Upregulation of adipocyte metabolism by agouti protein: possible paracrine actions in yellow mouse obesity BRYNN H. JONES, JUNG H. KIM, MICHAEL B. ZEMEL, RICHARD P. WOYCHIK, EDWARD J. MICHAUD, WILLIAM 0. WILKISON, AND NAIMA MOUSTAID Department of Nutrition and Physiology Program, University of Tennessee, Knoxville 37996-1900; Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8080; and Glaxo Research Institute, Research niangle Park, North Carolina 27709 Jones, Brynn H., Jung H. Kim, Michael B. Zemel, Richard I? Woychik, Edward J. Michaud, William 0. Wilkison, and Nai’ma Moustai’d. Upregulation of adipocyte metabolism by agouti protein: possible paracrine actions in yellow mouse obesity. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E192-E196, 1996.-Mutations leading to ectopic expres- sion of the murine agouti gene (a) result in progressive obesity. To further characterize this model, we analyzed adipose and hepatic mRNA levels for fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD), two key enzymes in de novo fatty acid synthesis and desaturation, respectively. FAS and SCD mRNA in both tissues of obese (Avy) mice were dramatically increased relative to lean (a/a) controls. Exces- sive expression of these genes in this model could be due to direct effects of the agouti gene product; to test this possibility we treated 3T3-Ll adipocytes in vitro with recombinant agouti protein. Agouti treatment increased FAS and SCD mRNAlevels by 1.5 and $-fold, respectively. In addition, FAS activity and triglyceride content were 3-fold higher in agouti- treated 3T3-Ll cells relative to controls; these effects were attenuated by simultaneous treatment with a calcium chan- nel blocker (nitrendipine). These data demonstrate that the agouti protein can directly increase lipogenesis in adipocytes and suggest that these effects are mediated through an intracellular calcium-dependent mechanism. 3T3-Ll adipocytes; fatty acid synthase; gene expression; intracellular calcium; lipogenesis; nitrendipine; stearoyl- coenzyme A desaturase; triglycerides IN WILD TYPE MICE the agouti gene is involved in coat color regulation. Expression of agouti in the hair follicle during early postnatal development causes a switch from black (eumelanin) to yellow (phaeomelanin) pig- ment synthesis, resulting in a subapical yellow band on otherwise dark hair (24). In several dominant mutants at the agouti locus (a), such as viable yellow (Avy) and lethal yellow (AY), regulated expression of agouti is disrupted, resulting in ectopic expression in almost all tissues examined throughout the life of the mouse. In addition to a distinctive yellow pelage, these mice are characterized by progressive obesity and develop body fat levels that are 3550% greater than those of wild type mice (4,30). The mechanisms that link this pigmentation protein to obesity have not been identified. Parabiosis experi- ments between AY and nonagouti (a/a) littermates demonstrate that the obese yellow mouse phenotype is not due to circulating agouti (25). Furthermore, the predisposition for AVy mice to become obese is not entirely dependent on pituitary, adrenocortical, or thy- roid hormones (11). These experiments suggest that agouti modifies adiposity through paracrine actions, consistent with its mechanism of action within the hair follicle. If the yellow mouse phenotype involves paracrine actions of agouti, lipogenic tissues are likely targets for the agouti gene product. Both elevated rates of hepatic lipogenesis (26) and increased adipocyte size (8) have been described in AVy mice relative to lean controls. These reports suggest that elevated rates of lipid synthesis and/or storage in liver and adipose tissue contribute to the obese phenotype of the yellow mouse. It is reasonable then to propose that regulatory en- zymes in lipid metabolic pathways may be elevated in this model and may play a role in its obesity. Fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD) are two key enzymes in fatty acid metabolism. FAS is the principal enzyme in long-term regulation of de novo fatty acid synthesis, whereas SCD catalyzes the initial reaction in desaturation of saturated fatty acids. Both FAS (l&23) and SCD (3,9) have been shown to be over- expressed in other genetic models of obesity. We were therefore interested to investigate whether expression of these genes was elevated inAVy mice relative to lean, nonagouti controls and, if so, whether similar increases could be elicited by direct treatment of adipocytes in vitro with the agouti gene product. We report that FAS and SCD mRNAs are overexpressed in liver and adi- pose tissue of Avy mice. Using 3T3-Ll adipocytes, we demonstrate that this effect is due to direct upregula- tion of these mRNAs by the agouti protein. Interest- ingly, our data suggest that agouti-mediated increases in FAS activity and cellular triglyceride content in adipocytes may be mediated by an intracellular calcium ( [Ca2+Ji)-dependent mechanism. MATERIALS AND METHODS Animals. C57BL/GJ-Avy mice were purchased from the Jackson Laboratory and maintained at the Oak Ridge Na- tional Laboratory (Oak Ridge, TN) by mating AVVa mice to El92 0193-1849/96 $5.00 Copyright o 1996’the American Physiological Society