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Effects of free fatty acids on insulin stimulated amino acid transport in cultured rat hepatocytes

Aim. The aim of this study was to determine the possible role of free fatty acids in insulin signalling. Methods. Hepatocytes were isolated by a modified collagenase perfusion technique of Berry and Friend and cultured for 24 h in M199 serum-free medium. To assess amino acid transport hepatocytes were incubated in Hanks-Hepes medium containing ?-amino14C-isobutyric acid (AIB) to the final concentration of 1 mmol/L. One hour later the medium was removed and hepatocytes were quickly frozen in liquid nitrogen after three washes with cold saline. The cells were digested in 0.2 mol/L NaOH and a aliquot were taken for determination of protein and radioactivity. Results. In hepatocytes obtained from rats on standard diet insulin produced almost 100% increase of AIB transport (P<0.01). Free fatty acids did not change basal AIB transport but strongly reduced insulin effect (> 50%) (P<0.01). Reduction of insulin-stimulated transport was achieved one hour after addition of oleic acid. On the other side removing of oleic acid also very quickly after 3 hours restore normal condition. Pretreatment with phorbol 12- myristate 13-acetate significantly reduced (TPA) (P<0.01) insulin effect in normal cells. 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H 7) partially antagonises (P<0.01) the oleic acid induced reduction of AIB transport. Conclusion. Although classical paradigm defining the pathophysiology of insulin resistance have focused on the abnormal regulation, our data also support a role for fatty acids as an primary physiological modulator of insulin action. Reduction of insulin effect by oleic acid was probably result of changes in insulin receptor-kinase (IRK) activity involving of protein kinase C (PKC).

amino acid transport; insulin; insulin resistance; free fatty acids; oleic acid; protein kinase C

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