Author
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Ramsay, Timothy |
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Rosebrough, Robert |
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Submitted to: Comparative Biochemistry and Physiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 12/23/2004 Publication Date: 5/1/2005 Citation: Ramsay, T.G., Rosebrough, R.W. 2005. Regulation of uncoupling proteins 2 and 3 in porcine adipose tissue. Domestic Animal Endocrinology. v.28:351-356. Interpretive Summary: Leptin is a hormone produced by adipose tissue that can affect feeding behavior, animal health and reproduction. Studies in pigs have demonstrated that leptin can affect food intake and the partitioning of energy in the body. First this research developed a method to examine hormonally regulated gene expression in intact adipose tissue over time. Secondly, this study examined whether genes associated specifically with energy loss, heat production, are expressed in adipose tissue and whether they are regulated by hormones. These uncoupling proteins were demonstrated in this study to be regulated by thyroid hormones and growth hormone. This suggests that these uncoupling proteins in swine could provide pharmacological targets for altering heat loss and wasted energy expenditure Technical Abstract: Uncoupling proteins 2 and 3 (UCP2, UCP3) are believed to be involved in changes in mitochondrial energy production and potentially cellular heat production or tissue specific energy metabolism/expenditure. The present study was performed to determine whether or not UCP2 and UCP3 expression in porcine adipose tissue are hormonally regulated and whether their expression is correlated with changes in metabolic activity. Briefly, dorsal subcutaneous adipose tissue samples were acquired and adipose tissue explants (approximately 100 mg) were prepared using sterile technique. Tissue slices were then 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 of interest. Triplicate tissue slices were incubated with either basal medium or hormone supplemented media in a tissue culture incubator at 37ºC with 95% air/5% CO2. Parallel cultures were maintained for either 2 hours or 24 hours to evaluate metabolic viability. At the end of the respective incubation period, tissue samples from these incubations were blotted and transferred to polypropylene 16 x 100 test tubes (Sarstedt) containing 1 mL of incubation medium (medium 199, 25 mM Hepes, 3% BSA, 5.5 mM glucose, 1 µCi/mL 14C-U-glucose) and then incubated for an additional two hours at 37° C in a shaking water bath (90 oscillations/min). Incorporation of label into CO2, total lipid and fatty acids was determined to evaluate the viability of the explant culture system. Insulin (100 nM) induced up to a doubling in glucose metabolism in these explant cultures (p < 0.05). Dexamethasone (1 µM) inhibited glucose metabolism by up to 80% (p<0.05), but insulin could overcome this inhibition (p < 0.05). Leptin (100 ng/mL) inhibited lipid synthesis in this explant system by approximately 22-30% (p < 0.05). Triiodothyronine (10 nM) had no effect on tissue metabolism (p > 0.05). Expression of UCP2 and UCP3 was assessed following 24 h of incubation with various combinations of these hormones. Following total RNA isolation, semiquantitive RT-PCR was performed in a single tube using the QuantiTect SYBR Green RT-PCR protocol with 1 'g of total RNA derived from the adipose tissue explants. UCP2 expression was induced by incubation with porcine growth hormone (100 ng/mL; p< 0.05), while UCP3 expression was induced by triiodothyronine (p < 0.05). Neither change in UCP expression could be associated with a change in metabolic response by the adipose tissue explants. |
