Neuronal nitric oxide synthase is phosphorylated in response to insulin stimulation in skeletal muscle

Abstract

Type 2 Diabetes (T2DM) is the seventh leading cause of death in the United States, and is quickly becoming a global pandemic. T2DM results from reduced insulin sensitivity coupled with a relative failure of insulin secretion. Reduced insulin sensitivity has been associated with reduced nitric oxide synthase (NOS) activity and impaired glucose uptake in T2DM skeletal muscle. Upon insulin stimulation, NO synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults. Endothelial NOS is activated by phosphorylation in the C-terminal tail in response to insulin. Neuronal NOS (nNOS), the primary NOS isoform in skeletal muscle, contains a homologous phosphorylation site, raising the possibility that nNOS, too, may undergo an activating phosphorylation event upon insulin treatment. Yet it remains unknown if or how nNOS is regulated by insulin in skeletal muscle. Data shown herein indicate that nNOS is phosphorylated in response to insulin in skeletal muscle and that this phosphorylation event occurs rapidly in C2C12 myotubes, resulting in increased NO production. In vivo phosphorylation of nNOS was also observed in response to insulin in mouse skeletal muscle. These results indicate, for the first time, that nNOS is phosphorylated in skeletal muscle in response to insulin and in association with increased NO production.

GSNOR Deficiency EnhancesIn SituSkeletal Muscle Strength, Fatigue Resistance, and RyR1 S-Nitrosylation Without Impacting Mitochondrial Content and Activity

“…Increases in RyR1-associated nNOSl activity may also contribute to RyR1 hypernitrosylation (21,39,67). To test the possibility that nNOSl was also driving RyR1 hypernitrosylation, we determined nNOSl protein expression and the fraction of active serine 1446 phosphorylated nNOSl (35,52). nNOSl expression and the fraction of ser 1446 phosphorylated nNOSl were unaffected in GSNOR -/-muscles, suggesting that nNOSl was not driving RyR1 hypernitrosylation ( Fig.…”

Neuronal nitric oxide synthase mediates insulin- and oxidative stress-induced glucose uptake in skeletal muscle myotubes

“…It has been reported that exercise induces the phosphorylation of nNOSμ at S1446 in skeletal muscle via AMP-activated protein kinase (AMPK), which is itself activated by exercise [43, 44]. Our previous work demonstrated that insulin signaling in myotubes and H 2 O 2 treatment of cardiomyocytes also leads to phosphorylation of nNOSμ S1446 and a concomitant increase in NO production (Figure 1; [28, 38]), so a similar mechanism was considered. As shown in Figure 6A, B, treatment with 400 µM H 2 O 2 resulted in a 2.3-fold increase in nNOSμ S1446 phosphorylation.…”

“…Previous results from our laboratory demonstrated that nNOSμ was phosphorylated at S1446 in C2C12 myotubes and in mouse skeletal muscle via the insulin-signaling cascade, resulting in increased NO synthesis [38]. A likely candidate for catalysis of this phosphorylation is the insulin-activated protein kinase B (Akt), more specifically, the Akt2 isoform.…”

“…Coupled with studies that NOS KO mice are insulin resistant [13], this suggests that insulin action in SkM requires NO production and that impaired NO synthesis contributes to insulin resistance in T2DM. Studies in our laboratory demonstrating that nNOSμ is phosphorylated in a key intrinsic regulatory region at S1446 in response to insulin treatment [14] suggest that the molecular mechanism for the response to insulin seen in the human studies involves phosphorylation of S1446. Protein kinase B (Akt) is an established signal transduction kinase that controls cellular energy homeostasis [15] and could be responsible for signal transduction controlling NO production through phosphorylation of S1446.…”

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms

“…Because it is not possible to specifically measure nNOS activity alone in vivo, we evaluated the fraction of active serine1446 phosphorylated nNOSl, the primary enzymatic NO source in muscle (34,61). Loss of GC1 decreased the fraction of active serine1446 phosphorylated nNOSl by 40% ( Supplementary Fig.…”