Since leptin levels are independently correlated with risk of coronary heart disease, we have identified signaling pathways important in mediating leptin production and lipogenesis in human preadipocytes. We used inhibitors of p70S6 kinase, p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K). Human preadipocytes were induced to differentiate in insulin, dexamethasone, triiodothyronine, and 3-isobutyl-1-methylxanthine in the presence or absence of inhibitors and the peroxisome proliferator-activated receptor (PPAR)-γ activator rosiglitazone. Differentiation was assessed by measuring leptin secretion, lipid content, and lipogenic activity. Rosiglitazone increased cell protein by 15%, the lipid content of the cell layer was doubled, and the lipogenic activity increased sevenfold but did not stimulate leptin secretion. None of the inhibitors significantly inhibited protein content over 20 days, but lipid content and lipogenic activity were inhibited by p70S6 kinase and p38 MAPK inhibition but not by p42/44 MAPK or PI3K inhibition. All of the inhibitors significantly decreased leptin secretion, and these inhibitory effects were increased by coincubation with rosiglitazone. We conclude that PI3K and p42/44 MAPK pathways are not critical to the differentiation program leading to lipid accumulation, but stimulation of leptin secretion is dependent on these as well as the p70S6 kinase and p38 MAPK signaling pathways.
Address correspondence and reprint requests to Dr. Margaret C. Eggo, The Medical School, Birmingham, B15 2TT, U.K. E-mail:.
Received for publication 6 June 2002 and accepted in revised form 23 September 2002.
N.G.P. and S.K. have received grant/research support from GlaxoSmithKline. J.C.H. and S.A.S. are employed by and hold stock in GlaxoSmithKline.
C/EBP, CCAAT/enhancer-binding protein; Dex, dexamethasone; ECL, enhanced chemiluminescence; ELISA, enzyme-linked immunosorbent assay; G3PDH, glycerol-3-phosphate dehydrogenase; FKBP12, FK-506-binding protein-12; HBSS, Hank’s balanced salt solution; HRP, horseradish peroxidase; IBMX, 3-isobutyl-1-methylxanthine; LPL, lipoprotein lipase; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PPAR, peroxisome proliferator-activated receptor; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; T3, triiodothyronine; TBS-T, Tris-buffered saline-Tween 20; TNF, tumor necrosis factor.