Expression of leptin mRNA and CCAAT-enhancer binding proteins in response to insulin deprivation during preadipocyte differentiation in primary cultures of porcine stromal-vascular cells

The aim of this study was to examine the correlation between CCAAT- enhancer binding proteins (C/EBPs) and leptin gene expression in response to insulin deprivation in preadipocytes and adipocytes. Adipose tissue from 7 d- old pigs was digested enzymatically and stromal-vascular (S-V) cells were seeded and plated for 3 d in fetal bovine serum (FBS) with dexamethasone (DEX) followed by 6 d (Days 3-9) in serum-free medium with insulin (850 nM or 10 nM), transferrin, and selenium. During FBS+DEX treatment (Days 0-3) a large number of preadipocytes develop with no lipid accretion. In contrast, preadipocyte number does not change with lipid accretion during insulin treatment (Days 3-9). Total RNA and cells were harvested from S-V cultures after periods with and without insulin after FBS+DEX. Northern-blotting and Western blot analysis were used to study leptin mRNA and C/EBP protein expression in cultures, respectively. Insulin deprivation from Days 3-4 reduced leptin mRNA and C/EBP-α protein expression: Treatment with 850 nM or 10 nM insulin from Days 3-9 induced leptin mRNA and C/EBP-α expression at a similar level. In cultures treated with 10 nM insulin from Days 3-7, leptin and C/EBP-α expression were reduced markedly by insulin deprivation from Days 7-9, but were restored by insulin treatment for 6 hr before harvesting. The restoration of leptin expression by insulin was blocked by cycloheximide treatment. However, C/EBP-β protein levels did not change regardless of insulin deprivation. Insulin deprivation from Days 7-9 in cultures treated with 850 nM insulin from Days 3-7 did not influence C/EBP-α or leptin mRNA expression, whereas C/EBP-α and leptin expression were reduced after treating these cultures with 1.5 uM okadaic acid for 45 min before harvesting on Day 9. However, cycloheximide treatment for 6 hr before harvesting did not reduce leptin mRNA expression. These results suggest that 1) leptin expression is positively correlated with C/EBP-α expression, and 2) the maintenance of leptin expression after insulin deprivation in 850 nM insulin- treated cultures on Day 9 may be associated with the presence of C/EBP-α expression and/or activation.