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Hexosamine Biosynthesis Pathway Flux Contributes to Insulin Resistance via Altering Membrane Phosphatidylinositol 4,5-Bisphosphate and Cortical Filamentous Actin

Departments of Cellular and Integrative Physiology (P.B., G.R.P., E.M.H., A.M.M., J.S.E.) and Biochemistry and Molecular Biology (K.M.H., J.S.E.), Indiana University School of Medicine, Center for Diabetes Research, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Jeffrey S. Elmendorf, Ph.D., Department of Cellular and Integrative Physiology, Indiana University School of Medicine, VanNuys Medical Science Building Room 308A, Indianapolis, Indiana 46202. E-mail: jelmendo{at}iupui.edu.

We recently found that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP₂)-regulated filamentous actin (F-actin) polymerization was diminished in hyperinsulinemic cell culture models of insulin resistance. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes the PIP₂/F-actin dysregulation and insulin resistance induced by hyperinsulinemia. Increased HBP activity was detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nM insulin for 12 h) and in cells where HBP activity was amplified by 2 mM glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP₂ and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP₂ corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP₂/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored PIP₂-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of glutamine:fructose-6-phosphate amidotransferase was associated with a loss of detectable plasma membrane PIP₂ and insulin sensitivity. A slight decrease in intracellular ATP resulted from amplifying HBP by hyperinsulinemia and GlcN. However, experimental maintenance of the intracellular ATP pool under both conditions with inosine did not reverse the PIP₂/F-actin-based insulin-resistant state. Furthermore, less invasive challenges with glucose, in the absence of insulin, also led to PIP₂/F-actin dysregulation. Accordingly, we suggest that the functionality of cell systems dependent on PIP₂ and/or F-actin status, such as the glucose transport system, can be critically compromised by inappropriate HBP activity.