Regulation of Neuronal Nitric Oxide Synthase by Insulin in Skeletal Muscle

Type 2 Diabetes (T2DM) is the seventh leading cause of death in the United States, and is quickly becoming a global pandemic. Its patients suffer either reduced insulin production or insulin sensitivity, the latter of which has been associated with reduced nitric oxide synthase (NOS) activity in T2DM skeletal muscle. NOS proteins produce the gaseous signaling molecule nitric oxide (NO), which has been implicated in glucose uptake and insulin sensitivity. In skeletal muscle, NO is produced by neuronal nitric oxide synthase (nNOS), the tissue's primary NOS isoform. Upon insulin stimulation, NO synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults. Additionally, the endothelial NOS (eNOS) isoform in the vasculature is activated via insulin-activated kinase phosphorylation. That nNOS contains a homologous phosphorylation site is suggestive that nNOS, too, may undergo an activating phosphorylation event with insulin treatment. Yet, it remains unknown if or how nNOS is regulated by insulin in skeletal muscle. Our working hypothesis is that skeletal muscle nNOS is phosphorylated and activated by an insulin-activated kinase(s) during insulin signaling, and that under insulin-resistant conditions, this phosphorylation is attenuated. This will be addressed by the following specific aims: 1) establish that skeletal muscle nNOS is phosphorylated in response to insulin in vitro, and that this phosphorylation affects NO production; 2) identify the kinase(s) activated by insulin that phosphorylates skeletal muscle nNOS under insulin treatment in vitro; and 3) demonstrate that skeletal muscle nNOS is phosphorylated in response to insulin in vivo using mouse models, and that this phosphorylation is attenuated under conditions of insulin resistance. These studies will provide training in molecular biochemistry (immunoblot analysis, mass spectrometry, fluorescent activity assays), cell culture (immunocytochemistry, pharmacological inhibitors, siRNA knockdown, dominant-negative inhibition), and animal models (immunohistochemistry, insulin clamping, diet-induced insulin resistance). These studies are innovative because the role of nNOS in skeletal muscle insulin signaling has yet to be explored, despite it being the primary skeletal muscle NOS isoform. The proposed research is significant because it will more fully reveal fundamental function(s) of NOSs in skeletal muscle insulin signaling. As a result, these studies will have a significant impact on our understanding of skeletal muscle insulin signaling and will lead to development of therapeutic targets to improve skeletal muscle insulin sensitivity, as well as a number of other disorders in which nNOS has been implicated. The applicant's career goal is to become an endocrinologist-investigator who utilizes biochemical approaches to metabolic disease. An integrated educational plan and supportive environment ensures the applicant's career development. PUBLIC HEALTH RELEVANCE: Type 2 diabetes (T2DM), the seventh leading cause of death in the USA, is a chronic disease that primarily affects insulin sensitivity in skeletal muscle. Neuronal nitric oxide synthase (nNOS) is an enzyme whose activity is decreased in T2DM patient's skeletal muscle. The planned studies will improve understanding of nNOS's role in skeletal muscle insulin signaling and will lead to development of therapeutic targets to improve skeletal muscle insulin sensitivity.