Hormonal regulation of leptin and leptin receptor expression in porcine subcutaneous adipose tissue.
The current study was performed to examine the response of the leptin gene to hormonal stimuli in porcine adipose tissue from finishing pigs. Yorkshire gilts (approximately 150 kg BW) were used in this study. Tissue from four to six pigs was used per experiment. Dorsal subcutaneous adipose tissue samples were acquired, and adipose tissue explants (approximately 100 mg) were prepared using sterile technique. Tissue slices were transferred to 12-well tissue culture plates containing 1 mL of Media 199 with 25 mM HEPES, 0.5% BSA, pH 7.4, and various hormone supplements. Triplicate tissue slices were incubated with either basal medium or hormone-supplemented media in a tissue culture incubator at 37 degrees C with 95% air:5% CO2. Hormones included insulin (100 nM), dexamethasone (1 microM), porcine GH, 100 ng/mL), triiodothyronine (T3, 10 nM), porcine leptin (100 ng/mL), or IGF-I (250 ng/mL). Following incubation for 24 h, tissue samples from the incubations were blotted and transferred to microfuge tubes, frozen in liquid N, and stored at -80 degrees C before analysis for gene mRNA abundance by reverse-transcription PCR and subsequent quantification of transcripts by capillary electrophoresis with laser-induced fluorescence detection. Media from the incubations were collected in microfuge vials and stored at -20 degrees C before analysis for leptin content by RIA. Insulin was required to maintain tissue and mRNA integrity; therefore, insulin was included in all incubations. The combination of insulin and dexamethasone stimulated leptin secretion into the medium by 60% (P < 0.05; n = 6). Porcine GH inhibited insulin induced leptin secretion by 25% (P < 0.05; n = 6). Dexamethasone in combination with insulin produced a 22% increase in leptin mRNA abundance relative to insulin (P < 0.05; n = 4), and T3 stimulated a 28% increase in insulin-induced leptin mRNA abundance (P < 0.05; n = 4). Leptin receptor mRNA abundance was decreased by 25% with the combination of insulin and dexamethasone, relative to insulin-treated adipose tissue slices (P < 0.05; n = 4). Porcine GH decreased leptin receptor mRNA abundance by 17% (P < 0.05; n = 6). These data suggest that leptin secretion is a regulated phenomenon and that posttranslational processing may be significant. Alternatively, transport and exocytosis of leptin containing vesicles in the pig adipocyte may be quite complicated, which could account for the differences in observed mRNA abundance and protein secretion.
Ramsay
T G
TG
Growth Biology Laboratory, USDA-ARS, Beltsville, MD 20705, USA. tramsay@anri.barc.usda.gov
Richards
M P
MP
D000273:Adipose Tissue / Q000187:drug effects* / Q000378:metabolism*; D000818:Animals; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006728:Hormones / Q000494:pharmacology*; D020738:Leptin / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D054411:Receptors, Leptin; D013552:Swine / Q000235:genetics* / Q000378:metabolism
D006728:Hormones; D020738:Leptin; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin