The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase

Unlike prototypical receptor tyrosine kinases (RTKs), which are single-chain polypeptides, the insulin receptor (InsR) is a preformed, covalently linked tetramer with two extracellular α subunits and two membrane-spanning, tyrosine kinase-containing β subunits. A single molecule of insulin binds asymmetrically to the ectodomain, triggering a conformational change that is transmitted to the cytoplasmic kinase domains, which facilitates their trans-phosphorylation. As in prototypical RTKs, tyrosine phosphorylation in the juxtamembrane region of InsR creates recruitment sites for downstream signaling proteins (IRS [InsR substrate] proteins, Shc) containing a phosphotyrosine-binding (PTB) domain, and tyrosine phosphorylation in the kinase activation loop stimulates InsR’s catalytic activity. For InsR, phosphorylation of the activation loop, which contains three tyrosine residues, also creates docking sites for adaptor proteins (Grb10/14, SH2B2) that possess specialized Src homology-2 (SH2) domains, which are dimeric and engage two phosphotyrosines in the activation loop.Insulin is a highly potent anabolic hormone that is critical for tissue development and for glucose homeostasis (Taniguchi et al. 2006). Released from the β cells of the pancreas, insulin regulates glucose output from the liver and glucose uptake into (primarily) skeletal muscle and adipose tissue. In addition, insulin promotes the synthesis and storage of carbohydrates, lipids, and protein. Insulin’s actions are mediated by the insulin receptor (InsR), a plasma membrane-resident glycoprotein and member of the receptor tyrosine kinase (RTK) family. Other members of the InsR subfamily of RTKs include the insulinlike growth factor-1 receptor (IGF1R) and insulin receptor-related receptor, the latter of which has no known ligand. As an RTK, InsR is ligand-activated through mechanisms that are both prototypical and atypical of RTKs. These mechanisms will be the focus of this article.     

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor^+/+ MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor^−/− MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.     

Ahsg-fetuin blocks the metabolic arm of insulin action through its interaction with the 95-kD β-subunit of the insulin receptor

We previously have shown that Ahsg, a liver glycoprotein, inhibits insulin receptor (InsR) tyrosine kinase (TK) activity and the ERK1/2 mitogenic signaling arm of insulin signaling. Here we show that Ahsg blocks insulin-stimulated GLUT4 translocation and Akt activation in intact cells (mouse myoblasts). Furthermore, Ahsg inhibits InsR autophosphorylation of highly-purified insulin holoreceptors in a cell-free, ATP-dependent system, with an IC₅₀ within the range of single-chain Ahsg concentrations in human serum. Binding of ¹²⁵I-insulin to living cells overexpressing the InsR shows a dissociation constant (K_D) of 250 pM, unaltered in the presence of 300 nM Ahsg. A mutant InsR cDNA encoding the signal peptide, the β-subunit and the furin processing site, but deleting the α-subunit, was stably expressed in HEK293 cells. Treatment with peroxovanadate, but not insulin, dramatically increased the 95 kD β-subunit tyrosine phosphoryation. The level of tyrosine phosphorylation of the 95-kD β-subunit can be driven down sharply by treatment of living HEK293 transfectant cells with physiological doses of Ahsg. Treatment of myogenic cells with Ahsg blunts insulin-stimulated InsR autophosphorylation and AKT phosphorylation. Taken together, we show that Ahsg antagonizes the metabolic functions initiated by InsR activation without interference in insulin binding. The experiments suggest a direct interaction of Ahsg with the InsR ectodomain β-subunit in a mode that does not significantly alter the high-affinity binding of insulin to the holoreceptor's two complementing α-subunits.     

Specific factors are required for kinase-dependent endocytosis of insulin receptors.

