Receptor/Ligand Binding

Insulin binds with high affinity (K_d < 0.5 nM) to the homodimeric IRB, which predominates in the classic insulin target tissues—adult liver, muscle and adipose tissues (see Fig. 8-2). IRB is selective for insulin because its K_d for IGF-1 and IGF-2 is at least 50- to 100-fold higher. Adult liver and adipose are purely insulin-responsive tissues, expressing IRB but lacking IGF1R.⁷ By comparison, IRA predominates in fetal tissues, the adult central nervous system, and hematopoietic cells.^49–52 IRA binds insulin almost as well as IRB but also binds IGF-2 with moderate affinity (see Fig. 8-2). IGF-1 and IGF-2 bind with high affinity (K_d < 1 nM) to the homodimeric IGF1R and to the hybrid receptors (IGF1R::IRA and IGF1R::IRB), whereas insulin barely binds to these proteins (see Fig. 8-2).^53,54 Thus, under ordinary conditions, insulin never activates the IGF1R tyrosine kinase, whereas IGF-1 and IGF-2 can activate the IR tyrosine kinase, especially when it forms a hybrid with the IGF1R.

Site-directed mutagenesis reveals the location of two insulin-binding sites in the α subunit of the insulin receptor.^55,56 Chimeric receptors between the α subunits of the insulin and IGF-1 receptors confirm that one of the sites is located within the first leucine-rich (L1) region of the insulin receptor.⁵⁷ Photo-affinity labeling of mutant insulin receptors reveals the second insulin binding site in the FnIII1 region.⁵⁸ High-affinity insulin binding depends upon α subunit dimerization in the IR tetramer, insofar as monomeric α subunits bind insulin with low affinity. Insulin bound to the IR tetramer at high affinity dissociates slowly unless the concentration of soluble insulin increases, explaining how the binding of a second insulin molecule to a low-affinity site can reduce the affinity of the first.^59,60

Insulin binding apparently begins through interactions between the S1 site on insulin and the FnIII1 region in the α subunit.³² Sixteen amino acid residues at the COOH-terminus (CT) of FnIII2 interacts with the L1::CR-region to create a composite insulin binding site—L1::CR::CT—that interacts with the classical receptor binding surface on insulin (S2) (see Fig. 8-1C). Although the α subunits are arranged symmetrically in the dimer, there is a sharp bend between the L2 and FnIII1 regions that juxtaposes the L1::CR::CT domain antiparallel to FnIII1 (see Fig. 8-1C).^61–63 Owing to this arrangement of α subunits, insulin binds to the L1::CR::CT domain of one α subunit and to FnIII1 of the adjacent α subunit, creating the cross-link that activates the kinase. Space constraints allow only one insulin molecule to bind with high affinity.^40,61,63 Inclusion of exon 11 in IRB lengthens the CT-region by 12 amino acids, which modifies the L1::CR::CT domain to exclude IGF-2 binding and increase the insulin-binding affinity.

Insulin Receptor Tyrosine Kinase

The tyrosine kinase activity of the insulin receptor was originally found by biochemical experiments using [³²P]-labeled hepatoma cells or partially purified insulin receptors incubated with insulin and [γ³²P]ATP.64–66 However, until the insulin receptor cDNA was isolated and sequenced,^34,35 many other mechanisms for signal transduction continued to be investigated.^67,68 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—supported the central role of tyrosyl phosphorylation in the mechanism of insulin action.⁶⁹

The intracellular portion of the insulin receptor β-subunit is composed of three distinct regions that contain tyrosyl phosphorylation sites: Y₉₆₅ and Y₉₇₂ in the juxtamembrane region (IRB) between the transmembrane helix and the cytoplasmic tyrosine kinase domain; Y₁₁₅₈, Y₁₁₆₂, and Y₁₁₆₃ in the activation loop (A-loop) of the core catalytic domain (IRB); and Y₁₃₂₈ and Y₁₃₃₄ in the COOH-terminus (IRB).41,70,71 The unphosphorylated Tyr₁₁₆₂ folds into the catalytic site to restrict ATP binding, which explains the high apparent K_m for ATP before insulin stimulation.^72,73 However, the closed A-loop is in equilibrium with an alternate conformation that allows occasional access by ATP to mediate basal autophosphorylation.⁷⁴ Infrequent oscillation from the inactive to the active conformation might be coupled to complementary changes in the α subunits, which are definitively stabilized upon insulin binding to accelerate ATP entry and autophosphorylation of Tyr₁₁₆₂ (Fig. 8-3A). Once initiated, the autophosphorylation cascade progresses rapidly to Tyr₁₁₅₈, resulting in a bis-phosphorylated and active kinase.⁷⁰ Although autophosphorylation of Tyr₁₁₆₃ is relatively slow, the resulting tris-phosphorylated A-loop stabilizes the open conformation to allow unrestricted access by Mg-ATP and protein substrates (see Fig. 8-3A). Autophosphorylation probably occurs through the interaction between adjacent β subunits, since the reaction progresses through first-order kinetics.⁶⁶ This model of kinase regulation is validated by activation of the kinase via substitution of Asp₁₁₆₁ in the middle of the A-loop with Ala₁₁₆₁—which shifts the steady-state conformation of the unphosphorylated A-loop toward the open configuration.⁷⁴ Substitution of Tyr₁₁₆₂ with phenylalanine also increases basal autophosphorylation, consistent with its role in stabilizing the closed conformation.⁷⁵

Regulation of the insulin receptor kinase is modulated by heterologous protein interactions with the phosphorylated A-loop. At least two phosphotyrosine residues in the A-loop are completely solvent-exposed, creating sites that bind to various src-homology-2 (SH2)-domain-containing proteins, including APS and Grb10/14.⁷⁶ Structural analysis reveals how the dimerized Src homology-2 (SH2) domains of APS can bind to the A-loop of adjacent β subunits to stabilize the active conformation (see Fig. 8-3B).⁷⁷ By contrast, binding of the dimeric SH2-domains of Grb10 or Grb14 to adjacent A-loops orients a 40-amino-acid motif into in the catalytic site, which inhibits the IR kinase.⁷⁸ These structural predictions are consistent with gene deletion studies that confirm tissue-specific negative regulation of the insulin receptor by Grb14.⁷⁹ Variable expression of these or other regulatory proteins—due to genetic variation or physiologic or environmental challenge—should be expected to modulate insulin signaling and glucose tolerance.⁷⁶

Introduction

Following discovery of the insulin receptor tyrosine kinase, many groups searched for insulin receptor substrates that might mediate downstream signals.^80,81 The “substrate” hypothesis was difficult to prove for any receptor tyrosine kinase, because the only known substrates were abundant proteins of unknown physiologic importance. The first evidence for a cellular substrate of a receptor tyrosine kinase came from antiphosphotyrosine antibody immunoprecipitates, which revealed a 185-kD phosphoprotein (pp 185) in insulin-stimulated hepatoma cells.⁸² This substrate seemed to be biologically important because it was phosphorylated immediately after insulin stimulation but not phosphorylated by catalytically and biologically inactive insulin receptors. Importantly, a few catalytically active but biologically inactive insulin receptor mutants fail to phosphorylate pp185.⁸³ Together, these data provided the first clue that substrate phosphorylation following receptor autophosphorylation are the initial steps in signal transduction.

