Clarke, Mark S. F.2019-06-242019-06-24December 22018-12December 2https://hdl.handle.net/10657/4036Insulin resistance is a condition characterized by the inability of insulin to stimulate glucose uptake in skeletal muscle. This can lead to serious diseases such as metabolic syndrome, diabetes mellitus 2, cancer and blindness. The onset and progression of insulin resistance is correlated with a variety of environmental-related conditions, such as a high fat diet, chronic physical inactivity and sarcopenia, as well as physiological conditions such as obesity, hypercholesterolemia and chronic inflammation. It is clear that insulin resistance in skeletal muscle and the onset of Type 2 diabetes is correlated with a number of conditions that exhibit abnormal regulation of lipid metabolism, such as obesity and hypercholesterolemia. However, it still remains unclear if and how elevated circulating levels of cholesterol impact myofiber function at a cellular level, resulting in the inhibition of insulin signaling and glucose uptake. Mounting evidence suggests that accrual of cholesterol within skeletal muscle myofiber membranes may be causally linked to skeletal muscle insulin resistance and the onset of Type 2 diabetes. Cholesterol is found in both the external sarcolemma/T-tubule and internal sarcoplasmic reticulum membranes of skeletal myofibers. In the case of sarcolemma/T-tubule membranes, cholesterol-enriched micro-domains called caveolae contain insulin receptors and glucose transporter 4 (GLUT4) docking proteins. Cholesterol-rich caveolae are the site of GLUT4 vesicle fusion with the sarcolemma/T-tubule membrane system during insulin-stimulated GLUT-4 mediated myofiber glucose uptake. Cholesterol is also found in sarcoplasmic reticulum membranes, a complex intra- membrane system from which GLUT-4 containing membrane vesicles are derived. The sarcoplasmic reticulum is also the source and storage organelle for the intracellular calcium required for SNARE-mediated GLUT4 vesicle fusion with the external sarcolemma/T-tubule membrane which is the final step in insulin stimulated myofiber glucose uptake. Evidence to suggest that overall myofiber membrane cholesterol content is an important regulator of insulin stimulated glucose uptake comes from in vivo and in vitro studies where increasing or decreasing myofiber membrane cholesterol content resulted in the inhibition or enhancement of insulin-stimulated glucose uptake. Here we demonstrate a reliable and reproducible tissue culture model of skeletal myofibers consisting of highly differentiated C2C12 myotubes in which to mechanistically investigate the effects of selectively enriching or depleting the sarcolemma/T-tubule and/or sarcoplasmic reticulum membrane alone on insulin-stimulated glucose uptake. Selective enrichment or depletion of C2C12 myotube membranes with native cholesterol was achieved by exposing differentiated C2C12 myotubes to a methyl-β-cyclodextrin/cholesterol complex or methyl-β-cyclodextrin alone, utilizing specific pulse-chase labeling conditions in tissue culture. Confirmation that these defined pulse-chase labeling conditions selectively enriched or depleted specific myotube membranes was confirmed using a fluorescent analog of cholesterol, 23-(dipyrrometheneboron-difluoride)-24-norcholesterol, which in turn was spatially resolved within myotube membranes using scanning confocal microscopy. The effects of selective cholesterol membrane enrichment in differentiated C2C12 myotubes on insulin-stimulated glucose uptake were determined by measuring the uptake of 2-NBDG, a fluorescent analog of glucose. A 5x6-way ANOVA was used to determine any differences in insulin stimulated glucose uptake between membrane enrichment/depletion conditions across a range of insulin concentrations, while one-way ANOVAs were used to determine the effects of insulin concentration within each enrichment condition, as well as any differences between enrichment conditions for particular insulin concentrations. Using this defined tissue culture model of insulin stimulated glucose uptake in skeletal muscle myotubes, we demonstrate that increased membrane cholesterol content is causally related to disruption of insulin receptor signaling and inhibition of insulin stimulated glucose uptake in C2C12 myotubes. We further demonstrate that this inhibition is greatest when the difference between the relative concentrations of cholesterol in the sarcolemma/T-tubule and the SR membranes is highest, rather than when the total cholesterol content of either membrane system is increased. Our findings provide direct experimental evidence that increased muscle membrane cholesterol content is causally related to inhibition of insulin signaling, insulin stimulated glucose uptake, and by extension GLUT4 translocation in skeletal muscle. These findings have significant physiological implications, especially as they relate to the underlying cellular mechanisms responsible for the development and onset of insulin resistance.application/pdfengThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. 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