deletion. POMC (pro-opiomelanocortin) and AgRP (Agouti-related protein) neurons play an important role in modulating satiety. In the postprandial state, POMC neurons release α-MSH (α-melanocyte-stimulating hormone), which activates MC3/4R (melanocortin 3 and 4 receptors) on neurons in the PVN (hypothalamic paraventricular nucleus). The activation of this circuit reduces food intake, increases energy expenditure, and reduces hepatic glucose production. During fasting, AgRP neurons suppress MC4R activity to increase feeding, inhibit energy expenditure, and regulate glucose metabolism.454 The deletion of insulin receptors in AgRP neurons—but not POMC neurons—reduces hepatic glucose production, at least in part, through efferent vagal innervation of the liver.455,457 However, this mechanism might be species specific, as hypothalamic insulin signaling in dogs suppresses net hepatic glucose output by augmenting hepatic glucose uptake and glycogen synthesis, rather than inhibiting gluconeogenesis.458 Regardless, deletion of insulin receptors in either neuronal subtype of mice has no effect on the feeding behavior or energy expenditure.459 Thus central insulin signaling alone does not modulate food intake and energy homeostasis in healthy mice.454 Moreover, deletion of insulin receptors in the VMH (ventromedial hypothalamus) using SF1cre (cre recombinase regulated by the steroidogenic factor-1) suggests that other regions of the hypothalamus also contribute to the effects of insulin on satiety and energy use.460 Systemic insulin sensitivity is closely associated with female fertility. Indeed, male and female NIRKO mice display low LH and FSH levels that are associated with reduced fertility; however, the effect of genetic neuronal insulin resistance is inconsistent with the physiology of PCOS (polycystic ovarian syndrome)—one of the most common causes of human female infertility— which is associated with elevated LH levels, metabolic insulin resistance, and compensatory hyperinsulinemia. Unexpectedly, deletion of insulin receptors in pituitary gonadotrophs improves reproductive function of obese/ hyperinsulinemia mice.461 Although based on NIRKO mice central insulin signaling is essential for fertility, exacerbated pituitary insulin signaling owing to compensatory hyperinsulinemia during metabolic insulin resistance might promote PCOS.

Insulin/IGF1→IRS2 Integrates Longevity and Metabolism Conventional wisdom suggests that insulin/IGF1 signaling promotes cognitive function in humans and animals.