Mouse B82 cells that support high affinity saturable endocytosis of epidermal growth factor receptors (EGFR) exhibited only low rates of nonsaturable internalization of insulin receptors (InsR). To investigate the defect in endocytosis of InsR in B82 cells, we examined the role of sequence motifs and tyrosine kinase, the two receptor components shown to be required for efficient saturable endocytosis of InsR in Rat 1 cells. Placement of residues encoded by exon 16 of the InsR onto an EGFR truncated to residue 958 restored EGF-induced internalization of this mutant receptor indicating that the sequence codes in exon 16 are recognized by B82 cells. To determine whether the kinase function could be provided in trans, a B82 cell expressing both receptors was established. EGF-activated EGFR kinase was not able to restore insulin-dependent rapid endocytosis to InsR. However, fusion of untransfected Rat1 cells with InsR-expressing B82 cells enabled rapid endocytosis of InsR, indicating that the internalization defect can be complemented. These results indicate that, although internalization codes can function in the context of other receptors, activation of tyrosine kinase receptors requires an additional specific component.     

Synthesis and biological evaluation of berberine analogues as novel up-regulators for both low-density-lipoprotein receptor and insulin receptor

Berberine (BBR) is a natural compound with up-regulating activity on both low-density-lipoprotein receptor (LDLR) and insulin receptor (InsR). This one-drug-multiple-target characteristic might be suitable for the treatment of metabolic syndrome. In searching for up-regulators effective for both LDLR and InsR expression, the structure–activity relationship (SAR) analysis for BBR analogues was done. Fourteen BBR analogues were designed, synthesized and biologically evaluated. SAR analysis revealed that appropriate modifications on the phenyl ring A or D of BBR might retain the up-regulatory activities on the expression of both LDLR and InsR. Among these compounds, compound 13a bearing 9-methoxy and 10-hydroxyl on the ring D showed promising activities on either LDLR or InsR gene expression. The 10-hydroxyl of 13a could be an arm to connect proper chemical groups for optimizing drug-bioavailability in vivo. Thus, 13a could be considered to be a parent compound to make pro-drugs for either blood lipids or glucose.     

Pathogenesis of Selective Insulin Resistance in Isolated Hepatocytes

The development of insulin resistance (IR) in the liver is a key pathophysiologic event in the development of type 2 diabetes. Although insulin loses its ability to suppress glucose production, it largely retains its capacity to drive lipogenesis. This selective IR results in the characteristic hyperglycemia and dyslipidemia of type 2 diabetes. The delineation of two branched pathways of insulin receptor (InsR) signaling to glucose versus triglyceride production, one through FoxO and the other through SREBP-1c, provides a mechanism to account for this pathophysiological abnormality. We tested the complementary hypothesis that selective IR arises due to different intrinsic sensitivities of glucose production versus de novo lipogenesis to insulin as a result of cell-autonomous down-regulation of InsR number in response to chronic hyperinsulinemia. We demonstrate in mouse primary hepatocytes that chronic hyperinsulinemia abrogates insulin''s inhibition of glucose production, but not its stimulation of de novo lipogenesis. Using a competitive inhibitor of InsR, we show that there is a 4-fold difference between levels of InsR inhibition required to cause resistance of glucose production versus lipogenesis to the actions of insulin. Our data support a parsimonious model in which differential InsR activation underlies the selective IR of glucose production relative to lipogenesis, but both processes require signaling through Akt1/2.     

Activation of G proteins by GIV-GEF is a pivot point for insulin resistance and sensitivity

Insulin resistance (IR) is a metabolic disorder characterized by impaired insulin signaling and cellular glucose uptake. The current paradigm for insulin signaling centers upon the insulin receptor (InsR) and its substrate IRS1; the latter is believed to be the sole conduit for postreceptor signaling. Here we challenge that paradigm and show that GIV/Girdin, a guanidine exchange factor (GEF) for the trimeric G protein Gαi, is another major hierarchical conduit for the metabolic insulin response. By virtue of its ability to directly bind InsR, IRS1, and phosphoinositide 3-kinase, GIV serves as a key hub in the immediate postreceptor level, which coordinately enhances the metabolic insulin response and glucose uptake in myotubes via its GEF function. Site-directed mutagenesis or phosphoinhibition of GIV-GEF by the fatty acid/protein kinase C-theta pathway triggers IR. Insulin sensitizers reverse phosphoinhibition of GIV and reinstate insulin sensitivity. We also provide evidence for such reversible regulation of GIV-GEF in skeletal muscles from patients with IR. Thus GIV is an essential upstream component that couples InsR to G-protein signaling to enhance the metabolic insulin response, and impairment of such coupling triggers IR. We also provide evidence that GIV-GEF serves as therapeutic target for exogenous manipulation of physiological insulin response and reversal of IR in skeletal muscles.     

