With the escalation of obesity-related disease, there is great interest in defining the mechanisms that control appetite and body weight. We have identified a link between anabolic energy metabolism and appetite control. Both systemic and intracerebroventricular treatment of mice with fatty acid synthase (FAS) inhibitors (cerulenin and a synthetic compound C75) Led to inhibition of feeding and dramatic weight Loss. C75 inhibited expression of the prophagic signal neuropeptide Y in the hypothalamus and acted in a leptin-independent manner that appears to be mediated by malonyl-coenzyme A. Thus, FAS may represent an important Link in feeding regulation and may be a potential therapeutic target.

Mounting evidence supports a 'lipostatic' model for the regulation of adipose mass. In such a model, signals are generated in the periphery in proportion to adipose mass that act on hypothalamic control centers in the brain to regulate food intake and energy expenditure. Two such signals, leptin and insulin, have been identified and found to dramatically lower food intake and body weight. Several signalling molecules in the effector pathways that mediate the response to these signals in the brain have also been identified. The regulation of these factors and the nature of the adipose-CNS regulatory loop will be discussed.

Mutation of the obese gene produces obesity, hyperinsulinemia, and compensatory “overexpression” of the defective gene. As insulin activates obese gene expression, it seemed possible that hyperinsulinemia might be responsible for overexpression of the gene. To address this question we rapidly neutralized circulating insulin by injection of an insulin antibody. Unexpectedly, insulin depletion in obese ( ob/ob or db/db ) mice caused massive adipose RNA degradation confirmed by histological analysis to result from adipocyte cell death by a largely necrotic mechanism. This effect was not observed in lean littermates and was completely corrected by coadministration of insulin. Comparison of multiple tissues demonstrated that the effect was restricted to adipose tissue. Insulin depletion in obese mice by administration of streptozotocin also led to cell death, but this death was less extensive and appeared to be apoptotic in mechanism. Thus insulin may promote the survival side of the physiological balance between adipocyte survival and death.

3T3-F442A preadipocytes implanted s.c. into athymic mice develop into fat pads that are indistinguishable from normal adipose tissue. Implanted preadipocytes harboring a beta-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed beta-galactosidase. This finding proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed "adipose tissue." 3T3-F442A preadipocytes, when differentiated into adipocytes in cell culture, express the obese gene at an unexpectedly low level, i.e., =1% the level in adipose tissue. However, adipose tissue derived from s.c. implanted 3T3-F442A preadipocytes expressed leptin mRNA at a level comparable to that in epididymal adipose tissue. These findings indicate that a factor(s) or condition, present in the tissue context and necessary for maximal obese gene expression, is lacking in cell culture. Furthermore, adipocytes derived from the implanted cells were hormonally responsive in that leptin mRNA levels were up-regulated 3- to 8-fold by glucocorticoid injection into the host animal. Thus, these findings indicate that adipose-specific promoter-reporter constructs, transfected into 3T3-F442A preadipocytes, can be tested in an in vivo context during and after development of these cells into adipose tissue. Furthermore, the effect of transgenes on the adipogenic development of the implanted preadipocytes can be assessed. Thus, this approach offers a faster and less costly alternative to the transgenic mouse method for assessing adipose gene function.

Current evidence indicates that much of the regulation of adipocyte differentiation serves to modulate a common adipogenic transcriptional control pathway, comprising members of the C/EBP and PPAR families. Hormonal regulators have been found to control expression of these factors and to alter their activity through ligand binding, post-transcriptional modification, and protein-protein interactions.

Adipose tissue has long been known to house the largest energy reserves in the animal body. Recent research indicates that in addition to this role, the adipocyte functions as a global regulator of energy metabolism. Adipose tissue is exquisitely sensitive to a variety of endocrine and paracrine signals, e.g. insulin, glucagon, glucocorticoids, and tumor necrosis factor (TNF), that combine to control both the secretion of other regulatory factors and the recruitment and differentiation of new adipocytes. The process of adipocyte differentiation is controlled by a cascade of transcription factors, most notably those of the C/EBP and PPAR families, which combine to regulate each other and to control the expression of adipocyte-specific genes. One such gene, i.e. the obese gene, was recently identified and found to encode a hormone, referred to as leptin, that plays a major role in the regulation of energy intake and expenditure. The hormonal and transcriptional control of adipocyte differentiation is discussed, as is the role of leptin and other factors secreted by the adipocyte that participate in the regulation of adipose homeostasis.

