Transient Activation of Skeletal Muscle Rac1 in Obesity-Related Insulin Resistance

Rac1 is required for normal insulin-stimulated glucose transport in skeletal muscle and may be negatively regulated by lipids. We investigated if insulin stimulation of Rac1 was impaired with the accumulation of lipids in skeletal muscle, serving as a mechanism of insulin resistance with obesity. Male C57BL/6J mice consumed a 60% high-fat diet (HFD) for 12 weeks to induce obesity compared with low-fat diet (LFD). Insulin sensitivity and signaling were assessed using hyperinsulinemic-euglycemic (INS) or saline (CON) clamps (n=7-10 per diet/clamp). Rac1 activation and other insulin signaling was measured in quadriceps muscle harvested following the clamp conditions. Muscle lipids were assessed via liquid chromatography, tandem mass spectrometry. Activation of Rac1 (via GTP binding) was not increased in INS compared with CON conditions (P = 0.60) in either LFD or HFD. Further, insulin-stimulated pAkt^Ser473 was similar between groups (P = 0.90) despite HFD mice having greater fat mass (P < 0.01), lower glucose infusion rates during a 2-hour INS clamp (P < 0.02), and greater skeletal muscle lipid content, including total ceramide (P = 0.03) and numerous 1,2 diacylglycerols (P < 0.05). We considered if Rac1 activation is transient since Rac1 activity was not changed after the 2-hour INS condition. Time-course experiments in cultured L6 myotubes revealed that 2-min of insulin stimulation was sufficient to increase Rac1-GTP binding (P < 0.01 vs. basal) and activation returned to basal by 10-min (P = 0.23 vs. basal). Modeling insulin resistance using overnight fatty acid treatments did not alter insulin-stimulated Rac1-GTP binding compared with no fatty acid control cells (P = 0.44).

In summary, skeletal muscle Rac1 activation by insulin is transient, but may be a key determinant of impaired glucose uptake with obesity in mice given the lack of impairment in Akt activation. In contrast, overnight fatty acid incubation was not sufficient to inhibit activation of Rac1 by insulin in L6 myotubes.