Purification and molecular cloning of pp185 revealed one of the first signaling scaffolds and the first insulin receptor substrate, called IRS1.⁸⁴ Four IRS-protein genes exist in rodents, but only three (IRS1, IRS2, and IRS4) are expressed in humans.⁸⁵ IRS1 and IRS2 are broadly expressed in mammalian tissues, whereas IRS4 is largely restricted to the hypothalamus.⁸⁶ The IRS proteins are adapter molecules that link the IR and IGF1R to common downstream signaling cascades and heterologous regulatory mechanisms (Fig. 8-4A). IRS proteins are targeted to the activated receptors through an NH₂-terminal pleckstrin homology (PH) domain and a phosphotyrosine-binding (PTB) domain (see Fig. 8-4A). During insulin and IGF-1 stimulation, tyrosines in the COOH-terminal portion of the IRS proteins are phosphorylated and bind to the SH2 domains in various signaling proteins (see Fig. 8-4A).⁸⁷ The interaction between IRS1 and the 85-kD regulatory subunit (p85) of the class 1A phosphatidylinositol 3-kinase (PI3K) was the first insulin signaling cascade to be reconstituted successfully in cells and test tubes.⁸⁸ In addition to the IRS proteins, the insulin receptor phosphorylates other proteins in various contexts, including Shc, APS, SH2B, Gab1/2, Dock1/2, and Cbl.^89–96 Quantitative mass spectrometry–based analysis of proteins extracted from insulin-stimulated 3T3-L1 adipocytes reveals 122 tyrosine phosphorylation sites on 89 proteins, including IRS1 and IRS2 (see Fig 8-4A).⁹⁷ A wide variety of proteins are revealed by this approach, including components of the trafficking machinery for the insulin-responsive glucose transporter, GLUT4.⁹⁷ Although the role of each of these substrates merits attention, work with transgenic mice reveals that many insulin responses, especially those associated with somatic growth and carbohydrate and lipid metabolism, are initiated through IRS1 and IRS2.³⁶

Insulin Receptor Substrate Recruitment and Phosphorylation Site Selection

The structure of the activated insulin receptor β subunit reveals a mechanism by which the activated IR kinase phosphorylates tyrosine residues within specific amino acid motifs, including the YMXM, YVNI, and YIDL motifs.98–101 These motifs are targeted as antiparallel β strands to the COOH-terminal end of the open A-loop, allowing hydrophobic residues in the Y+1 and Y+3 positions to occupy two small hydrophobic pockets on the activated kinase (see Fig. 8-3A). Tyrosine residues lying within amino acid motifs that contain charged or bulky side chains at the Y+1 and Y+3 positions fit poorly into this site, excluding them from phosphorylation.¹⁰¹

In addition to the phosphorylation of specific tyrosine-containing motifs, selective and regulated recruitment of cellular proteins to the activated IR and IGF1R establishes signaling specificity. In this regard, the phosphorylated NPEY₉₇₂ motif in the juxtamembrane region of the insulin receptor (IRB-numbering) is essential.^83,102 The juxtamembrane region is about 35 residues long and connects the transmembrane helix of the IR β subunit to the kinase domain (see Figs. 8-1B and C). Tyr₉₈₄ in the juxtamembrane region plays an important structural role to maintain the kinase in the basal state.⁷⁸ However, unlike other receptor tyrosine kinases, the insulin receptor kinase is not regulated by autophosphorylation in the juxtamembrane region.^103,104 Phosphorylation of Tyr₉₇₂ creates a docking site for the phosphotyrosine-binding (PTB) domains in the IRS proteins and SHC.⁸³ In intact cells, phosphorylation of the NPXY₉₇₂ motif is among the first sites of insulin-stimulated autophosphorylation.^83,105 The juxtamembrane region appears to compete with the A-loop for access to the catalytic site, supporting a model in which the juxtamembrane region is phosphorylated before tris-phosphorylation of the A-loop.⁷² In this mechanism, substrate recruitment can be initiated before the catalytic domain is maximally activated. The NPXY motif in the IR juxtamembrane region fills an L-shaped cleft on the PTB domain, while the N-terminal residues of the bound peptide form an additional strand in the β sandwich.¹⁰² These interactions can explain why the IR and IGF1R—and a few cytokine receptors, including the IL-4 receptor—select IRS1 for tyrosine phosphorylation.

The IRS proteins also contain a pleckstrin homology (PH) domain that is structurally similar but functionally distinct from the PTB domain.¹⁰⁶ Although the PH domain promotes the interaction between IRS proteins and the IR, it does not bind phosphotyrosine, and its mechanism remains poorly understood. PH domains are generally thought to bind phospholipids, but the PH domains in IRS1 and IRS2 are poor examples of this specificity.^107,108 Regardless, the PH domain in the IRS protein plays an important and specific role because it can be interchanged among the IRS proteins without noticeable loss of bioactivity. However, heterologous PH domains inhibit IRS1 function when substituted for the IRS1 PH domain, confirming a specific functional role.¹⁰⁹

IRS2 utilizes an additional motif located between amino acid residues 591 and 786—especially Tyr₆₂₄ and Tyr₆₂₈—to interact with the activated insulin receptor.^110,111 This binding region in IRS2 was originally called the kinase regulatory loop binding (KRLB) domain because tris-phosphorylation of the IR A-loop was required to observe its interaction.¹¹⁰ Autophosphorylation moves the A-loop out of the catalytic site so the functional part of the KRLB domain—residues 620 to 634 in murine IRS2—can fit into the catalytic site (see Fig. 8-4B-D).¹¹² This interaction aligns Tyr₆₂₈ of IRS2 for phosphorylation; however, it also inserts Tyr₆₂₁ into the ATP-binding pocket, which might attenuate signaling by blocking ATP access to the catalytic site. By contrast, this interaction might promote signaling by opening the catalytic site before tris-autophosphorylation. Interestingly, the KRLB motif does not bind to the IGF1R, which might explain signaling differences between IR and IGF1R, as well as the receptor hybrids.¹¹²

The IRS→PI3K→AKT Cascade

One of the best studied and most important signaling cascades activated by insulin involves the production of phosphatidylinositol lipids by the class 1A phosphatidylinositide 3-kinase (PI3K) (Fig. 8-5). The PI3K plays a role in many signaling cascades. It is composed of a regulatory subunit that contains two src-homology-2 (SH2) domains and a catalytic subunit that phosphorylates the D3 position of the inositol ring.¹¹³ The catalytic subunit is unstable and only detected in association with the regulatory subunit. The PI3K is inactive until both SH2 domains in the regulatory subunit are occupied by phosphorylated YXXM motifs, especially those in the IRS proteins.⁸⁸ 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.^114–116

PI-3,4,5-P₃ produced by the activated PI3K recruits several Ser/Thr-kinases to the plasma membrane, including the 3-phosphoinositide-dependent protein kinase-1 (PDK1) and v-akt murine thymoma viral oncogene (AKT, also known as PKB), where AKT is activated by PDK1-mediated phosphorylation of Thr₃₀₈ in its activation loop. AKT has a central role in cell biology, phosphorylating many proteins that control cell survival, growth, proliferation, angiogenesis, metabolism, and migration (see Fig. 8-5).¹¹⁷ Phosphorylation of several AKT substrates is especially relevant to insulin-like signaling: 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 and the hypothalamus), p21^CIP1 and p27^KIP1 (blocks cell cycle inhibition), eNOS (stimulates NO synthesis and vasodilatation), and PDE3b (hydrolyzes cyclic adenosine monophosphate [cAMP]) (see Fig. 8-5).

