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Title: HORMONAL REGULATION OF LEPTIN RECEPTOR EXPRESSION IN PRIMARY CULTURES OF PORCINE HEPATOCYTES

Author
item Caperna, Thomas
item Shannon, Amy
item Poch, Stephen
item Garrett, Wesley
item Richards, Mark

Submitted to: Domestic Animal Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/23/2005
Publication Date: 11/1/2005
Citation: Caperna, T.J., Shannon, A.E., Poch, S.M., Garrett, W.M., Richards, M.P. 2005. Hormonal regulation of leptin receptor expression in primary cultures of porcine hepatocytes. Domestic Animal Endocrinology. 29:582-592.

Interpretive Summary: Studies in many species suggest that leptin, a peptide hormone produced by fat cells, is involved in regulation of feed intake and also plays a role in energy metabolism in liver tissue. We recently showed that leptin had no direct effect on hepatic metabolism of the pig by assessment of liver cell energy metabolism. Since a specific receptor for leptin is required for leptin action in cells, a question remained as to whether pig liver cells maintain expression of the active form of the leptin receptor. In this investigation, we isolated liver cells from normal pigs and studied the expression of the leptin receptor gene using a highly sensitive and quantitative method (reverse transcriptase polymerase chain reaction; RT-PCR ). The complete intact form of the receptor, as well as, other short forms were indeed expressed in cultured hepatocytes. Expression of the receptor was found to be up-regulated by thyroid hormone (T3) but not influenced by glucagon, insulin, leptin glucagon-like peptide-1 or ghrelin. Following manipulation of the of the leptin receptor gene by preincubating the cells with T3, leptin failed to alter glycogen metabolism and the intracellular messenger, cyclic AMP. These data suggest that porcine hepatocytes may be insensitive to leptin stimulation even when leptin receptor expression is enhanced by T3. This indicates that the primary metabolic actions of leptin on the liver operate through other signaling mechanisms or by indirect actions via the central nervous system.

Technical Abstract: 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 one day and switched to a serum-free medium for the remainder of the three d 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 hr, 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 hr (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.