| The purpose of the current investigation was to determine whether; (1) Insulin, independently of glucose regulates LCFA metabolism and if so to determine the signaling pathway by which it has its effects, (2) Chronic AMPK activation enhances insulin-sensitive fatty acid metabolism, and (3) Increasing intracellular calcium effects LCFA metabolism. Three experimental series were conducted using L6 myotubes and provide novel information on the regulation of LCFA in muscle.; In the first set of experiments, L6 myotubes were incubated, in the presence or absence of insulin and with either an inhibitor of PI3K, PKB/Akt, or aPKC-zeta followed by incubation with 1[14C]palmitate for 30 min. PI3K inhibition prevented the insulin-induced increase in LCFA uptake and decrease in LCFA oxidation. While inhibition of aPKC-zeta abolished the insulin-induced increase in LCFA uptake, it did not prevent the insulin-induced decrease in LCFA oxidation. None of the variables were affected by Akt inhibition.; In experiment 2, L6 cells were treated with AICAR or vehicle for 5 hours/day for 5 days. On day 6 cells were pre-treated with AICAR for 60 min prior to exposure to insulin for 15 min, followed by incubation with 1[14C]palmitate for 30 min. Chronic plus acute AMPK activation resulted in enhancement of insulin-sensitive LCFA uptake and decrease in LCFA oxidation. Further, we found increases in total FAT/CD36, aPKC-zeta, cytochrome C and PGC-1 protein following chronic AICAR treatment.; Experiment 3 was conducted to determine the effects of raising intracellular calcium on LCFA uptake and oxidation and to delineate possible regulatory molecules. Multiple experimental conditions existed: acute, 5-day, and 5-day plus acute caffeine treatment with or without the inhibitors, dantrolene or KN-93. All three caffeine treatments increased LCFA uptake and oxidation. Dantrolene prevented the caffeine-induced increase in LCFA uptake and oxidation in all conditions; while, KN93 prevented the caffeine-induced increase in LCFA uptake and oxidation only in acute-caffeine treated cells. Chronic caffeine treatment resulted in increased expression FAT/CD36, ERK1/2 and aPKC-zeta, as well as PGC-1 and cytochrome C.; These results suggest that both insulin and contraction regulate LCFA metabolism in muscle and under certain experimental conditions induce expression of regulatory proteins. |