AKT directly stimulates cell growth through the activation of mTORC1 complex—composed of the mammalian target of rapamycin (mTOR), mLST8, and raptor—which phosphorylates the S6-kinase and eIF4E-BP1 (also known as PHAS-1) to stimulate protein synthesis (see Fig. 8-5).^117–119 The regulatory mechanism involves several steps, beginning with AKT-mediated 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). RHEB accumulates in its GTP-bound form when TCS1/2 is inhibited by AKT-mediated phosphorylation, which activates mTORC1 (see Fig. 8-5). In one possible regulatory mechanism, FKBP38 (FK506 binding protein 8) binds to mTORC1 and inhibits mTOR until RHEB-GTP binds to FKBP38 to disinhibit the kinase.^120–122 This complex regulatory cascade is augmented by other pathways, including the direct inhibition of the mTOR catalytic site by PRAS40 (the proline-rich AKT substrate of 40 kD) until AKT-mediated phosphorylation of PRAS40 reverses the inhibition.¹¹⁷ The mTORC2 complex is also composed of mTOR and mLST8, but instead of raptor, this rapamycin-insensitive complex contains rictor and mSIN1 and is mainly regulated by nutrients. Regardless of this complexity, the insulin-mediated control of mTORC1 is directly linked to the IRS→PI3K→AKT cascade and plays an important role in cell growth and feedback regulation.

Downstream effectors of mTORC1—p70^S6K and 4E-BP1—control protein synthesis and cell growth. The p70^S6K occurs as two isoforms, p70^S6K1 and p70^S6K2. Disruption of the S6k1 gene in mice causes glucose intolerance due to reduced size of pancreatic islet β cells; however, peripheral insulin action is enhanced, suggesting that p70^S6K1 contributes to feedback inhibition of insulin signaling.¹²³ Activation of p70^S6K1 is accomplished by multisite phosphorylation events from various kinase activities in response to insulin and other mitogens when amino acids are available.^124,125 Nutrient sensitivity might arise when mTORC1 mediates the phosphorylation of a hydrophobic motif needed for interaction of p70^s6k with PDK1.

Systemic Inactivation of Insulin or IGF-1 Receptors

Even though the insulin and IGF-1 receptors have some overlapping functions during development and adult life, complete deletion of the IR or the IGF1R has serious physiologic consequences that cause death shortly after birth.²¹ The IGF-1/2→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.¹²⁶ IGF1R-deficient mice are born 50% smaller than normal littermates and die after a few days, owing to developmental defects.¹²⁷ By contrast, mice lacking the insulin receptor are nearly normal size at birth, except for a reduced adipose tissue mass. Regardless, insulin receptor–deficient mice also die within a few days of birth, owing to severe hyperglycemia.¹²⁶ Mice retaining at least 20% of normal insulin-receptor expression throughout the body can survive, with severe postnatal growth retardation and hyperglycemia that resembles human leprechaunism.²¹ This growth defect might arise, at least in part, from elevated hepatic IGFBP1 that reduces IGF-1 bioavailability. Thus, small mice with severely reduced insulin or IGF-1 signaling have short lifespans due to developmental and metabolic defects. These mouse models reflect many but not all the features of humans with inactivating human insulin-receptor mutations.¹⁹

Insulin Receptor Substrate–Protein Signaling

Irs1 and Irs2 couple the receptors for insulin and IGF-1 to the PI3K→Akt cascade in mice and humans. Deletion of Irs1 produces small, insulin-resistant mice with nearly normal glucose homeostasis, owing to β-cell expansion and compensatory hyperinsulinemia.¹²⁸ Irs1 appears to mediate most of the Igf1 signal for somatic growth. Mice lacking Irs2 display nearly normal growth but develop life-threatening diabetes between 8 and 15 weeks of age as a result of reduced β-cell mass and insufficient compensatory insulin secretion.¹²⁹ Whereas the complete deletion of Irs1 and Irs2 is lethal, littermates retaining one allele of Irs1 (Irs1^+/−::Irs2^−/−) or one allele of Irs2 (Irs1^−/−::Irs2^+/−) can be born alive.¹³⁰ 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. 8-6). 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 age¹³⁰ (see Fig. 8-6). Regardless, the small Irs1^−/−::Irs2^+/− mice are very fragile and require extraordinary care to live beyond this age. Thus, healthy glucose tolerance and small size are not definitive determinants of lifespan.

The central role of IRS proteins in the PI3K→AKT signaling cascade is validated by a wide array of cell-based and mouse-based experiments. Although IRS1 was originally purified and cloned from rat hepatocytes, the principle role of Irs1 and Irs2 during insulin signaling in hepatocytes in vivo was verified only recently.131–134 The simplest experiments employ an intraperitoneal injection of insulin into ordinary mice or mice lacking hepatic Irs1, Irs2, or both. In ordinary mice, insulin rapidly stimulates Akt phosphorylation and the phosphorylation of its downstream substrates, Foxo1 and Gsk3α/β. However, both Irs1 and Irs2 must be deleted before insulin receptors are uncoupled from the PI3K→AKT cascade.¹³⁵ These results confirm the shared but absolute requirement for IRS1 or IRS2 for the hepatic insulin response.

Downstream Insulin Signaling Components

Except for PDK1, the IRS→PI3K→AKT cascade is robust as a result of the expression of multiple isoforms of the proximal components. The type 1_A PI3K is a dimer composed of a catalytic subunit—either p110α, p110β, or p110δ—and one of five regulatory subunit isoforms encoded by three different genes—Pik3r1, Pik3r2, and Pik3r3.¹¹⁷ In most cells, including the liver, products of Pik3r1—p85α, p55α, and p50α—and Pik3r2 (p85β) stabilize and inhibit the catalytic subunits.^136,137 Partial loss of the regulatory subunits increases insulin sensitivity, which appears to be related to diminished negative feedback to the IRS proteins.¹³⁸ By contrast, 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 subunits—which dysregulates glucose and lipid homeostasis, hepatic size, and function.¹¹⁶ Thus, PI3K activity is responsible for nearly all of insulin’s actions in the liver in vivo.