Genetic analysis of type-1 insulin-like growth factor receptor signaling through insulin receptor substrate-1 and -2 in pancreatic beta cells

Signaling by receptor tyrosine kinases regulates pancreatic β cell function. Inactivation of insulin receptor (InsR), IGF1 receptor (Igf1r), or Irs1 in β cells impairs insulin secretion. Conversely, Irs2 ablation impairs β cell replication. In this study, we examined aspects of the Igf1r regulatory signaling cascade in β cells. To examine genetically the involvement of Irs1 and Irs2 in Igf1r signaling, we generated double mutant mice lacking Igf1r specifically in pancreatic β cells in an Irs1- or Irs2-null background. We show that Igf1r/Irs1 double mutants do not differ phenotypically from Irs1 single mutants and exhibit hyperinsulinemia, while maintaining normal β cell mass and glucose tolerance. In contrast, lack of Igf1r function in β cells aggravates the consequences of Irs2 ablation in double mutants and results in lethal diabetes by 6 weeks of age. This additivity of phenotypic manifestations indicates that Irs2 serves a pathway that is largely independent of Igf1r signaling. Consistent with the view that the latter is the InsR pathway, we show that combined β cell-specific knock-out of both Insr and Igf1r results in a phenocopy of double mutants lacking Igf1r and Irs2. We conclude that Igf1r signals primarily through Irs1 and affects insulin secretion, whereas β cell proliferation is mainly regulated by InsR using Irs2 as a downstream signaling effector. The insulin and IGF pathways appear to control β cell functions independently and selectively.     

Effect of Fluoride on Insulin Level of Rats and Insulin Receptor Expression in the MC3T3-E1 Cells

Studies on the role of insulin and insulin receptor (InsR) in the process of skeletal fluorosis, especially in osteogenic function, are rare. We evaluated the effect of increasing F^? doses on the marker of bone formation, serum insulin level and pancreatic secretion changes in vivo and mRNA expression of InsR and osteocalcin (OCN) in vitro. Wistar rats (n?=?50) were divided into two groups, i.e. a control group and fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100?mg?F^?/L. The fluoride ion-selective electrode measured the fluoride concentrations of femurs. The alkaline phosphatase (ALP), OCN, insulin and glucagon of serum were tested to observe the effect of fluoride action on them. Meantime, the pancreas pathological morphometry analysis via ?? cells stained by aldehyde fuchsin showed the action of fluoride on pancreas secretion. MC3T3-E1 cells (derived from newborn mouse calvaria) were exposed to varying concentrations and periods of fluoride. The mRNA expression of InsR and OCN was quantified with real-time PCR. Results showed that 1-year fluoride treatment obviously stimulated ALP activity and OCN level along with increase of bone fluoride concentration of rats, which indicated that fluoride obviously stimulated osteogenic action of rats. In vitro study, the dual effect of fluoride on osteoblast function is shown. On the other hand, there was a significant increase of serum insulin level and a general decrease of glucagon level, and the histomorphometry analysis indicated an elevated insulin-positive area and increase in islet size in rats treated with fluoride for 1?year. In addition, fluoride obviously facilitated the mRNA expression of InsR in vitro. To sum up, there existed a close relationship between insulin secretion and fluoride treatment. The insulin signal pathway might be involved in the underlying occurrence or development of skeletal fluorosis.     

