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International Textbook of Diabetes Mellitus, 4th Ed., Excerpt #63: Mechanisms of Insulin Signal Transduction Part 7 of 8

Insulin resistance in humans: abnormalities in insulin signaling and in the glucose transport effector system

Abnormalities in insulin signaling

Although the key elements of the insulin signaling network have been defined by studies employing molecular and cell biology techniques, a number of studies have focused on translating this knowledge to human studies in a clinical research setting. Insulin resistance in peripheral tissues characterizes obesity and T2DM, and is involved in the pathogenesis of diabetes [187]. Studies using hyperinsulinemic, euglycemic clamps have demonstrated that systemic insulin resistance is associated with reduced stimulation of glucose transport in muscle and adipose tissues biopsied from these same subjects [188]. Further studies in these insulin target tissues have elucidated defects in insulin signaling as well as defects intrinsic to the glucose transport system in insulin-resistant humans.

Regarding insulin signal transduction, investigators have demonstrated reductions in insulin-stimulated tyrosine phosphorylation of both insulin receptor and IRS-1, decreased association of PI-3 kinase with IRS-1, and more modest decrements in insulin-stimulated Akt phosphorylation [189,190]. Despite these defects, insulin stimulation of ERK2 phosphorylation was normal or even elevated in muscle from patients with T2DM [189,190]. This dichotomy between insulin-stimulated PI-3 kinase and MAP kinase signaling later was extended to insulin signaling in vascular cells under conditions that produce insulin resistance [191]. The implications of this dichotomy remain unclear, but it is conceivable that maintenance of this imbalance could lead to worsening of insulin resistance through serine phosphorylation of IRS-1 by ERK [192]. To probe causal relationships, other studies employed perturbations to either enhance or diminish in vivo insulin sensitivity to determine if there were commensurate changes in phosphorylation/dephosphorylation of the insulin receptor, IRS-1, and PI-3 kinase. Using muscle biopsies and euglycemic clamps, investigators have shown beneficial effects of treatment with thiazolidinediones [193], exercise [194], caloric restriction [195], and tight glucose control [196]. In contrast, insulin resistance provoking treatments like infusion of a triglyceride emulsion worsened insulin signaling defects [197].

One of the methodological breakthroughs that enabled investigators to define mechanisms of phosphorylation-induced activation or inactivation of insulin signaling proteins was the use of motif-specific antibodies directed against phosphopeptide motifs present in these proteins. Although this provided a powerful and facile method approach in in vivo studies, there always remain questions of specificity and quantification. Furthermore, with these techniques, only changes in known phosphorylation events can be assessed. A relatively new addition to the tool kit used to analyze insulin signaling is mass spectrometry based proteomics techniques, especially applied to posttranslational modifications, and in particular, phosphorylation, due to its importance in regulating activity of signaling proteins. Mass spectrometry allows for identification of novel phosphorylation sites in known insulin signaling proteins and the discovery of new insulin signaling proteins that exhibit insulin-induced changes in phosphorylation. These techniques have proven to be invaluable for deciphering the complexity of serine and threonine phosphorylation sites in IRS proteins [198,199] and AS160 [130]. These methods allow for simultaneous quantification of many phosphorylation sites at the same time, unlike immunoblot analysis. For example, Humphries et al. were able to identify 37,248 phosphorylation sites on 5705 proteins in 3T3-L1 adipocytes, with approximately 15% responding to insulin. This led to the discovery that SIN1, a core component of the mTORC2 complex, is an Akt substrate, and the phosphorylation of SIN1 by Akt regulates mTORC2 activity in response to growth factors [200].

The glucose transport system

Given that plasma membrane glucose transport is rate-limiting for insulin-stimulated glucose metabolism in insulin target cells, defects in GLUT4 expression, diminished functional activity, or impaired translocation could readily explain insulin resistance [201]. The multicompartmental nature of GLUT4 cellular trafficking, requiring a multiplicity of regulatory proteins, indicates that there are many potential sites for defects that could impair GLUT4 translocation. Indeed, defects intrinsic to the glucose transport system have been shown to be an important cause of insulin resistance in muscle and fat tissues from insulin-resistant and diabetic patients. In adipocytes, cellular depletion of GLUT4 transporters is a major mechanism of insulin resistance in obesity and T2DM [202], while GLUT4 expression is relatively normal in skeletal muscle [203]. However, in both muscle and fat, GLUT4 accumulates or is redistributed to a dense membrane compartment under basal conditions, and this abnormality is linked to impaired GLUT4 translocation to the plasma membrane in response to insulin [204 – 206] (see Figure 12.6). These data are indicative of a trafficking or targeting abnormality impairing GLUT4 translocation in tissues derived from insulin-resistant subjects. Additional study of pathways and factors involved in abnormal GLUT4 traffic may elucidate mechanisms causing human insulin resistance and establish targets for drug development.

ITDM Fig. 12.6

Modulation of insulin action

Important recent advances in the study of insulin signaling include the elucidation of multiple mechanisms for dampening or inhibiting insulin signal transduction. The studies have provided insight into the extensive regulation of insulin action, and have also identified potential therapeutic targets, since blocking these inhibitory mechanisms could enhance insulin sensitivity. These mechanisms are summarized in Figure 12.7.

ITDM Fig. 12.7

Serine-threonine phosphorylation of the insulin receptor and IRS proteins

Serine-threonine phosphorylation of insulin receptors and insulin substrate docking proteins has emerged as a major mechanism for modulation of insulin signal transduction. Serine phosphorylation of the insulin receptor diminishes its tyrosine kinase activity [10]. Serine phosphorylation of IRS-1 (and other IRSs) decreases receptor-IRS coupling by inhibiting insulin-mediated receptor phosphorylation, tyrosine phosphorylation of IRS-1, binding and activation of PI-3 kinase, and stimulation of glucose transport [207 – 209]. There are consensus sequences in IRS-1 that make it susceptible to a wide variety of serine-threonine kinases including PKC, PKA, Akt/PKB, MAP kinase, GSK3, casein kinase II, Cdc2 kinase, and JKN. Several of these kinases have been shown to function as physiologic modulators causing desensitization of insulin signaling pathways [210].

Protein kinase C

Protein kinase C isoforms are categorized as classic (α,β,γ), novel (δ,θ,ε,η,μ), and atypical (ζ,λ,ι) depending on their ability to be activated by calcium and diacylglycerol (DAG). These serine-threonine kinases can act on multiple substrates, including IRS docking proteins and the insulin receptor [10,207,208]. Serine-threonine phosphorylation of IRS impairs its ability to associate with the insulin receptor and with PI-3 kinase, resulting in desensitization of the PI-3 kinase pathway. Hyperinsulinemia, hyperglycemia, and elevated circulating free fatty acids increase intracellular DAG, which in turn activates conventional and novel PKC isoforms principally via recruitment to the plasma membrane. These conditions are associated with increased serine-threonine phosphorylation and diminished function of insulin receptors and IRS proteins. In addition, insulin activates atypical PKCs, such as PKCζ, via the PI-3 kinase pathway, and atypical PKCs are then also capable of phosphorylating and desensitizing IRS [211,212]. Thus, PKCζ could be involved both in direct promulgation (Figure 12.2) and in feedback inhibition (Figure 12.7) of insulin signal transduction.

Tumor necrosis-