The PI3K catalytic subunits, p110α and p110β, are ubiquitously expressed, but p110δ is predominantly expressed in leukocytes. Whereas deletion of either p110α or p110β causes embryonic lethality,^139,140 deletion of p110δ causes immune-system defects.¹⁴¹ Mice heterozygous for deletion of either p110α or p110β show no phenotypic abnormalities, but combined heterozygosity for p110α and p110β causes mild glucose intolerance and fasting hyperinsulinemia.¹⁴² Mutation of Asp₉₃₃ of p110α to alanine abolishes its lipid kinase activity, and knock-in mice homozygous for this mutation (D933A) die during embryogenesis.¹⁴³ By contrast, heterozygous D933A mice are viable and fertile but develop severe insulin resistance, glucose intolerance, hyperphagia, and adiposity, indicating a function of p110α not shared by p110β. This unique function derives from the highly selective recruitment of p110α to IRS signaling complexes following insulin stimulation.¹⁴³

The mammalian genome contains three genes that separately encode the AKT isoforms, AKT1, AKT2, and AKT3. The analysis of knockout mice confirms that AKT can regulate important biological functions, including cell proliferation, growth, survival, and differentiation and glucose metabolism in vivo; however, the AKT isoforms are not redundant components of the insulin-like signaling cascade.¹⁴⁴ AKT1 has a major role in embryonic development, growth, and survival but minor effects upon metabolism.¹⁴⁵ By comparison, AKT2-deficient mice display metabolic defects, and AKT3-deficient mice display neural defects.¹¹⁷ AKT2 rather than AKT1 is primarily responsible for insulin-stimulated GLUT4 translocation.¹⁴⁶ Targeted disruption of AKT impairs insulin-stimulated glucose uptake in muscle and adipocytes and prevents the suppression of hepatic glucose output by insulin, which causes glucose intolerance and insulin resistance that progresses to diabetes and β-cell failure.¹⁴⁷ Two human subjects with a dominant-negative mutation in AKT2 display many features of type 2 diabetes—hyperglycemia, increased lipogenesis, elevated liver fat content, TG-enriched VLDL, hypertriglyceridemia, and low HDL cholesterol levels.^19,148

Although PDK1 is a master upstream kinase controlling the activation of numerous kinases including AKT, it occurs as a single isoform encoded by one gene. Unlike most kinases, PDK1 is constitutively active, so phosphorylation of its substrates is regulated by mechanisms that control the interaction of substrates with PDK1.¹⁴⁹ PDK1 is required for normal development, inasmuch as mouse embryos lacking PDK1 die at day E9.5; however, PDK1 hypomorphic mice with 90% reduction of PDK1 expression in all tissues are viable, fertile, and small.¹⁵⁰ Surprisingly, activation of AKT1 and S6K1 by insulin is largely normal in PDK1 hypomorphic mice. Moreover, liver-specific PDK1^−/− mice display normal blood glucose and insulin concentrations under ordinary fasting and postprandial conditions; however, they are markedly glucose intolerant and paradoxically fail to normalize their blood glucose after injection of insulin.¹⁵¹ Mistargeted PDK1 due to mutation in the pleckstrin homology domain causes insulin resistance.¹⁵² Thus, PDK1 might not be a rate-limiting step in the insulin signaling cascade; however, excess capacity might be important during acute metabolic challenge when definitive activation of AKT is required for tight metabolic control.

Introduction

Chronic metabolic or inflammatory stress dysregulates IRS proteins through various mechanisms, including inhibition of transcription, phosphatase-mediated dephosphorylation, Ser/Thr-phosphorylation, and proteasome-mediated degradation. The regulation of IRS-protein signaling might be one of the most important sites to coordinate the intensity and duration of the insulin response among various tissues.

Transcriptional Control

Transcription of the IRS1 gene is generally stable, but details of its regulation are unclear. By contrast, experiments conducted in various murine tissues suggest that production of IRS2 is regulated by multiple nutrient-sensitive transcription factors, including cAMP response element binding protein (CREB) and its binding partner CRTC2, forkhead box O 1 (FOXO1), transcription factor E3 (TFE3), and the sterol regulatory element binding protein SREBP1.^200,201 Interestingly, the CREB/CRTC2 transcriptional complex—which binds to cAMP response elements (CRE)—has opposite effects upon IRS2 expression in β cells and liver. After a meal, the production of ATP from glucose oxidation depolarizes β cells, which promotes both Ca²⁺ influx and cAMP production that has many important effects, including the activation of CREB/CRTC2.^200,202 Thus glucose is coupled directly to IRS2 expression in β cells, which stimulates β-cell growth and compensatory insulin secretion. By contrast, CREB/CRTC2 promotes IRS2 expression in the fasting liver, which can inhibit the gluconeogenic program by augmenting the basal insulin response.²⁰¹ In this regard, the KRLB motif in IRS2 might play an important role to engage the transiently activated insulin receptor catalytic domain, leading to an especially strong basal insulin response to attenuate the action of counterregulatory hormones.

In addition to CRE sequences responsive to cAMP, the promoter region of the IRS2 gene includes insulin response elements that bind FOXO family members, an E-box that binds TFE3, and a sterol response element (SRE) recognized by SREBP-1c.203–205 FOXO1 links the PI3K→AKT cascade to the expression of genes important in cell growth, survival, and metabolism (see Fig. 8-5). In liver, IRS1 and IRS2 promote FOXO1 phosphorylation, which causes its export from the nucleus and degradation in the cytosol. Deletion of hepatic FOXO1 attenuates CREB/CRTC2-mediated gene expression—including that of the IRS2 gene—and inhibits gluconeogenesis while promoting glycogen synthesis.²⁰⁶ 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.²⁰⁴ 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 that binds the SREBP-1c. SREBP-1c is an important transcriptional regulator of lipid synthesis.²⁰⁷ SREBP-1c concentrations increase during nutrient excess and chronic insulin stimulation.^205,208 The increase in hepatic SREBP-1c expression is associated with a decrease in IRS2 mRNA, which might be explained by competition between overlapping binding sites for SREBPs (SRE) and FOXO1 (IRE).²⁰⁵ Up-regulation of IRS2 expression by TFE3/FOXO and down-regulation by SREBP-1c parallels the switch from glycogenolysis and gluconeogenesis during fasting to lipogenesis following feeding. An imbalance in this reciprocal regulation may ultimately contribute to pathophysiologic effects of overnutrition, leading to the development of the metabolic syndrome and diabetes. More work is needed to establish the relevance of this regulatory mechanism.

Multisite Ser/Thr Phosphorylation of IRS Proteins

Heterologous signaling cascades initiated by proinflammatory cytokines or metabolic excess—including tumor necrosis factor α (TNF-α), endothelin-1, angiotensin II, excess nutrients (free fatty acids, amino acids, and glucose) or endoplasmic reticulum stress—can promote Ser/Thr-phosphorylation of Irs1 and cause insulin resistance.^209,210 Many biochemical and genetic experiments show that individual Ser/Thr-phosphorylation sites throughout the structure of Irs1 can reduce its insulin-stimulated tyrosine phosphorylation by up to 50%.²¹¹ This level of inhibition is sufficient to cause glucose intolerance that progresses to diabetes, especially if pancreatic β cells fail to provide adequate compensatory hyperinsulinemia.²¹² Moreover, hyperinsulinemia itself can exacerbate Ser/Thr-phosphorylation of Irs1 through the PI3K→Akt or the mTORC1→p70^s6k cascades.

