Effects of anti-diabetic agent troglitazone on insulin resistance induced with oleic acid in cultured rat hepatocytes

Aim: The aim of this study was to elucidate the mechanism of oleic acid induced post-binding changes and effects of new oral anti-diabetic drug troglitazone on insulin resistance in cultured rat hepatocytes. 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 alpha-amino(14)C-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 aliquots were taken for determination of protein and radioactivity. Results: Insulin increased AIB transport almost two fold in hepatocytes obtained from rats on standard diet (5.76+/-0.21 vs. 11.05+/-0.41; p<0.01). Oleic acid did not change basal AIB transport but reduced insulin effect to less than a half value (5.26+/-0.17 vs. 6.57+/-0.29; p<0.01). Pretreatment with phorbol 12- myristate, 13-acetate (TPA) significantly stimulated basal AIB transport, but insulin effect did not change. Polymyxin B partially antagonized the oleic acid induced reduction of AIB transport (5.01+/-0.1 vs. 7.11+/-0.13; p<0.01). Troglitazone did not produce any increase in basal and insulin stimulated AlB transport in normal hepatocytes, but normalized insulin-stimulated transport in oleic acid-induced transport (5.73+/-0.19 vs. 9.05+/-0.21; p<0.01). Conclusion: Troglitazone is able to prevent and reverse insulin resistance induced by oleic acid. Insulin resistance was probably result of changes in receptor-kinase activity involving protein kinase C.