Hormonal regulation of leptin receptor expression in primary cultures of porcine hepatocytes.
A study was conducted to elucidate hormonal control of leptin receptor gene expression in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (52 kg) and seeded into collagen-coated T-25 flasks. Monolayer cultures were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the 3-day culture period. To establish basal conditions hepatocytes were maintained in serum-free William's E medium containing 10 nM dexamethasone and 1 ng/ml insulin. For the final 24 h, insulin (1 or 100 ng/ml) or glucagon (100 ng/ml), were added in the presence or absence of 100 nM triiodothyronine (T3). RNA was extracted and quantitative RT-PCR was performed with primers specific for the long form and total porcine leptin receptors. Leptin receptor expression was calculated relative to co-amplified 18S rRNA. Expression of the long form of the leptin receptor was confirmed under basal conditions. Insulin, glucagon and synthetic human proteins (ghrelin and GLP-1) at 100 ng/ml had no influence on leptin receptor expression; the addition of T3 was associated with a marked increase (P < 0.001) in expression of total and long forms of the leptin receptor by 1.6 and 2.4-fold, respectively. Addition of leptin to cells which were pre-treated with T3 for 24 h (to up-regulate leptin receptor expression), confirmed the lack of a direct effect of leptin on glucagon-induced glycogen turnover and cAMP production. These data suggest that porcine hepatocytes may be insensitive to leptin stimulation even when leptin receptor expression is enhanced by T3.
Caperna
T J
TJ
Growth Biology Laboratory, Animal and Natural Resources Institute, Beltsville Agricultural Research Center, USDA-ARS, Bulding 200, Room 202, BARC-East, Beltsville, MD 20705, USA. caperna@anri.barc.usda.gov
Shannon
A E
AE
Poch
S M
SM
Garrett
W M
WM
Richards
M P
MP
D000818:Animals; D000242:Cyclic AMP / Q000096:biosynthesis; D054439:Ghrelin; D005934:Glucagon / Q000378:metabolism / Q000494:pharmacology*; D052216:Glucagon-Like Peptide 1; D004763:Glucagon-Like Peptides / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D008112:Liver Glycogen / Q000378:metabolism; D008297:Male; D010446:Peptide Fragments / Q000494:pharmacology; D036361:Peptide Hormones / Q000494:pharmacology; D020033:Protein Isoforms; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D054411:Receptors, Leptin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014284:Triiodothyronine / Q000378:metabolism / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D054439:Ghrelin; D007328:Insulin; C515448:LEPR protein, human; D008112:Liver Glycogen; D010446:Peptide Fragments; D036361:Peptide Hormones; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D014284:Triiodothyronine; C055453:glucagon-like peptide 1 (7-36)amide; D004763:Glucagon-Like Peptides; D012313:RNA; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D000242:Cyclic AMP