1. Effect of Peripheral Melanotan-II Administration on Adipose Stearoyl-CoA Desaturase 1 Expression in Rats Ji Lin, Yang-Ho Choi, Mary Anne Della-Fera, Diane L. Hartzell, ChangLong Li and Clifton A. Baile Depts. of Animal & Dairy Science and Foods & Nutrition, University of Georgia, Athens, GA 30602 Abstract The melanocortin (MC) system plays an important role in the regulation of food intake and energy balance. Melanotan-II (MTII), a synthetic agonist of MC receptors, inhibits food intake and causes body weight loss. In this study we investigated the effect of MTII on adipose stearoyl-CoA desaturase-1 (SCD1) expression, because SCD1 functions as a key lipogenic enzyme in the synthesis of monounsaturated fatty acids, it is expressed in adipose tissue, and knockout of SCD1 in mice results in reduced adiposity. Male Sprague-Dawley rats (~210 g; N=24) were injected IP for 4 successive days with either MTII (2 mg/2 ml/kg) or saline (2 ml/kg). A third group was pair fed to the MTII group and injected IP with saline (2 ml/kg). Rats were killed on day 5 and epididymal (Epi), retroperitoneal (Rp) and inguinal (Ing) fat depots were collected and total RNA was isolated. SCD1 mRNA expression was determined by real time RT-PCR using fluorogenic LUX™ primers. MTII treatment, as well as pair-feeding, suppressed SCD1 expression in Epi (-26 % and –18 %, respectively, P<0.05), but not in Rp or Ing, as compared to controls. These observations suggest that suppression of SCD1 expression, which in turn results in reduced triglyceride synthesis and storage, is not a specific effect of MC receptor activation, but rather is a consequence of reduced food intake and weight loss. The Epi fat pad appears to be more sensitive to this effect after 4 days of treatment, compared to Rp and Ing fat pads; thus, Epi SCD1 expression may be an early indicator of whole body energy homeostasis. Methods Male Sprague-Dawley rats (~210 g, N=24) were injected IP daily with either MTII (2 mg/2 ml/kg) or saline (2 ml/kg) for 4 successive days. A third group was pair-fed to the MTII group and injected IP with saline (2 ml/kg). On day 5, rats were killed and retroperitoneal, inguinal and epididymal fat pads were collected. About 20 mg fat tissue was homogenized, supernatants were transferred to a 96 well deep plate and total RNA was isolated using Qiagen 96 RNeasy kit via BioRobot 3000 system with on-column DNase digestion. One-half microgram of total RNA from each sample was reverse transcribed, and SCD1 mRNA expression was determined by real time RT-PCR using fluorogenic LUX™ primers (Invitrogen, Carlsbad, CA) and an ABI 7700 sequence detector (Applied Biosystems, Foster City, CA ). A standard curve of SCD1 expression was generated by using a series of known amounts of larger PCR fragments. Results Conclusion Suppression of SCD1 mRNA expression in the Epi fat depot after 4 days of either IP MTII treatment or pair-feeding suggests that it is not a specific action of the melanocortin system, but rather a consequence of reduced feed intake and body/fat weight loss. Supported in part by the Georgia Research Alliance Eminent Scholar endowment held by CAB. Fig. 1. SCD1 mRNA expression in Epi fat using Real time RT-PCR. Both MTII treatment and pair-feeding resulted in suppression of SCD1 expression of 26% and 18%, respectively. *P<0.05. Fig. 2. SCD1 mRNA expression in Ing fat using Real time RT-PCR. Neither MTII treatment nor pair-feeding affected SCD1 expression. Fig. 3. SCD1 mRNA expression in Rp fat using Real time RT- PCR. Neither MTII treatment nor pair-feeding affected SCD1 expression. Lux Primer set: 5’-gacagcGGGTTGGTTGTTGGGCTGtC-3’, 5’-CACTCCCTGCCTGCCTTGAT-3’ Reference Standard Primer set: 5’-GCTAGTTACCCGAGTGTATCA-3’, 5’-ACTATACCACTCCCTGCCTG-3’ Table 1. Lux Primer set (FAM) for real time PCR and primer set used for generating larger PCR fragment (408bp) as known standard. Introduction The melanocortin system is an important component in the regulation of feed intake and energy homeostasis. The synthetic melanocortin agonist, melanotan-II (MTII), reduces food intake and body weight and also affects lipid metabolism. Stearoyl- CoA desaturase 1 (SCD1) is a key lipogenic enzyme in the synthesis and regulation of monounsaturated fatty acids, which are used as substrates for the synthesis of triglycerides and membrane phospholipids. Knockout of the SCD1 gene in mice results in reduced adiposity with increased lipid oxidation. In this study we investigated whether SCD1 mRNA expression in adipose tissue was suppressed as a result of peripheral administration of MTII in rats.