One of the best-studied regulatory phosphorylation sites in Irs1 is Ser307 (S307) in the rodent protein (S312 in human IRS1).213–218 Phosphorylation of S307 might be a common mechanism of insulin resistance (Fig. 8-8). S307 phosphorylation inhibits tyrosine phosphorylation of Irs1 and inhibits the activation of the PI3K/Akt pathway in response to insulin.^219,220 Insulin itself promotes S307 phosphorylation through activation of the PI3K, revealing feedback regulation that can be mediated by many kinases—PKCζ, IKKβ, JNK, mTOR, and S6K1.²¹¹ JNK can bind directly to Irs1, which appears to facilitate S307 phosphorylation during stimulation of cells with proinflammatory cytokines. S307 is poorly phosphorylated in ob/ob (obese) mice that lack Jnk1, suggesting that this mechanism of inhibition has physiologic significance.²²¹ Free fatty acids that contribute to insulin resistance promote S307 phosphorylation, possibly through PKCθ.²¹⁵ Hyperactivated mTOR also promotes S307 phosphorylation, which is diminished in mice lacking S6K.^120,222,223 IKKβ inhibitors (aspirin and salicylates) block S307 phosphorylation,²¹⁴ which improves insulin sensitivity in obese rodents and in type 2 diabetes patients.^224–226 Phosphorylation of S307—located near the PTB domain—inhibits insulin-stimulated IRS1 tyrosine phosphorylation by disrupting the association between the insulin receptor and IRS1,^220,227 although this disruption might depend upon the phosphorylation of additional sites.²²⁸

The use of siRNA to suppress the expression of more than 600 kinases in HepG2 cells revealed many kinases that can regulate IRS1 signaling—including Pim2, PDHK, PKC isoforms, CaMKI-like, DAPK2, DCAMKL1, STK10, S-T kinase 25, MKK4, 6, or 7, LIMK2, SIK, CGPK1, et al.²²⁹ PKCδ phosphorylates several serine residues that inhibit Irs1 tyrosine phosphorylation: S307, S323, and S574.²³⁰ The Akt cascade can mediate negative-feedback inhibition of insulin signaling through S522 phosphorylation²³¹ (see Fig. 8-8). Irs1 contains 6 canonical AKT kinase phosphorylation motifs (RXRXXS). Four of these RXRXXS motifs occur in or near the Irs1 PTB domain (S265, S302, S325, and S358), whereas S522 and S1100 reside among the tyrosine phosphorylation sites in the tail of Irs1. S1100 is phosphorylated by PKCθ, whereas the sites near the PTB domain are phosphorylated by Akt and possibly other kinases.^232,233 Atypical protein kinase C isoforms—including PKCθ or PKCζ—are activated by the PI3K cascade and could promote S522 phosphorylation (see Fig. 8-8); rapamycin or glucose starvation does not inhibit insulin-stimulated S522 phosphorylation, excluding the mTORC1→p70^s6k cascade.

Some Ser/Thr phosphorylation sites on IRS1 appear to promote insulin-stimulated tyrosine phosphorylation. AMP kinase, which is activated by increased AMP levels during energy depletion, associates with Irs1 and phosphorylates S789, increasing insulin-stimulated tyrosine phosphorylation.²³⁴ Under certain experimental conditions, S302 appears to promote Irs1 tyrosine phosphorylation.²³⁵ S302 phosphorylation is strongly inhibited by amino acid or glucose starvation, which also attenuates insulin-stimulated tyrosine phosphorylation in cell-based assays. Moreover, S302→A302 substitution inhibits insulin-stimulated tyrosine phosphorylation in cell-based assays. Rapamycin inhibits S302 phosphorylation, but the phosphorylation of other inhibitory sites is also reduced, which generally increases tyrosine phosphorylation.²³⁵ More work is needed to understand how multi-site Ser/Thr phosphorylation mediated by various kinases is integrated to regulate tyrosine phosphorylation and glucose tolerance in an intact animal.

Degradation of IRS1

Significant regulation of IRS1 and occasionally IRS2 is thought to occur by ubiquitin-mediated degradation.^236,237 Degradation of IRS proteins is stimulated in cultured cells by tumor necrosis factor α (TNF-α), interferon γ, insulin and IGF-1, platelet-derived growth factor (PDFGF), free fatty acids (FFA), 12-myristate 13-acetate (PMA), inhibitors of Ser/Thr phosphatases, and inhibitors of calcineurin.²³⁸ Activation of mTORC1 is at least partially responsible for mediating IRS1 degradation, especially during extreme conditions of mTORC1 activation.^{222,236,239,240} Hyperactivation of the mTORC1 complex in cells lacking TSC1 or TSC2 results in increased serine phosphorylation of IRS proteins^222,240; however, there is some evidence that IRS-protein degradation in these cells may be due to coincident activation of endoplasmic reticulum (ER) stress-signaling pathways and is partially separable from the effects of IRS serine phosphorylation.²⁴¹ Thus Ser/Thr phosphorylation of IRS1 does not appear to be an absolute requirement. Although the mechanism of mTORC1-mediated IRS1 degradation is not completely clear, the E3 ubiquitin-ligase CUL7/Fbw8 might play an important role.²⁴² However, CUL7/Fbw8 does not target IRS2, so other mechanisms must also be involved.²⁴³ More work is needed to determine the relevance of IRS protein degradation in various experimental systems to the development of insulin resistance in humans.

SOCS proteins also participate in cross-talk with insulin-like signaling and contribute to insulin resistance (Fig. 8-9). SOCS1 and SOCS3 can bind to distinct domains of the insulin receptor, which influences IR-mediated phosphorylation of IRS1 and IRS2.²⁴⁴ Overexpression of SOCS1 in the liver preferentially inhibits IRS2 tyrosine phosphorylation, whereas SOCS3 overexpression decreases tyrosine phosphorylation of both IRS1 and IRS2.^244,245 Resistin can increase SOCS3 in H4IIE hepatocytes, which might explain the inhibitory effect of resistin upon insulin action.^245,246 SOCS1 and SOCS3 also targets IRS1—and possibly IRS2—for ubiquitinylation by the elongin BC-containing E3 ubiquitin-ligase complex.²⁴⁷ Overexpression of SOCS1/3 in the liver causes insulin resistance and up-regulation of key regulators involved in fatty acid synthesis and SREBP-1c, whereas inhibition of SOCS1/3 by anti-sense oligonucleotides in obese and diabetic mice improves insulin sensitivity and normalizes SREBP-1c expression.²⁴⁸ In this manner, SOCS1 and SOCS3 can link infection, inflammation, or metabolic stress to insulin resistance and glucose intolerance. Interestingly, the core protein of hepatitis C virus up-regulates SOCS3, which may help explain why infected patients have increased fasting insulin levels compared to patients with other chronic liver disease.²⁴⁹

Other Posttranslational Modifications

Acetylation and O-linked beta-N-acetylglucosamine (O-GlcNAc) also regulate insulin signaling. O-GlcNAc was first identified in the 1980s in rat liver subcellular organelles,²⁵⁰ and increased O-GlcNAc of IRS1 and AKT2 in primary rat adipocytes is accompanied by a partial reduction in insulin-stimulated phosphorylation of IRS1 and AKT2.²⁵¹ At the same time, the NAD⁺-dependent deacetylase, SirT1, the mammalian homolog of Sir2, may positively regulate insulin-induced IRS2 but not IRS1 tyrosine phosphorylation.²⁵² Sir2 regulates lifespan in C. elegans, Drosophila, and Saccharomyces cerevisiae (where it was first discovered), but the role of SirT1 in lifespan regulation is less clear. SirT1 is involved in glucose homeostasis,²⁵³ insulin secretion,^254,255 and lipid mobilization.²⁵⁶ These observations illustrate how various metabolic processes such as aging and diabetes could be linked.

