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-alpha protein expression. Treatment with 850 nM or 10 nM insulin from Days 3-9 induced leptin mRNA and C/EBP-alpha expression at a similar level. In cultures treated with 10 nM insulin from Days 3-7, leptin and C/EBP-alpha 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-beta protein levels did not change regardless of insulin deprivation. Insulin deprivation from Days 7-9 in cultures treatedwith 850 nM insulin from Days 3-7 did not influence C/EBP-alpha or leptin mRNA expression, whereas C/EBP-alpha 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-alpha 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-alpha expression and/or activation.
Chen
X L
XL
Department of Foods and Nutrition, University of Georgia, Athens 30602, USA.
Dean
R G
RG
Hausman
G J
GJ
D017667:Adipocytes / Q000166:cytology*; D000273:Adipose Tissue / Q000254:growth & development*; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D002417:Cattle; D002454:Cell Differentiation; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D007328:Insulin / Q000502:physiology*; D020738:Leptin / Q000235:genetics*; D009687:Nuclear Proteins / Q000096:biosynthesis*; D019319:Okadaic Acid / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis*; D013552:Swine / Q000254:growth & development*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007328:Insulin; D020738:Leptin; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D019319:Okadaic Acid; D003513:Cycloheximide