Cell volume regulation and signaling in 3T3-L1 pre-adipocytes and adipocytes: on the possible roles of caveolae, insulin receptors, FAK and ERK1/2

Caveolae have been implicated in sensing of cell volume perturbations, yet evidence is still limited and findings contradictory. Here, we investigated the possible role of caveolae in cell volume regulation and volume sensitive signaling in an adipocyte system with high (3T3-L1 adipocytes); intermediate (3T3-L1 pre-adipocytes); and low (cholesterol-depleted 3T3-L1 pre-adipocytes) caveolae levels. Using large-angle light scattering, we show that compared to pre-adipocytes, differentiated adipocytes exhibit several-fold increased rates of volume restoration following osmotic cell swelling (RVD) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required for either RVD or RVI in pre-adipocytes. The insulin receptor (InsR) localizes to caveolae and its expression dramatically increases upon adipocyte differentiation. In pre-adipocytes, InsR and its effectors focal adhesion kinase (FAK) and extracellular signal regulated kinase (ERK1/2) localized to focal adhesions and were activated by a 5 min exposure to insulin (100 nM). Osmotic shrinkage transiently inhibited InsR Y(146)-phosphorylation, followed by an increase at t=15 min; a similar pattern was seen for ERK1/2 and FAK, in a manner unaffected by cholesterol depletion. In contrast, cell swelling had no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity is not required for volume regulation. Given the relationship between hyperosmotic stress and insulin signaling, the finding that cell volume regulation is dramatically altered upon adipocyte differentiation may be relevant for the understanding of insulin resistance and metabolic syndrome.     

Comparative analysis of the signaling capabilities of the insulin receptor-related receptor

Although insulin receptor (InsR) and type I insulin-like growth factor receptor (IGF-IR) elicit different physiological effects in their target tissues, their signaling capabilities are similar to a large extent. In the present work, we investigated the potential of the third member of the family, insulin receptor-related receptor (IRR), to associate with known interaction partners of the InsR and the IGF-I receptor in a yeast two-hybrid assay. Using the intracellular part of the IRR we found no association with any of the tested signaling molecules. Phosphotyrosine detection revealed a lack in the constitutive activation of the IRR described for analogous constructs of the two other members of the family. Replacement of the kinase domain of the IGF-IR or its C-terminal lobe alone into the IRR caused a complete restoration of the tyrosine phosphorylation of the IRR. The reestablishment of autophosphorylation was paralleled by restoration of interaction with a specific range of signaling molecules.     

Reconstitution of insulin action in muscle, white adipose tissue, and brain of insulin receptor knock-out mice fails to rescue diabetes

Type 2 diabetes results from an impairment of insulin action. The first demonstrable abnormality of insulin signaling is a decrease of insulin-dependent glucose disposal followed by an increase in hepatic glucose production. In an attempt to dissect the relative importance of these two changes in disease progression, we have employed genetic knock-outs/knock-ins of the insulin receptor. Previously, we demonstrated that insulin receptor knock-out mice (Insr(-/-)) could be rescued from diabetes by reconstitution of insulin signaling in liver, brain, and pancreatic β cells (L1 mice). In this study, we used a similar approach to reconstitute insulin signaling in tissues that display insulin-dependent glucose uptake. Using GLUT4-Cre mice, we restored InsR expression in muscle, fat, and brain of Insr(-/-) mice (GIRKI (Glut4-insulin receptor knock-in line 1) mice). Unlike L1 mice, GIRKI mice failed to thrive and developed diabetes, although their survival was modestly extended when compared with Insr(-/-). The data underscore the role of developmental factors in the presentation of murine diabetes. The broader implication of our findings is that diabetes treatment should not necessarily target the same tissues that are responsible for disease pathogenesis.     

Inhibition of RPS6K reveals context-dependent Akt activity in luminal breast cancer cells

Aberrant signaling through insulin (Ins) and insulin-like growth factor I (IGF1) receptors contribute to the risk and advancement of many cancer types by activating cell survival cascades. Similarities between these pathways have thus far prevented the development of pharmacological interventions that specifically target either Ins or IGF1 signaling. To identify differences in early Ins and IGF1 signaling mechanisms, we developed a dual receptor (IGF1R & InsR) computational response model. The model suggested that ribosomal protein S6 kinase (RPS6K) plays a critical role in regulating MAPK and Akt activation levels in response to Ins and IGF1 stimulation. As predicted, perturbing RPS6K kinase activity led to an increased Akt activation with Ins stimulation compared to IGF1 stimulation. Being able to discern differential downstream signaling, we can explore improved anti-IGF1R cancer therapies by eliminating the emergence of compensation mechanisms without disrupting InsR signaling.