Introduction

Type 2 diabetes is the result of insulin resistance that is uncompensated by increased insulin secretory capacity.²⁵⁷ Clinical staging of type 2 diabetes suggests that early insulin resistance in skeletal muscle, which is the primary site of glucose disposal in humans is important in disease initiation.^258,259 Compatible with this hypothesis, healthy human subjects that display skeletal muscle insulin resistance exhibit decreased incorporation of glucose into muscle glycogen that is paralleled by increased liver triglyceride synthesis.¹⁸ However, animal models of pure skeletal muscle insulin resistance, including mice that lack the insulin receptor in muscle,²⁶⁰ show surprisingly mild phenotypes. Regardless, many features of type 2 diabetes and the associated metabolic syndrome are recapitulated by deletion of the insulin receptor—or the combined deletion of IRS1 and IRS2—in hepatocytes.^261–263 These and other experiments, including those delineating the liver transcriptional control network (Fig. 8-10), support the importance of “selective” insulin resistance in diabetes etiology.²⁶⁴ Although adipose tissue is not the primary site of glucose disposal, insulin resistance in visceral adipocytes in particular gives rise to increasing circulating free fatty acids that further contribute to insulin resistance.²⁶⁵

Nutrient Excess and Insulin Resistance

Excess metabolites promote the development of type 2 diabetes by both direct and indirect means. The progressive rise in compensatory insulin concentrations during development of type 2 diabetes activates negative feedback of insulin signaling by homologous pathways (e.g., PI3K→AKT→p70^S6K; see Fig. 8-5). By contrast with hyperinsulinemia, the accumulation of excess triglycerides within insulin-sensitive tissues inhibits insulin signaling through heterologous down-regulation of IRS1 and IRS2 via pathways involving atypical protein kinase C and JNK kinases (see Fig. 8-8). Increased adipose tissue mass resulting from increased triglyceride storage is also associated with macrophage infiltration into white adipose tissue depots and production of inflammatory cytokines, which may contribute to insulin resistance in other tissues. Serum branched-chain amino acids in conjunction with obesity promote insulin resistance in humans and rodents, owing at least in part to increased muscle mTORC1 activity.²⁶⁶ Increased activation of the mTOR pathway due to nutrient excess contributes to endoplasmic reticulum (ER) stress-mediated insulin resistance (see Fig. 8-8).

The ER is a luminal network where protein synthesis, maturation, folding, and transport occur. The ER is also an important site for lipid and cholesterol biosynthesis.^267,268 Perturbation of ER function by various means (e.g., glucose and energy deprivation, viral infection, increased protein trafficking, accumulation of unfolded proteins, cholesterol accumulation, exposure to chemical agents such as tunicamycin and thapsigargin) causes ER stress. Hyperactivation of mTOR due to nutrient excess and hyperinsulinemia seems likely to cause ER stress through increased flux of newly synthesized proteins through the ER lumen. ER stress activates a set of signaling pathways collectively known as the unfolded protein response (UPR).^267,268 Distinct branches of the UPR are initiated by the two type-I transmembrane kinases, PERK (PKR-like endoplasmic reticulum kinase) and IRE1 (inositol requiring enzyme-1); and by ATF6 (activating transcription factor-6), a type-II transmembrane protein.^267,268

ER stress likely causes heterologous down-regulation of IRS-protein signaling through activation of the IRE1→JNK branch of the UPR (see Fig. 8-8). Activation of IRE1→JNK signaling in the liver and adipose tissues creates insulin resistance concomitant with an increase in serine phosphorylation of IRS1.²⁶⁹ UPR activation owing to mTORC1 hyperactivation also appears to be largely responsible for the resulting ubiquitin/proteasome-dependent degradation of IRS1.²⁴¹ Recent observations further indicate that PERK-mediated phosphorylation of eIF2 contributes to accumulation of lipids in the liver, which may indirectly inhibit IRS-protein function.²⁷⁰

Genetically engineered mouse models susceptible to development of ER stress gain more weight and develop more severe insulin resistance than controls. For example, mice lacking one allele of X-box binding protein 1 (XBP1), a transcription factor that regulates ER folding capacity, develop higher levels of ER stress, obesity, and insulin resistance.²⁶⁹ Furthermore, when ER stress is attenuated in the obese and diabetic mice through the use of chemical chaperones—agents that have ability to increase ER folding capacity—insulin resistance is abolished, glucose tolerance is greatly enhanced, and blood glucose concentration returns to normal.²⁷¹ The presence of ER stress in the liver and fat tissues of obese patients strengthens the conclusion that this system plays a significant role in development of insulin resistance.²⁷² Future studies are needed to investigate whether chemical chaperones will have antidiabetic effects in humans.

Inflammation and Insulin Resistance

Adipose tissue functions not only as a passive depot for energy storage but as an active secretory organ that modifies insulin sensitivity through production of several adipocyte-derived cytokines, or adipokines.²⁷³ Important adipokines that contribute to normal physiology include leptin, which regulates energy balance through hypothalamic control of satiety and energy expenditure, and adiponectin, the circulating concentration of which is positively correlated with insulin sensitivity in humans. By contrast with these molecules, factors secreted by adipose tissue during obesity can promote insulin resistance by generation of persistent low-grade inflammation.^17,274

Increased triglyceride storage during obesity gives rise to an expanded white adipose tissue compartment characterized by the presence of hypoxia and infiltrating macrophages.²⁵⁷ Hypoxia likely contributes to the generation of a microenvironment conducive to macrophage infiltration,²⁷⁵ which results in increased local and circulating concentrations of proinflammatory cytokines, including monocyte chemoattractant protein (MCP1), IL-6, and TNF-α, each one of which is up-regulated transcriptionally by the immunomodulatory transcription factor nuclear factor (NF)-κB.²⁷⁴ MCP1 expression causes increased recruitment of macrophages to adipose tissue. In agreement with a general role of adipose tissue inflammation in causing insulin resistance, mice lacking MCP1 show increased insulin sensitivity.²⁷⁶ Although serum IL-6 concentration is positively correlated with human obesity, mice lacking the IL-6 gene are obese and insulin resistant²⁷⁷; thus, the role of this cytokine in modification of insulin sensitivity is still debated.

TNF-α activates signaling cascades in insulin-sensitive tissues that result in activation of Jun N-terminal kinase 1 (JNK1) and the inhibitor of kappa light polypeptide gene enhancer in β cells, kinase beta (IKKβ).²⁷⁴ In addition to cytokines, JNK1 and IKKβ can be stimulated by FFAs or by pattern-recognition receptors of the innate immune system, such as toll-like receptor 4 (TLR4).²⁷⁸ JNK1-deficient mice are protected from obesity and insulin resistance caused by high-fat-diet feeding, indicating an important role of this kinase in development of insulin resistance.²²¹ Compatible with phosphorylation and inhibition of IRS proteins by JNK kinases, mice lacking JNK1 also show decreased phosphorylation of IRS1 at Ser307. Inhibition of JNK activity using JNK inhibitors in obese mice increases insulin sensitivity and improves glucose homeostasis.²⁷⁹ Paradoxically, however, increased circulating TG concentration and hepatic TG accumulation are observed in this model, reflecting the complex role of JNK in mediation of insulin resistance due to fatty acids and inflammation.²⁸⁰

TNF-α signaling also interacts with excess fatty acids to stimulate the production of ceramide. Accumulation of ceramide in insulin-responsive tissues also causes insulin resistance, likely through a mechanism involving serine phosphorylation of IRS proteins; however, the relevant kinases have not been determined.²⁸¹ Further demonstrating the connection between lipids and inflammation, in rodents, the inhibitory effects of lipid or free fatty acid infusion are reduced by agents that prevent accumulation of ceramide.²⁸²

Although increased circulating TNF-α concentration is generally accepted to cause insulin resistance, there is substantial disagreement about how common this might be in human insulin resistance.²⁷⁴ Moreover, the improved insulin sensitivity reported for mice lacking TNF-α is not, in at least one report, shared by mice that lack the two TNF-α receptors.²⁸³ Similarly, the administration to insulin resistant humans of a highly effective TNF-α inhibitor for 3 months does not improve glucose homeostasis, despite reduction in some parameters of systemic inflammation.²⁸⁴ Thus, despite some very strong experimental evidence, the physiologic relevance of TNF-α as a cause of insulin resistance in humans remains unclear.

