Activation of mammalian target of rapamycin complex 1 and insulin resistance induced by palmitate in hepatocytes

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Abstract

Excessive supply of fatty acids to the liver might be a contributing factor to hepatic insulin resistance associated with obesity and type 2 diabetes mellitus. The aim of this study was to investigate direct effects of palmitate on insulin signaling in hepatocytes. The ability of metformin to reverse changes induced by palmitate was also studied. Rat hepatocytes in primary culture exhibited a rightward shift of the insulin dose–response curve for PKB phosphorylation during culture with palmitate. The insulin-stimulated phosphorylation of GSK-3β, a metabolic substrate of PKB, was diminished in palmitate hepatocytes. By contrast, the mTOR protein kinase was overstimulated in cells incubated with palmitate. Hepatocytes cultured with palmitate displayed hyperphosphorylation of IRS-1 at Ser residues 632/635, known to be phosphorylated by mTOR. Metformin treatment of the hepatocytes resulted in activation of the AMP-activated kinase, attenuation of the mTOR/S6K1 pathway, reduction of IRS-1 phosphorylation, and a leftward shift in the insulin dose–response curve for PKB activation. These data suggest a link between an oversupply of fatty acid to hepatocytes, a disproportionate stimulation of mTOR/S6K1, and resistance to insulin.

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Materials and methods

Materials. Cell culture materials, antibodies and other reagents were obtained from sources listed in Supplementary methods.

Isolation and culture of hepatocytes. Male Wistar rats weighing between 190 and 270 g were deprived of food 68 h before hepatocyte isolation and resupplied with standard chow 23 h before hepatocyte isolation. The drinking water was replaced by a glucose solution (20% w/v) for the last 17 h before the isolation of hepatocytes. The animals were anesthetized with thiopental and

Insulin resistance in hepatocytes cultured with palmitate and re-sensitization by metformin

Insulin triggers a rapid and sustained phosphorylation of S473 in PKB in primary rat hepatocytes, an effect accompanied by activation of the kinase [12], [13]. The degree of PKB phosphorylation at S473 was measured in hepatocytes cultured in presence of 0.4 mM palmitate prior to stimulation with a range of insulin concentrations from 0.39 nM to the near-maximal concentration of 6.25 nM (Fig. 1A). Compared to control cells without fatty acid supplement, hepatocytes in the presence of palmitate

Discussion

Culture of primary rat hepatocytes in medium supplemented with palmitate was sufficient to induce insulin resistance. This effect was manifested by a rightward shift in the dose–response curve for insulin stimulation of PKB phosphorylation and activity. By contrast with the inhibition of insulin-dependent PKB activation, the mTOR/S6K1 kinase cascade downstream of PKB appeared to be overstimulated in palmitate-treated hepatocytes. Metformin, apparently acting via stimulation of AMPK, was able to

Acknowledgments

We thank Asllan Gjinovci for assistance in the isolation of hepatocytes and Françoise Assimacopoulos-Jeannet for advice. Research supported by Grant 310000–111797 from Swiss NSF.

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