Downregulation of Grb2 contributes to the insulin-sensitizing effect of calorie restriction

Calorie restriction (CR) alleviates insulin resistance and has a beneficial effect on numerous metabolic disorders, yet the underlying mechanism has not been fully elucidated. In the present study, we found that CR of mice (60% of the diet consumption compared with ad libitum mice) reduces the expression levels of Grb2 in skeletal muscle, an insulin target tissue that accounts for 85% of insulin-stimulated blood glucose clearance. To determine whether Grb2 downregulation contributes to increased insulin sensitivity in the regulation of glucose metabolism, we generated C₂C₁₂ cell lines in which the expression of Grb2 is suppressed by RNA interference. Suppressing Grb2 expression in C₂C₁₂ myoblasts enhances insulin-stimulated insulin receptor substrate (IRS)-1, tyrosine phosphorylation, and Akt phosphorylation, which is associated with decreased IRS-1 serine phosphorylation at residues 307, 612, and 636/639. In addition, reducing Grb2 expression levels increased insulin-stimulated glucose uptake in C₂C₁₂ myotubes. Reduced IRS-1 serine phosphorylation is also found in Grb2 ^+/- heterozygous knockout mice, which is associated with enhanced insulin signaling and resistance to high-fat diet-induced glucose and insulin intolerance. All together, our results suggested that reducing the expression levels of Grb2 provides a mechanism by which CR increases insulin sensitivity in vivo.