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However, experiments in mice generally point to the brain as the site where reduced insulin-like signaling has a consistent effect to extend mammalian life span—as it does in worms and flies.3,462 The insulin/IGF→IRS2 cascade in the brain provides a link between life-span extension, metabolic regulation, and cognition. Reduced IRS2 signaling in all tissues or just in the brain (IRS2L/L•creNestin) increases by nearly 6 months the life span of mice maintained on a high-energy diet.463 However, brain-specific, long-lived IRS2 knockout mice consume approximately the same or slightly more food than the short-lived controls. At 22 months of age, brain IRS2 knockout mice are overweight, hyperinsulinemic, and glucose intolerant.463 Thus, less brain IRS2 signaling is associated with increased life span regardless of the peripheral insulin sensitivity and weight gain. The impact of altered insulin/IGF1 signaling on aging and metabolism in the brain might prove to be related to its action in separate sets of neurons. For nutrient homeostasis, a great deal of attention has been paid to distinct populations of arcuate (ARC) nucleus neurons that express POMC or AgRP; however, deletion of IRS2 or IR from these neurons in mice negligibly impacts metabolism.464-466 Similarly, disruption of the PI3K→AKT─┤FoxO1 pathway in POMC and AgRP neurons presents minor effects on energy balance and glucose homeostasis.467-469 By comparison, deletion of the IR from SF1 (steroidogenic factor 1) neurons—a specific set of neurons of the VMH—promotes modest weight gain on an HFD without altering energy balance or insulin sensitivity in chow-fed animals.460 Although insulin signaling in tyrosine hydroxylase (TH)-expressing catecholaminergic neurons is important for the control of mesolimbic dopamine (DA) signaling, lack of IR signaling in these neurons also fails to change measures of adiposity or glucose homeostasis.470 Thus, the relevant site at which insulin signaling contributes to metabolic regulation must be a distinct or broader set of neurons. LEPRB (long-form leptin receptor) neurons include those that express either AgRP, POMC, SF-1, TH (thyroid hormone), as well as other metabolically important neurons in the ARC, and elsewhere.471 Deletion of IRS2L/L from LEPRB neurons dysregulates gene expression within the hypothalamic melanocortin system—which increases feeding, decreases energy expenditure, and promotes insulin resistance. Whereas the neurons that mediate the metabolic effects of IRS2 signaling in the brain are by definition the same ones that mediate leptin actions, IRS2 is not required for leptin action.465 Deletion of IRS2 and interference with insulin/IGF1 signaling in LEPRB neurons does not alter the thyroid, adrenal, or reproductive axes.472 Whereas leptin might promote some IRS2→PI3K signaling,473 leptin signaling is largely normal in the absence of IRS2. Thus IRS2 functions primarily to mediate insulin/ IGF1 signaling in the leptin-responsive neurons, rather than functioning as a direct mediator of LEPRB signaling. Indeed, whereas the hypothalamic application of PI3K inhibitors or the genetic blockade of PI3K in some leptinresponsive neurons impairs the acute anorectic response to leptin, these manipulations have little effect on baseline leptin action or energy balance.140-142 Thus, although IRS2

signaling in LEPRB neurons is crucial for metabolic signaling, it does not appear to play a direct role in leptin action. Moreover, there is no reduction in the life span of mice lacking IRS2 in LEPRB neurons (Myers MG, Miller RA, and White MF, unpublished data). Thus neurons that mediate metabolic effects of the insulin/IGF1 signaling from CNS appear to be distinct from those that mediate IRS2 effects on the life span.

The Protective Effects of Reduced Insulin/IGF1→IRS2 Signaling in Mouse Models of AD Tg2576 transgenic mice expressing the human APP (amyloid precursor protein) gene carrying the Swedish mutation (APPsw) were used to investigate the role of reduced insulin/ IGF signaling in AD. Deletion of IGF1R from the hippocampus of Tg2576 mice—by using the cre-recombinase under control of the synapsin-1 promoter (Tg2576•IGF1RL/L •creSyn1)—completely prevented APPsw lethality. Interestingly, transgenic AD mice heterozygous for the IGF1R are long-lived and stress-resistant. They are protected from AD-associated memory and orientation impairments, and they display reduced neuroinflammation, neuronal, and synaptic losses.474 The protective mechanism involves the appearance of smaller and highly dense Aβ plaques in the IGF1R+/- brain. Thus less IGF1R appears to protect the brain by sequestering highly toxic Aβ oligomers.474 Tg2576 transgenic mice were also crossed with mice lacking IRS2 to generate AD animals that lack the IRS2 gene (Tg2576•IRS2−/−). IRS2 deletion causes a significant reduction in Aβ plaque burden in the brain. Furthermore, Tg2576•IRS2−/− mice exhibit improved learning and memory when compared with the Tg2576 controls.475 In a separate study with Tg2576 mice, the deletion of IRS2 reduces the premature lethality of female Tg2576 mice; however, male Tg2576•IRS2−/− mice have a shortened life span owing to hyperglycemia caused by β-cell failure. This example illustrates how IRS2 signaling might show opposing effects in the CNS compared against the peri­pheral tissues, which must be integrated properly to prevent disease and to promote a normal life span.

