We have generated a transgenic mouse with no white fat tissue throughout life. These mice express a dominant-negative protein, termed A-ZIP/F, under the control of the adipose-specific aP2 enhancer/promoter. This protein prevents the DNA binding of B-ZIP transcription factors of both the C/EBP and Jun families. The transgenic mice (named A-ZIP/F-1) have no white adipose tissue and dramatically reduced amounts of brown adipose tissue, which is inactive. They are initially growth delayed, but by week 12, surpass their littermates in weight. The mice eat, drink, and urinate copiously, have decreased fecundity, premature death, and frequently die after anesthesia. The physiological consequences of having no white fat tissue are profound. White adipose tissue (WAT) is the major organ for regulated storage of triglycerides for use as metabolic energy. WAT helps control energy homeostasis, including food intake, metabolic efficiency, and energy expenditure, via its secreted hormone, leptin, and possibly additional unknown hormones. The quantity of body fat varies widely in mammals, ranging from 2% to >50% of body mass, typically from 10% to 20% in mice and humans. Much of this variability can be observed within a single individual, highlighting the delicate balance of factors controlling fat deposition. The huge variation in fat mass is unlike that of any other organ in the body and is determined by both an individual's genetic background and environmental factors including diet and physical activity (Comuzzie and Allison 1998; Hill and Peters 1998). Excess body fat, or obesity, is a major health problem, particularly in America, increasing the risk of diabetes, hypertension, and coronary artery disease (Thomas 1995). The mechanisms by which obesity causes these diseases, however, are unclear. To understand better the contribution of adipose tissue to diabetes and metabolism, it would be valuable to examine a mouse with no adipose tissue. To this end, we produced a transgenic mouse with essentially no white adipose tissue and examined the contribution of WAT to energy metabolism, reproductive function, and disease susceptibility.Mutant mice with either increased or decreased levels of WAT have been reported. For example, two mutations that disrupt signaling between WAT and the brain (ob/ ob and db/db, affecting leptin and its receptor, respectively) cause an increase in WAT amount leading to diabetes (Coleman 1978;Zhang et al. 1994; Chen et al. 1996). These mice have increased food intake and decreased physical and sympathetic nerve activity, all contributing to obesity. Adipose-specific expression of a diphtheria toxigene resulted in mice with either a severe phenotype including neonatal death or a mild phenotype, characterized by resistance to induced obesity or delayed loss of WAT at 10 months (Ross et al. 1993;Burant et al. 1997). These results suggest that WAT may be an essential organ for life. At present, there are no mice, from either knockout or transgene technologies that are devoid of WAT throughout develop...

Insulin-expressing beta cells, found in pancreatic islets, are capable of generating more beta cells even in the adult. We show that fibroblast-like cells derived from adult human islets donated postmortem proliferate readily in vitro. These mesenchymal-type cells, which exhibit no hormone expression, can then be induced to differentiate into hormone-expressing islet-like cell aggregates, which reestablishes the epithelial character typical of islet cells. Immunohistochemistry, in situ hybridization, and messenger RNA measurements in single cells and cell populations establish the transition of epithelial cells within islets to mesenchymal cells in culture and then to insulin-expressing epithelial cells.

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Uncoupling protein-3 (UCP3) is a mitochondrial protein that can diminish the mitochondrial membrane potential. Levels of muscle Ucp3 mRNA are increased by thyroid hormone and fasting. Ucp3 has been proposed to influence metabolic efficiency and is a candidate obesity gene. We have produced a Ucp3 knockout mouse to test these hypotheses. The Ucp3 (؊/؊) mice had no detectable immunoreactive UCP3 by Western blotting. In mitochondria from the knockout mice, proton leak was greatly reduced in muscle, minimally reduced in brown fat, and not reduced at all in liver. These data suggest that UCP3 accounts for much of the proton leak in skeletal muscle. Despite the lack of UCP3, no consistent phenotypic abnormality was observed. The knockout mice were not obese and had normal serum insulin, triglyceride, and leptin levels, with a tendency toward reduced free fatty acids and glucose. Knockout mice showed a normal circadian rhythm in body temperature and motor activity and had normal body temperature responses to fasting, stress, thyroid hormone, and cold exposure. The base-line metabolic rate and respiratory exchange ratio were the same in knockout and control mice, as were the effects of fasting, a ␤3-adrenergic agonist (CL316243), and thyroid hormone on these parameters. The phenotype of Ucp1/Ucp3 double knockout mice was indistinguishable from Ucp1 single knockout mice. These data suggest that Ucp3 is not a major determinant of metabolic rate but, rather, has other functions.Human obesity is the result of energy intake greater than metabolic expenditure and is increasing in incidence (1). On an evolutionary time scale, obesity is a recent development, attributed to the interaction of predisposing genetic backgrounds with a sedentary lifestyle and an abundance of food (2, 3). Little is known about the molecular mechanisms and genes that contribute to the regulation of metabolic rate. For example, metabolic efficiency decreases with increased food intake, and it increases with lowered food intake (4), but the mechanistic details are unknown.The discovery of uncoupling protein (UCP, 1 now named UCP1) illustrated one way to regulate metabolic efficiency. UCP1 uncouples oxidative phosphorylation by allowing leakage of protons into the mitochondrial matrix without the phosphorylation of ADP (5). Heat is released because UCP1 degrades the proton gradient energy without storing it chemically or using it to perform physical work. At the whole-body level, this shows up as metabolic inefficiency. Ucp1 is expressed only in brown adipose tissue (BAT), which is a major heat-producing tissue in small mammals. In addition to cold-induced thermogenesis, BAT and UCP1 have been implicated in diet-induced thermogenesis, the increased energy expenditure that accompanies increased food intake (6). Activation of BAT and increased expression of Ucp1 cause reduced adiposity (7-9). However, BAT is present in only small amounts in large mammals, so its role in regulating energy homeostasis in adult humans is problematic (10).Interest in UCPs incr...

