9 - Therapeutic Targeting of Skeletal Muscle Nix in Early-Onset Insulin Resistance

Therapeutic targeting of skeletal muscle Nix in early-onset insulin resistance lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that Nix, a lipotoxicity-responsive gene, accumulates in response to diacylglycerols induced by high-fat and sucrose (HFS) feeding and exacerbated by exposure to gestational diabetes (GDM) during fetal development. Here we identify a novel phosphorylation residue, activated by cilomilast treatment that can prevent Nix-induced mitochondrial dysfunction in muscle cells. In a series of gain- and loss-of-function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium-dependent activation of DRP-1 and mitophagy. In addition, Nix-induced mitophagy leads to myotube insulin resistance through activation of mTOR-S6K inhibition of IRS-1. Through detailed phospho-peptide mapping of Nix, we identified a novel phosphorylation residue within the transmembrane domain, modulated by PKA activating agents, such as adrenergic agonist clenbuterol and the phosphodiesterase-4 inhibitor cilomilast. Treatment of myotubes with these agents serves to prevent Nix-induced mitochondrial dysfunction and restore insulin sensitivity. Furthermore, Nix knock-down or clenbuterol/cilomilast treatment rescued palmitate-induced phosphorylation of Ser1101 on the insulin receptor substrate-1 (IRS-1) and prevented insulin resistance. These findings provide insight into the role of Nix-induced mitophagy and muscle insulin resistance during an overfed state. Finally, our data supports the hypothesis that Nix regulates mitochondrial metabolism and insulin signalling in myotubes and suggests a mechanism by which pharmacological activation of PKA may circumvent the mitochondrial dysfunction characteristic of insulin resistance.