Reduced Insulin/IGF1→IRS2 Signaling in Huntington’s Disease Compared to the lengthy degenerative processes associated with mortality in AD, neurodegeneration in Huntington’s disease (HD) progresses at a faster pace and predictably leads to death from a single cause.476 HD results from an expanded CAG triplet repeat in the HD gene that encodes a mutant form of huntingtin (HTT). Mutant HTT produces intranuclear and cytoplasmic inclusions that kill cells in striatum and cerebral cortex— although other areas are also affected.477 As HD and AD arise from dysregulated protein aggregation, both diseases appear to provide suitable models to investigate the effects of the insulin/IGF1→IRS2 signaling on degenerative processes in the CNS. The R6/2-mouse is a well-studied model of HD.478,479 Increasing IRS2 levels in the brains of R6/2 mice by twofold accelerates neurodegeneration and significantly reduces the life span.480 By contrast, reducing IRS2 levels throughout the body—except in β cells where IRS2 expression is needed to prevent diabetes

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onset—extends the life span of the R6/2 mice.480 Thus, modulating IRS2 signaling can influence the progression of Huntington’s disease. The predictable progression of HD in R6/2 mice provides an opportunity to investigate the molecular relationship between insulin-like signaling and neurodegeneration. Mitochondrial dysfunction and oxidative stress increase significantly in the CNS of R6/2 with elevated IRS2 levels, showing a consequence of excess insulin/IGF signaling on acute neurodegeneration and early death of HD mice.480 By comparison, the slower progression of HD-like symptoms in R6/2 mice with less IRS2 expression is associated with increased nuclear localization of the transcription factor FOXO1, which increases the expression of several target genes, including Ppargc1α and SOD2 that have strong positive effects on energy homeostasis and attenuation of oxidative stress.481,482 Whether neuronal mitochondrial damage and oxidative stress during normal aging is modulated by IRS2 signaling is an important question to investigate, as it can inform strategies to attenuate the progression of debilitating neurodegenerative disease in an aging population. PolyQ-HTT causes pathologic sequela in R6/2-mice that develop progressively until death, including abnormal behavior and movement owing to the accumulation of protein aggregates in the CNS, mitochondrial dysfunction and oxidative stress, and neuroinflammation.479,483,484 Aggregate accumulation is thought to reflect cellular damage, as cells that form aggregates tend to die.485 R6/2 mice with reduced IRS2 signaling display more autophagosomes with fewer polyQ-HTT aggregates.480,486 This protective effect of reduced IRS2 is associated with increased nuclear FOXO, which can increase the expression of genes that mediate autophagy and promote autophagosome formation. Thus, increased autophagy might be responsible for the reduced HTT aggregates and slower HD progression in mice with reduced IRS2 signaling. Whereas increasing insulin signaling in peripheral tissues might be necessary to avoid the consequences

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of insulin resistance—hyperglycemia, dyslipidemia, and cardiovascular disease—it is critical to understand detrimental and life-threatening consequences of insulin/ IGF1→IRS2 signaling in the brain.

SUMMARY AND PERSPECTIVES The investigation of the insulin-signaling cascade shows a highly integrated multisystemic network that extends far beyond the classic insulin target tissues of liver, muscle, and adipose. It is now clear that insulin-like signaling plays a major role in pancreatic β-cells and in the central nervous system. Peripheral insulin resistance is ordinarily opposed by increased insulin secretion from pancreatic β-cells. Although this compensatory response can prevent the effects of acute hyperinsulinemia, it might include negative consequences in the vasculature and the brain. The tools now available to probe the insulin-like signaling cascades in healthy and diabetic tissues provide a rational platform to develop new strategies to treat insulin resistance and to prevent its progression to type 2 diabetes and its life-threatening sequela. Understanding how the insulin/IGF→IRS cascade integrates the conflicting signals generated during insulin and cytokine action might be a valuable starting point. Whether better management of inflammatory responses can attenuate insulin resistance and diminish its sequela continues to be a fruitful direction for investigation in humans.487 Future work must better resolve the network of insulin responses that are generated in various tissues, because too much insulin action might also shorten our lives.

  

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