Biochemical and Biophysical Research Communications
Activation of mammalian target of rapamycin complex 1 and insulin resistance induced by palmitate in hepatocytes
Section snippets
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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2016, Journal of Lipid ResearchCitation Excerpt :In hepatocytes, palmitate-induced apoptosis was associated with inhibition of PKB activation (115, 116) and decreased insulin-induced PKB phosphorylation (99, 117). In additional cell types, such as myocytes, insulin resistance due to palmitate is also manifest as PKB inhibition (118). Whether palmitate inhibition of PKB signaling contributes to progression of NAFLD is not known.