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Leptin is a hormone produced in adipose cells that regulates energy expenditure, food intake, and adiposity. In mice, we observed that circulating leptin levels increase 20 -40-fold during pregnancy. Pregnant ob/ob females had no detectable serum leptin, demonstrating that the heterozygous conceptus was not the source of the leptin. However, leptin RNA and protein levels in maternal adipose tissue were not elevated. The circulating leptin was in a high molecular weight complex, suggesting that the rise in leptin was due to expression of a binding protein. Indeed, quantitative assays of serum leptin binding capacity revealed a 40-fold increase, coincident with the rise in serum leptin. Leptin binding activity reached a capacity of 207 ؎ 15 nmol/liter of serum at day 18 of gestation, and half-maximal binding was observed with ϳ3 nM leptin. The binding protein was purified and partially sequenced, revealing sequence identity to the extracellular domain of the leptin receptor. We found that the placenta produces large amounts of the OB-Re isoform of leptin receptor mRNA, which encodes a soluble binding protein. Thus, the extreme hyperleptinemia of late pregnancy is attributable to binding of the leptin by a secreted form of the leptin receptor made by the placenta.Leptin, a hormone produced in adipose cells, is important in the regulation of metabolic efficiency, energy expenditure, food intake, and adiposity (1-3). It serves as a signal reporting the degree of adiposity: circulating leptin levels correlate best with the amount of body fat (4, 5). Mice lacking a functional leptin (formerly ob or obese) gene become massively obese and develop diabetes mellitus due to overeating and decreased metabolic expenditure (6). These mice are also hypogonadal and hypercorticosteronemic, presumably on a hypothalamic basis. Leptin treatment of ob/ob (lep ob /lep ob ) mice reverses all of these abnormalities, and in normal mice it causes decreased food intake, increased energy expenditure, weight loss, and precocious sexual maturity (7-11). Although much studied for its role in regulation of adiposity, leptin is probably of even greater importance in the metabolic adaptation to inadequate food intake (12).Metabolism during pregnancy is quite different from metabolism in the nongravid state. It is altered to provide rapid growth of the fetus and placenta and to prepare the mother for nursing. Some species, including humans and mice, accumulate maternal fat during gestation, and then use it during lactation (13, 14). The importance of leptin in the regulation of adiposity and energy metabolism led us to investigate the physiology of leptin during gestation in the mouse. In addition, the observation that leptin RNA is made by the human placenta (15, 16) suggested a role for leptin during pregnancy. To our surprise, we observed that, although mice have a profound hyperleptinemia in the third trimester of gestation, the murine placenta does not make leptin. There is, however, a massive increase in a circulating leptin-binding protein ...

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.

We tested the effect of chronic leptin treatment on fasting-induced torpor in leptin-deficient A-ZIP͞F-1 and ob͞ob mice. A-ZIP͞F-1 mice have virtually no white adipose tissue and low leptin levels, whereas ob͞ob mice have an abundance of fat but no leptin. These two models allowed us to examine the roles of adipose tissue and leptin in the regulation of entry into torpor. Torpor is a short-term hibernation-like state that allows conservation of metabolic fuels. We first characterized the A-ZIP͞F-1 animals, which have a 10-fold reduction in total body triglyceride stores. Upon fasting, A-ZIP͞F-1 mice develop a lower metabolic rate and decreased plasma glucose, insulin, and triglyceride levels, with no increase in free fatty acids or ␤-hydroxybutyrate. Unlike control mice, by 24 hr of fasting, they have nearly exhausted their triglycerides and are catabolizing protein. To conserve energy supplies during fasting, A-ZIP͞F-1 (but not control) mice entered deep torpor, with a minimum core body temperature of 24°C, 2°C above ambient. In ob͞ob mice, fasting-induced torpor was completely reversed by leptin treatment. In contrast, neither leptin nor thyroid hormone prevented torpor in A-ZIP͞F-1 mice. These data suggest that there are at least two signals for entry into torpor in mice, a low leptin level and another signal that is independent of leptin and thyroid hormone levels. Studying rodent torpor provides insight into human torpor-like states such as near drowning in cold water and induced hypothermia for surgery.fasting ͉ lipoatrophic diabetes ͉ body temperature ͉ hypothermia ͉ A-ZIP͞F-1 mice L iving organisms must cope with food scarcity. The capabilities to store excess fuel and regulate energy expenditure were thus crucial evolutionary adaptations. In higher organisms, white adipocytes store triglycerides that are burned during fasting and starvation (1, 2). These adipocyte triglycerides account for the vast majority of the body's fuel reserves (3).Adipose tissues also contribute to energy homeostasis in an endocrine͞paracrine manner. Leptin is secreted by adipocytes in direct proportion to body fat mass and regulates energy expenditure, metabolic efficiency, and food intake (4-6). Leptin binds to receptors in the hypothalamus and other sites and conveys the size of adipose tissue lipid stores. Adipocytes also make additional hormones, including tumor necrosis factor ␣, which affects insulin sensitivity (7,8). Thus it is clear that adipose tissues contribute to energy metabolism in two ways, as a metabolic regulator and fuel depot.To analyze the physiologic roles of fat, we generated a transgenic mouse, named A-ZIP͞F-1, which has virtually no white adipose tissue and a reduced amount of brown adipose tissue (9). These mice express, selectively in adipose tissue, a dominant negative protein that heterodimerizes with certain basic leucine zipper transcription factors. The A-ZIP͞F-1 phenotype strikingly resembles that of humans with severe lipoatrophic diabetes mellitus, a disease characterized by reduced amounts of...