Porcine leptin alters insulin inhibition of lipolysis in porcine adipocytes in vitro

The present study determined whether porcine leptin can alter the lipolytic rate in porcine adipocytes produced in vitro. The stromal-vascular cell fr action of neonatal subcutaneous adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. These stromal-vascula r cells were seeded on 25-cm(2) tissue culture flasks and proliferated to c onfluency in 10% fetal bovine serum in DMEM/ F12 (50:50). Cultures were dif ferentiated using 2% pig serum + 10 mM isobutyl methylxanthine + 1 muM dexa methasone far 48 h. This medium was replaced with 5% pig serum + 1 muM insu lin to promote lipid filling of adipocytes for 7 d. Adipocyte-containing cu ltures were incubated overnight in serum-free medium and then used for expe riments. Acute experiments assessed lipolysis in cultures exposed to porcin e leptin (0 to 1,000 ng/ nit medium) for 2 h. Chronic experiments used cult ures incubated with 100 ng porcine leptin/mL of medium for 72 h prior to li polysis measurements. Direct effects of leptin were examined by incubating cultures in DMEM/ F12, 25 mM HEPES, 3% bovine serum albumin, 20 mU of adeno sine deaminase/mL of medium in the presence of 0 to 1,000 ng of porcine lep tin/mL of medium. Indirect effects of leptin were examined using the same i ncubation medium but also supplemented with 1 muM isoproterenol + 10 nM ins ulin in the presence of 0 to 1,000 ng of porcine leptin/mL of medium. Media glycerol concentration was measured at the end of 2-h inc Libations. Acute leptin exposure induced up to a 76% increase in lipolysis (P < 0.05) but h ad no effect on insulin's inhibition of lipolysis. Chronic exposure to lept in produced up to a 56% increase in lipolysis (P < 0.05) and reduced insuli n's inhibition of isoproterenol-stimulated lipolysis by up to 31% (P < 0.05 ). These data demonstrate leptin functions to promote the partitioning of e nergy away from lipid accretion within porcine adipose tissue by promoting lipolysis directly and indirectly by reducing insulin-mediated inhibition o f lipolysis.