3T3-F442A preadipocytes implanted s.c. into athymic mice develop into fat pads that are indistinguishable from normal adipose tissue. Implanted preadipocytes harboring a b-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed b-galactosidase. This finding proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed ‘‘adipose tissue.’’ 3T3-F442A preadipocytes, when differentiated into adipocytes in cell culture, express the obese gene at an unexpectedly low level, i.e.,<1% the level in adipose tissue. However, adipose tissue derived from s.c. implanted 3T3-F442A preadipocytes expressed leptin mRNA at a level comparable to that in epididymal adipose tissue. These findings indicate that a factor(s) or condition, present in the tissue context and necessary for maximal obese gene expression, is lacking in cell culture. Furthermore, adipocytes derived from the implanted cells were hormonally responsive in that leptin mRNA levels were up-regulated 3to 8-fold by glucocorticoid injection into the host animal. Thus, these findings indicate that adipose-specific promoter–reporter constructs, transfected into 3T3-F442A preadipocytes, can be tested in an in vivo context during and after development of these cells into adipose tissue. Furthermore, the effect of transgenes on the adipogenic development of the implanted preadipocytes can be assessed. Thus, this approach offers a faster and less costly alternative to the transgenic mouse method for assessing adipose gene function. The positional cloning of the obese gene led to the identification of its gene product (1), i.e., leptin, a peptide hormone produced by adipocytes that is involved in the regulation of food intake and energy expenditure. Cloning of the leptin receptor gene and characterization of its RNA transcripts revealed multiple splice variants (2, 3), one of which encodes a receptor isoform that transmits its ‘‘signal’’ via the JAKy STAT system (4). This, along with earlier evidence (5), indicates that the interaction of leptin with receptors of this type located in the hypothalamus triggers a response that leads to appetite suppression and increased energy expenditure (5). These findings provided new insight into how adipose tissue mass is regulated. The expression of leptin by the adipocyte appears to be subject to both positive and negative control. Insulin (6, 7), glucocorticoid (8, 9), and certain endotoxins and cytokines (10) potently up-regulate expression of the gene whereas cAMP and b-adrenergic agonists down-regulate its expression (11, 12). In obyob mice, which possess a mutated obese gene, and in dbydb mice, which possess a mutated leptin receptor gene, the obese gene is markedly overexpressed (6). These findings suggest that the gene is under negative feedback control by the leptin ‘‘signal’’ and that disruption of the leptin signaling pathway increases expression of the obese gene. Recent studies in this (13) and other laboratories (14–16) have shown that the proximal promoter of the obese gene possesses a CyEBP binding site that mediates transactivation by CyEBPa. This and other evidence (13) indicate that, like many other adipocyte genes (17), the obese gene is transcriptionally activated by CyEBPa during adipocyte differentiation. Although attempts have beenmade to identify other functional regulatory elements in the obese gene promoter, to our knowledge, none has been reported. Promoter analysis of the obese gene with differentiated preadipocyte cell lines in culture is complicated by the fact that the level of leptin expression is extremely low [#1% the level of expression of the endogenous gene (6)]. Although adipocytes in primary culture have proven useful in identifying exogenous agents (e.g., hormones) that affect leptin expression (8, 9), these cells lose their capacity to express leptin when carried in culture and, therefore, are of limited use for promoter analysis. We have sought to develop a methodology by which the obese gene promoter, as well as other adipocyte gene promoters, can be analyzed in a more appropriate adipose tissue context. Green and Kehinde (18) found that s.c. injection of 3T3F442A preadipocytes into athymic mice gave rise to fat pads resembling normal adipose tissue. We have exploited this approach and now show that the obese gene of 3T3-F442A preadipocytes differentiated in this in vivo context is expressed at a high level (i.e., comparable to that in white adipose tissue) and is responsive to hormonal stimulation. EXPERIMENTAL PROCEDURES Cell Culture. 3T3-F442A and 3T3-L1 cells were cultured and differentiated as described (19, 20), respectively. 89CRIP cells stably transfected with pLLZ (89CRIRynls-lacZ) (21) were from Nicolas Ferry (Rennes, France). Proliferating 3T3F442A cells were stably transfected by incubation with 20 ml of undiluted 89CRIPynls-lacZ conditioned medium six times over 5 days. b-Galactosidase was detected by staining fixed cells with 1% 5-bromo-4-chloro-3-indolyl b-D-galactoside. Clones were selected for high levels of b-galactosidase expression and efficient differentiation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y944300-6$2.00y0 PNAS is available online at http:yywww.pnas.org. †Present address: Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark. ‡Present address: Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622. ¶To whom reprint requests should be addressed at: Department of Biological Chemistry, The John Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205.