Excess triglycerides not stored in adipose tissues accumulate within the muscle, liver, and other tissues, impairing insulin signaling by heterologous pathways involving IRS1 (and possibly IRS2) Ser/Thr phosphorylation or by other inflammatory signaling pathways.257,274,285 A variety of clinical and experimental data support the hypothesis that intramyocellular triglyceride (IMTG) causes skeletal muscle insulin resistance in humans and rodents.²⁸⁵ Infusion of triglyceride/heparin solutions into humans or rodents—which transiently increases circulating free fatty acid concentration—inhibits insulin-stimulated glucose disposal into muscle by a mechanism involving decreased Irs1-associated PI3K activity.^215,286,287 In mice and rats, this resistance is associated with increased muscle Ser307 phosphorylation,²¹⁵ which (based on studies of knockout mice) occurs via PKCθ- and IKKβ-dependent pathways.^224,288 In liver, PKCε functions similarly to PKCθ in muscle²⁸⁵ and may contribute to the loss of inhibition of hepatic gluconeogenesis by insulin caused by FFA in human studies.^289,290 For both PKCθ and PKCε, the relevant activating ligands are diacylglycerols (DAG) present in increased concentration intracellularly as a result of decreased fatty acid oxidation or increased TG synthesis.²⁸⁵

In the liver, Kupffer cells are the equivalent of adipose tissue macrophages and are of equal or greater influence in the regulation of insulin sensitivity.²⁷⁴ Importantly, both of these types of myeloid-derived tissue-resident macrophages can undergo either classical (M1) activation by inflammatory cytokines, such as IL-1, or alternative (M2a) activation in response to IL-4 or IL-13, resulting in the production either more (M1) or less (M2a) inflammatory cytokines; moreover, it is clear that the cytokines released by adipose tissue macrophages during high-fat feeding or obesity result from M1 macrophage infiltration, whereas adipose tissue macrophages in lean animals are M2a macrophages.²⁹¹ Recent data indicate that the presence of tissue resident macrophages with the M2a profile is not merely neutral with respect to insulin sensitivity. Rather, the blockade of alternative macrophage activation—for example, by deletion of peroxisome proliferation activator delta (PPARδ) in myeloid cells^292,293—results in impaired insulin sensitivity in mice challenged with high-fat feeding. Further experiments are necessary to establish whether activation of alternative macrophage activation by manipulation of PPARδ or other means might be an effective adjunct to current therapies for treatment of insulin resistance.

The IR→IRS Signaling Cascade in Liver

Hyperglycemia and dyslipidemia due to hepatic insulin resistance are key pathologic features of type 2 diabetes.^294,295 In mice, near-total hepatic insulin resistance can be introduced via the systemic or liver-specific knockout of key insulin signaling genes.^{132,147,151,261,296} Among these approaches, the compound suppression or deletion of the insulin receptor substrates, Irs1 and Irs2, are the least complicated by defective insulin clearance or liver failure.^131,132

Hepatic deficiency of Irs1 and Irs2 leads to a significant growth retardation in both prediabetic and diabetic mice. Consistent with this phenotype, the expression of several hepatic genes that promote organismic growth (Igf1, Igfals, and Ghr) are significantly reduced in mice lacking Irs1 and Irs2.²⁹⁷ Moreover, the expression of Igfbp1 increases significantly in Irs1/2-deficient liver, which leads to increased circulating concentrations of Igfpb1 that inactivate Igf1.⁷ The deletion of hepatic Foxo1 restores normal expression of these genes in the Irs1 and Irs2-deficient liver, restoring growth of the mice to a normal size. Stat5b is well known to regulate Igf1 and Igfals expression, but whether Foxo1 directly modulates their expression requires additional work (see Fig. 8-10).²⁹⁸ Regardless, the postprandial inactivation of Foxo1 by the IR→IRS→PI3K→AKT cascade appears to be essential for the control of body growth by growth hormone and endocrine Igf1.

The program of gene expression directed by Foxo1 and its cofactors ordinarily protects cells, as well as whole organisms, from the life-threatening consequences of nutrient deprivation and oxidative and genotoxic stresses.²⁹⁹ This effect is especially important during prolonged starvation, because hepatic Foxo1 ensures the production of sufficient glucose to prevent life-threatening hypoglycemia.²⁰⁶ In healthy animals, the decreased insulin concentration during fasting promotes the nuclear localization of Foxo1, where it interacts with Ppargc1a and Creb/Crtc2 to increase the expression of the key gluconeogenic enzymes G6pc and Pck1^300–305 (see Fig. 8-10). In conjunction with Creb/Crtc2, Foxo1 also increases the expression of Irs2 and reduces the expression of the Akt inhibitor Trib3, which together can enhance fasting insulin sensitivity and augment the insulin response upon eventual feeding.^201,306 Although Foxo1 can be regulated by many mechanisms, its inactivation in postprandial liver appears to be under the exclusive control of the IRS1/2→Akt cascade (see Fig. 8-10).

In addition to the inactivation of Foxo1, inhibition of hepatic gluconeogenesis also involves the inactivation of a glucagon-stimulated nuclear transcription complex assembled from CREB (cAMP response element binding protein), CBP (CREB-binding protein) and CRTC2 (CREB-regulated transcription coactivator 2) (Fig. 8-11). During starvation, glucagon promotes the assembly of the transcriptionally active CREB-CBP-CRTC2 complex, which increases the expression of PPARGC1A, Pck1 (phosphoenolpyruvate carboxykinase), and G6pase (glucose 6-phosphatase).³⁰⁷ Inactivation of the CREB-CBP-CRTC2 complex involves two branches of the insulin signaling cascade. Atypical PKC-mediated phosphorylation of CBP appears to be one of the first steps to disassemble the active complex.³⁰⁸ Insulin appears to activate atypical PKC exclusively through the IRS2 branch of the signaling cascade.³⁰⁹ How this signaling specificity arises is unknown, but it is probably very important, because IRS2 expression is ordinarily augmented during starvation by CREB-CBP-CRTC2 itself. The second step is mediated by the PI3K→AKT cascade that stimulates SIK2 (salt inducible kinase 2), which inactivates the CREB-CBP-CRTC2 complex by phosphorylating CRTC2 at Ser171 and targeting it for degradation in the cytosol (see Fig. 8-11).³⁰⁰

Metformin, a first-line treatment for type 2 diabetes, also activates atypical PKC in liver to phosphorylate CBP and disassemble the CBP:CRTC2 dimer from CREB (see Fig. 8-11). Since nutrient excess and compensatory hyperinsulinemia preferentially suppresses hepatic IRS2, metformin can circumvent the block imposed by hepatic insulin resistance and activate a common signaling intermediate that reduces the expression of gluconeogenic enzymes under conditions of obesity, insulin resistance, and hyperglycemia.

