Insulin is the major hormone controlling critical energy functions such as glucose and lipid metabolism. Insulin elicits a diverse array of biological responses by binding to its specific receptor (Ref.1). The insulin receptor belongs to a subfamily of receptor tyrosine kinases that includes the IGF (Insulin-like Growth Factor) receptor and the IRR (Insulin Receptor-Related Receptor). These receptors are tetrameric proteins consisting of two α and two β subunits that function as allosteric enzymes in which the α subunit inhibits the tyrosine kinase activity of the β subunit. Insulin has diverse effects on cells including stimulation of glucose transport, gene expression and alterations of cell morphology. The hormone mediates these effects by activation of signaling pathways which utilize, i) adaptor molecules such the IRS (Insulin Receptor Substrates), the SHC (Src and Collagen Homologues) and the GRB2 (Growth Factor Receptor Binding protein-2), ii) lipid kinases such as PI3K (Phosphatidylinositol 3-Kinase), iii) small G-proteins like Rac, and iv) serine, threonine and tyrosine kinases (Ref.2).
Tyrosine-phosphorylated IRS then displays binding sites for numerous signaling partners. PI3K has a major role in insulin functions. It regulates three main classes of signaling molecules: the AGC family of serine/threonine protein kinases, guanine nucleotide-exchange proteins of the Rho family of GTPases, and the Tec family of tyrosine kinases. The best characterized of the AGC kinases is PDK-1 (Phosphoinositide-Dependent Kinase-1), one of the serine kinases that phosphorylates and activates the serine/threonine kinase Akt/PKB (Protein Kinase-B). Akt possesses a PH domain that also interacts directly with PIP3 (Phosphatidylinositol -3, 4, 5-Triphosphate), promoting membrane targeting of the protein and catalytic activation. Akt has been suggested to be important in transmission of the insulin signal, by phosphorylation of the enzyme GSK3 (Glycogen Synthase Kinase-3), the FKHRL1 (Forkhead-Related Family of Mammalian Transcription Factor) and cAMP (Cyclic Adenosine Monophosphate) response element-binding protein. Akt inhibits apoptosis by phosphorylating the BAD (BCL2 Antagonist of Cell Death) component of the BAD/BCLXL complex. Phosphorylated BAD binds to 14-3-3 causing dissociation of the BAD/BCLXL complex and allowing cell survival, and Akt activates IKK, which ultimately leads to NF-κB (Nuclear Factor-κB) activation and cell survival. Akt also activates the mTOR (Mammalian Target of Rapamycin)/FRAP pathway. Activation of mTOR results in the phosphorylation of ribosomal protein S6 kinase, p70S6K, which is also regulated by phosphorylation by PDK-1. Rapamycin (FRAP) interactions with mTOR also regulate the activity of p70S6K, the kinase that phosphorylates the 40S ribosomal protein S6. S6 is thought to be the only p70S6K substrate, and by controlling S6 phosphorylation, p70S6K regulates the translation of an essential family of mRNAs that contain an oligopyrimidine tract at their transcriptional start site. Activation of mTOR also results in phosphorylation and inactivation of eIF4EBP (Eukaryotic Initiation Factor 4EBinding Protein), also known as PHAS, an inhibitor of the translation initiation factor eIF4E (Eukaryotic Initiation Factor-4E). Insulin induces dephosphorylation of eEF2 (Eukaryotic Elongation Factor-2) and inactivation of eEF2K (Eukaryotic Elongation Factor-2 Kinase), and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR.
Akt and/or the atypical PKCs (Protein Kinase-C) seem to be required for insulin-stimulated glucose transport. The ability of insulin to stimulate glucose uptake relies on a complex signaling cascade that leads to the translocation of GLUT4 (Glucose Transporter Protein-4) from an intracellular compartment to the plasma membrane, which results in increased glucose uptake. While the PI3K/Akt cascade participates in this process, another major pathway leading to GLUT4 translocation involves the insulin receptor-mediated phosphorylation of CAP (c-Cbl Associated Protein) and formation of the CAP:Cbl:CrkII complex. This complex, through its interaction with flotillin, localizes to lipid rafts facilitating GLUT4 translocation, using in the final step a Synip-containing specialized Snare complex (Ref.3). The signaling contributions of other proteins bound by phosphorylated IRSs, including the phosphotyrosine phosphatase SHP2, Fyn, and the SH3- containing adaptor Nck, are yet to be clearly defined. Another important protein involved in insulin signaling is GRB10 (Growth Factor Receptor-Bound Protein 10). GRB10 interacts directly with IR (Insulin Receptor). IR does not phosphorylate GRB10, but phosphorylation by Tyrosine Kinases of the Src family negatively regulates binding to IR. GRB10 is believed to interact with MEK and play a role in signaling. As is the case for other growth factors, insulin stimulates the MAPK (Mitogen-Activated Protein) ERK (Extracellular Signal Regulated Kinase). This pathway involves the tyrosine phosphorylation of IRS proteins and/or SHC, which in turn interact with the adapter protein GRB2, recruiting the SOS (Son of Sevenless) exchange protein to the plasma membrane for activation of Ras. The activation of Ras also requires stimulation of the tyrosine phosphatase SHP2, through its interaction with receptor substrates such as GAB1 (GRB2 Associated Binding Protein-1) or IRS1/2. Once activated, Ras operates as a molecular switch, stimulating a serine kinase cascade through the stepwise activation of Raf, MEK and ERK. Activated ERK can translocate into the nucleus, where it catalyses the phosphorylation of transcription factors such as p62TCF, initiating a transcriptional programme that leads to cellular proliferation or differentiation (Ref.4).
Signal transduction by the insulin receptor is not limited to its activation at the cell surface. The activated ligand receptor complex, initially at the cell surface, is internalized into endosomes, and this process is dependent on tyrosine autophosphorylation. Endocytosis of activated receptors has the dual effect of concentrating receptors within endosomes and allowing the insulin receptor tyrosine kinase to phosphorylate substrates that are spatially distinct from those accessible at the plasma membrane. Acidification of the endosomal lumen, due to the presence of proton pumps, results in dissociation of insulin from its receptor. The endosome constitutes the major site of insulin degradation by the EAI (Endosomal Acidic Insulinase). This loss of the ligand-receptor complex attenuates any further insulin-driven receptor rephosphorylation events and leads to receptor dephosphorylation by extra lumenal endosomally associated PTPs.
PTPase (Protein Tyrosine Phosphatases) catalyze the dephosphorylation of insulin receptor and its substrates, leading to attenuation of insulin action. A number of PTPases have been implicated as the negative regulator of insulin signaling. Among them, the intracellular PTPase, PTP1B, has been shown to function as the insulin receptor phosphatase. PTEN (Phosphatase and Tensin Homolog Deleted On Chromosome-10) negatively regulates insulin signaling. SHIP2 (SH2-containing Inositol Phosphatase-2) is another negative regulator of insulin signaling and such negative regulation depends on its 5'-phopshatase activity. Overexpression of SHIP2 protein decreases Insulin-dependent PIP3 production as well as insulin-stimulated Akt activation, GSK3 inactivation, and glycogen synthetase activation. Insulin increases glucose uptake in muscle and fat, and inhibits hepatic glucose production, thus serving as the primary regulator of blood glucose concentration. Insulin also stimulates cell growth and differentiation, and promotes the storage of substrates in fat, liver and muscle by stimulating lipogenesis, glycogen and protein synthesis, and inhibiting lipolysis, glycogenolysis and protein breakdown. Insulin resistance or deficiency results in profound dysregulation of these processes, and produces elevations in fasting and postprandial glucose and lipid levels.
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