H. Stierwalt1, B. Bergman2, M. Robinson1, S. Newsom1

1Oregon State University, Corvallis, OR; 2University of Colorado Denver, Denver, CO

PURPOSE: Rac1 is required for normal insulin-stimulated glucose transport in skeletal muscle. Evidence indicates that Rac1 may be negatively regulated by lipids, and this may be a novel mechanism of insulin resistance in obesity. The purpose of this study was to investigate if obesity-related accumulation of bioactive signaling lipids might impair Rac1 activation in skeletal muscle. METHODS: Male C57BL/6J mice consumed a 60% high-fat diet (HFD) for 12 weeks to induce obesity and insulin resistance compared to low-fat diet (LFD). Insulin sensitivity and skeletal muscle insulin signaling were assessed using hyperinsulinemic-euglycemic (INS) or saline-control (CON) clamps (n=7-10/condition). Rac1 activation (i.e. Rac1-GTP binding) and other steps of insulin signaling were measured in quadriceps muscles using ELISA and western blotting, respectively. Intramyocellular lipids were assessed using liquid chromatography, tandem mass spectrometry. Cultured L6 myotubes were also used to assess insulin-stimulated Rac1 activation in the presence or absence of various fatty acid treatments. RESULTS: Compared with LFD, HFD resulted in greater fat mass (P < 0.01), insulin resistance as evidenced by lower glucose infusion rates during a 2-hour INS clamp (P < 0.02), and greater skeletal muscle lipid content, including total ceramide (+13.9%, P = 0.03) and numerous 1,2 diacylglycerols (P < 0.05). Nevertheless, insulin-stimulated Rac1-GTP binding was not influenced by diet (P = 0.45 for HFD vs. LFD). However, Rac1-GTP binding was not increased in INS compared with CON conditions (P = 0.60), indicating that insulin stimulation of Rac1 may have been transient. Time-course experiments in L6 myotubes revealed that 2-min of insulin stimulation was sufficient to significantly increase Rac1-GTP binding (P < 0.01vs. basal), and this effect was almost completely attenuated by 10-min (P = 0.23vs. basal). Importantly, acute insulin-stimulated Rac1-GTP binding was not attenuated by overnight fatty acid treatment (P = 0.44). CONCLUSION: Our results indicate that skeletal muscle Rac1 activation by insulin is transient, with a measurable increase perhaps limited to < 10-min. Overnight fatty acid incubation was not sufficient to inhibit transient activation of Rac1 by insulin in cultured L6 myotubes.

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