Evidence for a novel, reversible mechanism of mitochondrial dysfunction in insulin-resistant skeletal muscle

Insulin resistance and type 2 diabetes are associated with a decrease in mitochondrial function. Recent studies have demonstrated that an early etiological feature of diet-induced skeletal muscle (SkM) insulin resistance is increased plasma membrane (PM) cholesterol that impairs insulin-stimulated glucose transporter GLUT4-mediated glucose transport. Here we tested whether accompanying buildup of SkM mitochondrial membrane cholesterol manifests and contributes to mitochondrial dysfunction. Using genetically modified L6 SkM myotubes that allow for the rapid isolation of mitochondria, we found that 200 mM palmitate, a physiologically relevant hyperlipidemia model system demonstrated to cause de novo cholesterol biosynthesis, PM accumulation, and cellular insulin resistance, also increased mitochondrial cholesterol content. Paired analyses indicated a 25% ( p =0.02) reduction in cellular respiration which was fully reversed in cells treated with the cholesterol-lowering agent methyl-beta-cyclodextrin (MbCD). In contrast, we found experimental loading of L6 SkM myotubes with exogenous cholesterol coupled to MbCD increased mitochondrial cholesterol content and reduced mitochondrial respiration. Consistent with previous study which demonstrated that increased hexosamine biosynthesis pathway (HBP) activity mediates this palmitate induced cholesterolgenic response, siRNA knockdown of the rate limiting enzyme in the HBP pathway (GFPT1), reduced mitochondrial cholesterol accumulation and dysfunction under palmitate culturing conditions. In agreement with this derangement, we also measured increases in key mitochondrial dysfunction readouts ( e.g., lipid peroxidation, ROS ) during palmitate challenge, which were reversed with MbCD or GFPT1 knockdown. Ongoing studies of primary human SkM from diabetic donors that maintain ‘diabetic memory’ reveal an increase in mitochondrial cholesterol content and impaired mitochondrial respiration compared to that measured in SkM from non-diabetic donors. Together, these data provide evidence for a novel diet-induced etiology of mitochondrial dysfunction entailing excess nutrient flux through the HBP triggering cholesterol biosynthesis and accumulation in the mitochondria. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.