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 fraction of neonatal subcutaneous adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. These stromal-vascular cells were seeded on 25-cm(2) tissue culture flasks and proliferated to confluency in 10% fetal bovine serum in DMEM/ F12 (50:50). Cultures were differentiated using 2% pig serum + 10 mM isobutyl methylxanthine + 1 muM dexamethasone far 48 h. This medium was replaced with 5% pig serum + 1 muM insulin to promote lipid filling of adipocytes for 7 d. Adipocyte-containing cultures were incubated overnight in serum-free medium and then used for experiments. Acute experiments assessed lipolysis in cultures exposed to porcine leptin (0 to 1,000 ng/ nit medium) for 2 h. Chronic experiments used cultures incubated with 100 ng porcine leptin/mL of medium for 72 h prior to lipolysis measurements. Direct effects of leptin were examined by incubating cultures in DMEM/ F12, 25 mM HEPES, 3% bovine serum albumin, 20 mU of adenosine deaminase/mL of medium in the presence of 0 to 1,000 ng of porcine leptin/mL of medium. Indirect effects of leptin were examined using the same incubation medium but also supplemented with 1 muM isoproterenol + 10 nM insulin 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 had no effect on insulin's inhibition of lipolysis. Chronic exposure to leptin produced up to a 56% increase in lipolysis (P < 0.05) and reduced insulin's inhibition of isoproterenol-stimulated lipolysis by up to 31% (P < 0.05). These data demonstrate leptin functions to promote the partitioning of energy away from lipid accretion within porcine adipose tissue by promoting lipolysis directly and indirectly by reducing insulin-mediated inhibition of lipolysis.