IRS-Protein Signaling in Pancreatic β Cells

Mice lacking the Irs1 or Irs2 genes are insulin resistant, with impaired peripheral glucose utilization.129,310,311 Both types of knockout mice display metabolic dysregulation, but only Irs2^−/− mice develop diabetes between 10 and 15 weeks of age, owing to a near-complete loss of pancreatic β cells.¹²⁹ These results position the insulin-like signaling cascade through IRS2 at the center of β-cell function (Fig. 8-12). Among 4-week-old mice, β-cell mass is nearly normal in Irs2^−/− mice, whereas it is reduced by 50% in Igf1r^+/−::Irs2^+/− mice and nearly undetectable in Igf1r^+/−::Irs2^−/− mice.³¹² However, the targeted deletion of the Igf1r in β cells has insignificant effects upon β-cell growth and survival, suggesting that Igf1→Irs2 signaling might not be β-cell autonomous.^313,314 The β-cell-specific deletion of both IR and IGF1R in β cells causes loss of β-cell mass by 2 weeks of age, suggesting that both receptor kinases—and possibly receptor hybrids—promote the IRS2 signaling needed for β-cell growth and survival.³¹⁵

Many factors are required for proper β-cell function, including the homeodomain transcription factor, 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 (MODY)^316,317 (see Fig. 8-12). 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 restores β-cell function and normalizes glucose tolerance, linking the insulin-like signaling cascade to the network of β-cell transcription factors.^318,319 Transgenic expression of Irs2 or suppression of Foxo1 increases Pdx1 concentrations in Irs2^−/− mice, supporting the hypothesis that Pdx1 is modulated by the Irs2→Foxo1 cascade in β cells^319,320 (see Fig. 8-12). Haploinsufficiency for PTEN also prevents β-cell failure in Irs2^−/− mice, due at least in part to the stimulation of the PI3K→PKB/AKT cascade that inhibits Foxo1 (see Fig. 8-5).

Glucose and glucagon-like peptide-1 (GLP1) have strong effects upon β-cell growth, which depend upon the Irs2 signaling cascade. Irs2 is strongly up-regulated by these cAMP and Ca²⁺ agonists, which activate cAMP responsive element binding protein (CREB) and the CREB-regulated transcription coactivator 2 (CRTC2).³²¹ While many cAMP-mediated pathways oppose the action of insulin, the upregulation of Irs2 by glucose and GLP1 reveals an unexpected intersection of these important signals (see Fig. 8-5). Thus, hyperglycemia resulting from the daily consumption of high-caloric food promotes β-cell growth by increasing IRS2 expression.³²² Mice expressing low concentrations of glucokinase in β cells display impaired intracellular calcium responses to glucose, which suppresses the phosphorylation of CREB that induces Irs2 expression. Similarly, GLP1 couples into this mechanism by directly increasing the concentration of cAMP in β cells.³²¹ Glucose and GLP1 have no effect upon β-cell growth in Irs2^−/− mice.³²³ Thus, the Irs2-branch of the insulin-like signaling cascade—rather than Irs1 or the upstream receptor kinases—is an important “gatekeeper” for β-cell plasticity and function (see Fig. 8-10).

ATF3, a stress-inducible proapoptotic gene, can inhibit IRS2 expression in β cells, revealing a direct inverse link between the stress response and β-cell survival.²⁰² ATF3 is a member of the ATF/CREB family of transcription factors but has an opposite effect from CREB. Thus, the CRE/ATF-like site on the IRS2 promoter acts as an integration point for survival signals (such as GLP-1) and stress signals (such as proinflammatory cytokines) to exert their opposite effects on IRS2 transcription. Because ATF3 is induced by a variety of stress signals, it can function as a mechanism to suppress β-cell expansion when it is physiologically important to suppress the production of excess insulin that could oppose the release of glucose and other nutrients needed for longer-term repair.

Central Control of Nutrient Homeostasis and Lifespan

Work with lower metazoans—mainly C. elegans and Drosophila—has drawn attention to the idea that less insulin-like signaling extends healthy lifespan.³ Female fruit flies live up to 80% longer when the activity of the Drosophila insulin-like receptor is reduced by 80%.³²⁴ Moreover, ablation of neurosecretory cells in adult Drosophila that produce three of the seven insulin-like peptides also extends lifespan. Although the long-lived flies show resistance to oxidative stress, they also accumulate excess lipids and carbohydrate.³²⁵ Thus, reduced insulin-like signaling can extend lifespan in lower metazoans but cause paradoxically physiologic alterations that are associated with life-threatening diseases in rodents and humans.

Recent results point to the brain as the site where reduced insulin-like signaling might have a consistent effect to extend mammalian lifespan—as it does in C. elegans and D. melanogaster.^3,326 Irs1 and Irs2 are expressed in neurons throughout the brain, and Irs4 is expressed in the hypothalamus. However, Irs2 appears to play a major role for brain growth, fertility,^327,328 and nutrient homeostasis.^329–331 Proper regulation of Irs2 signaling in the brain might also play a role in mouse lifespan.³³² At 22 months of age, brain Irs2 knockout mice are slightly hyperphagic, overweight, hyperinsulinemic, and glucose intolerant; however, compared to control mice, brain Irs2 knockout mice display stable superoxide dismutase-2 (SOD) concentration in the hypothalamus during meals. Reduced Irs2 signaling in the brain increases by nearly 6 months the lifespan of mice maintained on a high-energy diet.³³²

As mammals age, compensatory hyperinsulinemia usually develops to maintain glucose homeostasis and prevent the progression toward life-threatening type 2 diabetes.³⁶ While this response is important at middle age, the chronic elevation of circulating insulin might have negative effects upon the brain that can reduce lifespan.^28,333,334 By directly attenuating brain Irs2 signaling, an aging brain can be shielded from the negative effects of overweight and hyperinsulinemia that ordinarily develop with advancing age. This hypothesis helps explain why lifespan correlates with reduced insulin signaling in C. elegans and Drosophila but correlates with peripheral insulin sensitivity in rodents—and probably in humans. Caloric restriction and exercise are the usual ways to increase peripheral insulin sensitivity and reduce the amount of circulating insulin needed to regulate circulating glucose throughout life. Moreover, these strategies might increase lifespan by reducing the activation of brain IRS2. Other genetic strategies that improve peripheral insulin sensitivity, including reduced growth hormone or adipocyte insulin signaling, can be explained by a similar mechanism.^335,336 Consistent with this hypothesis, human centenarians display increased peripheral insulin sensitivity and reduced circulating insulin levels.²⁸ Hence, less insulin-like signaling in the brain increases mammalian lifespan, just as reduced insulin-like signaling extends the lifespan of C. elegans and Drosophila.

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