Neuronal Nitric Oxide Synthase Phosphorylation Under Normal and Insulin‐Resistant Conditions in Mouse Skeletal Muscle

Type 2 Diabetes (T2DM) is the seventh leading cause of death in the United States, and results from reduced insulin sensitivity coupled with a relative failure of insulin secretion. Reduced insulin sensitivity itself has been associated with decreased nitric oxide synthase (NOS) activity and impaired glucose uptake in T2DM human skeletal muscle. Upon insulin stimulation, NO synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults. We have previously shown that neuronal NOS (nNOS) is phosphorylated at Ser1446, resulting in its activation, in response to insulin in mouse skeletal muscle in vitro and in vivo . But it is still unclear which kinase is responsible, and how insulin resistance in skeletal muscle affects nNOS Ser1446 phosphorylation. In C2C12 myotubes, nNOS phosphorylation was dependent on one or more AKT isoforms, as AKTi, which prevents AKT1/2 activation, and the AKT2‐specific inhibitor, AKT2i, both inhibited nNOS phosphorylation. As AKT's activity is inhibited in insulin resistance, we utilized a 45% high fat diet (HFD) mouse model of obesity‐induced insulin resistance. In this HFD model, basal levels of phospho‐nNOS increased compared to those in a control diet. However, insulin failed to stimulate nNOS phosphorylation in skeletal muscle of the HFD mice, whereas insulin robustly increased phospho‐nNOS in control diet mice. Taken together, these results suggest that while nNOS may depend on AKT2 for Ser1446 phosphorylation, this phosphorylation is attenuated under insulin resistance in mice. However, basal nNOS phosphorylation overall is increased, suggesting that alternative mechanisms for nNOS phosphorylation exist in mice compared to humans.