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Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/21/2005 Publication Date: 9/1/2005 Citation: Ramsay, T.G. 2005. Porcine preadipocyte proliferation and differentiation: A role for leptin? Journal of Animal Science. 83(9): 2066-2074. Interpretive Summary: Leptin is a hormone produced by pig adipose tissue that can affect feeding behavior, animal health and reproduction. Studies in pigs have demonstrated that leptin can reduce feed intake. This experiment was designed to determine if leptin can affect the recruitment of fat cells. Secondly, this study attempted to determine whether leptin can alter the recruitment of fat cells in response to other adipogenic hormones. The data demonstrate that leptin cannot affect the recruitment of pig adipocytes. Secondly, leptin did not interfere with or promote the ability of other hormones to stimulate fat cell formation. However leptin was able to stimulate the proliferation all of the cells comprising the body fat, including the adipocyte precursor cells. While leptin did not affect the formation of fat cells, it could stimulate the proliferation of the cells which become fat cells. These data suggest that leptin may have a role in promoting the proliferation of adipose tissue, but is not a requirement for the formation of adipocytes. The implications of this study suggest a potential target for future attempts to regulate the growth of adipose tissue by affecting a factor that promotes the proliferation of adipose precursor cells. Technical Abstract: The present study was designed to determine whether porcine leptin can alter the proliferation and differentiation of the porcine preadipocyte. The stromal vascular cell fraction of neonatal pig subcutaneous adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. For differentiation studies, cells were seeded on 6 well tissue culture plates and proliferated to confluency in 10% (vol:vol) fetal bovine serum (FBS) in Dulbecco's modified Eagle medium/F12 (DMEM/F12, 50:50). Cultures were differentiated using 2.5% pig serum (vol:vol), and recombinant porcine leptin at concentrations of 0-1000 ng/mL alone or in combination with porcine insulin (100 nM), dexamethasone (1 µM), or IGF-1 (250 ng/mL). After 7 d of lipid filling, cultures were harvested for analysis of sn-glycerol 3 phosphate dehydrogenase (GPDH), lipoprotein lipase (LPL), and lactate dehydrogenase (LDH). GPDH and LPL activity are measures of preadipocyte differentiation. Data were corrected for protein content of the cultures. For proliferation experiments, 24 hours after seeding cells with 10% FBS in DMEM/F12 in 25 cm2 tissue culture flasks, cells were switched to 2.5% FBS and supplemented with 0-1000 ng porcine leptin or 1000 ng murine leptin. Cell proliferation was measured by 3H-thymidine incorporation in preconfluent cultures over 24 hours on d4 of culture. At confluency, cells were switched to a medium to promote differentiation and lipid filling (2.5% pig serum, 100 nM insulin, 1µM dexamethasone) for 7 days. Cells were harvested from the flasks and adipocytes were separated from stromal cells by Percoll gradient centrifugation. In a series of experiments, leptin alone or in combination with insulin, dexamethasone or IGF-I did not affect differentiation as measured by the activity of GPDH and LPL (p > 0.05). Leptin at any concentration did not inhibit differentiation induced by insulin, dexamethasone or IGF-I (p>0.05). However, leptin at 1000 ng/mL stimulated a 30% increase in preadipocyte proliferation (p < 0.05, n=6) and a 27% increase in stromal cell proliferation (p < 0.05, n = 6). These data suggest that porcine leptin may contribute to the recruitment of new adipocytes within the adipose tissue. |
