We recently found that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP 2)-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 PIP 2/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 PIP 2 and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP 2 corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP 2/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored PIP 2-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of glutamine:fructose-6-phosphate amidotransferase was associated with a loss of detectable plasma membrane PIP 2 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 inosinedid not reverse the PIP 2/F-actin-based insulin-resistant state. Furthermore, less invasive challenges with glucose, in the absence of insulin, also led to PIP 2/F-actin dysregulation. Accordingly, we suggest that the functionality of cell systems dependent on PIP 2 and/or F-actin status, such as the glucose transport system, can be critically compromised by inappropriate HBP activity.
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