NAASO Meeting 2005, Vancouver #3
Profound Insulin Resistance of PKB (Akt) Activation in Intra-abdominal Adipocytes from Obese Hypertensive SHROB rats
Zheng Sun, B.S. and Paul Ernsberger, Ph.D.. 1Cleveland, OH, United States.
Background: The obese spontaneously hypertensive rat (SHROB) is an established model for human metabolic syndrome X, expressing insulin resistance, hypertension, obesity, and hyperlipidemia. Previous studies showed reduced cellular responses to insulin in liver and skeletal muscle in SHROB relative to lean SHR littermates. Specifically, tyrosine phosphorylation of the insulin receptor and its substrate are reduced. We now tested adipocytes and examined a further downstream step insulin signaling through PKB (Akt), which has been implicated in human insulin resistance. SHROB and SHR rats were matched for gender and age, and 4 g of the gonadal depot was digested 1h at 37oC with collagenase. Floating cells were filtered through 200 m mesh, preincubated, then exposed to insulin concentrations ranging from 0 to 100nM insulin for 0 to 90 min. Cells were lysed by free-thaw in medium containing detergent. Aliquots of cell extracts were analyzed by Western blot. PKB activation was defined as the ratio of immunoreactivity for phosphorylated PKB to total PKB in each sample. Lean SHR adipocytes showed peak 25-fold activation of PKB at 3 min. SHROB adipocytes, in contrast, showed peak activation of only 4-fold, maintained for 90 min. In dose-response experiments, SHR and SHROB adipocytes showed large differences in maximum fold response (Emax) at 10 min: 18.8 +/- 2.3 vs. 3.7 +/- 0.8. Also consistent with reduced insulin sensitivity, the ED50 for insulin was lower in SHR than in SHROB (3.5 +/- 0.5 vs. 29 +/- 3.8 nM). Thus, the PKB activation step of the insulin signaling pathway in adipocytes from SHROB shows profound resistance to activation by insulin, suggesting that this step could be an important indicator or target for the treatment of insulin resistance.
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