Downregulation of muscle cell Insr and insulin receptor signaling by hyperinsulinemia in vitro and in vivo

Hyperinsulinemia is often viewed as compensatory to insulin resistance, but studies have shown that high levels of insulin may also contribute to insulin resistance. The precise mechanisms by which hyperinsulinemia contributes to insulin resistance remain poorly defined. To understand the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated differentiated C2C12 mouse myotubes with elevated insulin for 16 hours, followed by 6 hours serum starvation, before examining key insulin signaling nodes. Using this model, we found that prolonged high insulin treatment significantly increased the phosphorylation of insulin receptor (INSR) and AKT, but not ERK. After serum starvation, acute AKT and ERK signaling stimulated by 0.2 - 20 nM insulin was attenuated. INSR protein levels were significantly downregulated by hyperinsulinemia in an insulin-dose-dependent manner. Surface INSR was reduced proportionally to total INSR levels. Mechanistically, we found that hyperinsulinemia strongly downregulated Insr mRNA which was correlated with increased threonine 24 phosphorylation of FOXO1. Interestingly, 6h serum starvation reversed the effects of high insulin on basal phosphorylation of INSR, AKT and FOXO1, and Insr transcription. Using RNA sequencing, bioinformatic analysis, and follow-up RNAi studies, we identified SIN3A as a negative regulator of Insr mRNA levels and JUND, MAX and MXI as positive regulators of Irs2 mRNA. We validated our in vitro results by determining that INSR levels in mouse skeletal muscle were negatively correlated with circulating insulin in vivo. Together, our findings shed new light on the mechanisms underlying hyperinsulinemia-induced insulin resistance in muscle cells, which are likely to be relevant in the pathogenesis of type 2 diabetes.