Phosphoinositide 3-Kinase-mediated Reduction of Insulin Receptor Substrate-1/2 Protein Expression via Different Mechanisms Contributes to the Insulin-induced Desensitization of Its Signaling Pathways in L6 Muscle Cells

Abstract

Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/ protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviraldriven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts.

Specificity of insulin signalling in human skeletal muscle as revealed by small interfering RNA

“…This finding of differential signalling responses would suggest that IRS1 relays signals to the metabolic arm of the insulin signalling cascade, whereas IRS2 relays signals along gene regulatory pathways. Our results in human myotubes are consistent with earlier studies in L6 muscle cells [27,35], in which siRNA directed against either Irs1 or Irs2 reduced insulin action on AKT phosphorylation and siRNA targeting Irs2 reduced insulin action on ERK1/2 MAPK. However, in L6 myotubes, the link between AKT activation and IRS isoforms is less clear.…”

“…In one study [27], depletion of IRS1 reduced AKT1 and AKT2 phosphorylation, whereas depletion of IRS2 reduced only AKT2 phosphorylation. Conversely, in another study [35], IRS2, not IRS1, was shown to be the primary mediator of insulin-stimulated AKT phosphorylation. The reason for the differences between these studies is unclear; however, the introduction of siRNA into a cell may also have an unknown 'off-target' effect that influences cellular signalling and metabolism above and beyond any effect associated with the gene of interest.…”

Insulin Receptor Substrate-2-dependent Interleukin-4 Signaling in Macrophages Is Impaired in Two Models of Type 2 Diabetes Mellitus

“…New work by Pirola et al (55) shows that in L6 muscle cells, prolonged insulin treatment leads to a reduction in IRS-2 protein levels which is dependent on the PI3-kinase/mTOR pathway. In primary skeletal muscle cells from patients with impaired glucose tolerance, however, IRS-2 protein levels were unaffected by high glucose, although insulin-dependent IRS-2 tyrosine phosphorylation and associated PI3-kinase activity were decreased significantly (56).…”

Insulin Resistance Due to Phosphorylation of Insulin Receptor Substrate-1 at Serine 302

“…The antibodies were prepared against a phosphopeptide sequence corresponding to the region of IRS-1 surrounding Ser-302 (RRSRTEpSITATSP) that is identical in rats (Ser-302), mice (Ser-302), and humans (Ser-307) (45)(46)(47). The affinity-purified antibodies were used for Western blotting experiments.…”