Transcriptional activation of the mouse obese ( ob ) gene by CCAAT / enhancer binding protein a ( 3 T 3-Ll / preadipocytes and adipocytes / leptin / differentiation / obesity )

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein a (C/EBPa) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBPa. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBPa, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBPa. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBPa expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBPa Taken together, these findings implicate C/EBPa as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter. Since the discovery of the obese (ob) mutation (1), the ob/ob mouse has been widely investigated as a model of genetic obesity and type II diabetes. Recessive mutations at the ob locus produce a phenotype characterized by obesity, hyperglycemia, hyperinsulinemia, hyperphagia, and lowered basal metabolic rate (reviewed in refs. 2-5). The obese gene, which was recently identified by positional cloning (6), encodes a 16-kDa secretory protein, referred to as leptin (or OB protein), that is expressed only in white and brown adipose tissue (6-9). Mutation of the obese gene in ob/ob mice gives rise to premature termination of translation of the obese message, producing a nonfunctional protein (6). The db/db mouse exhibits an obese phenotype similar to that of the ob/ob mouse (10). Parabiosis experiments with db/db, ob/ob, and lean mice showed that a blood-borne factor produced by lean mice and overproduced by db/db mice can reverse the effects of the obese mutation in ob/ob mice (10, 11). This led to the hypothesis that ob/ob mice lacked the factor, while db/db mice lacked the ability to respond to this factor. Consistent with this hypothesis, the effects of administration of recombinant leptin to ob/ob and db/db mice are 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. strikingly different. Administration of recombinant leptin causes a dramatic decrease in food intake and a profound loss of body weight by ob/ob mice but is without effect on db/db mice (12-15). Thus, db/db mice appear to lack either the receptor or another component of the signal transmission system for leptin. As might be expected, treatment of lean mice with leptin also reduces food intake and body weight (12, 13, 16). Relevant to transmission of the leptin signal is the observation that ob/ob mice overexpress the obese message (6, 7), while db/db mice overexpress both the obese message and leptin (13, 17, 18). Taken together these results suggest that an interruption of the leptin signaling system prevents negative feedback of obese gene expression. It has been postulated that db/db mice lack a leptin receptor located in the brain, presumably the hypothalamus. A large body of evidence indicates that the hypothalamus possesses a satiety center that is responsive to signals indicative of the global energy status of the animal (3, 4). Consistent with the occurrence of a leptin receptor in this area of the brain, injection of leptin into the intracerebroventricular region of the brain reduced food intake and weight gain by ob/ob mice (14). Moreover, leptin was recently shown to bind with high affinity to hypothalamic plasma membranes (15). Recent studies show that expression of the obese gene is markedly altered by changes in the nutritional-hormonal status of the animal. Thus, fasting or diabetes drastically lower the level of obese mRNA in adipose tissue, whereas refeeding or insulin administration, respectively, rapidly reverse these effects (7, 18-20). In addition, it has been shown that glucocorticoid administration increases expression of the obese message in adipose tissue (21) and isolated adipocytes in culture (22). Consistent with a tissue distribution limited to adipose depots, obese mRNA is expressed in adipocyte cell lines (e.g., the 3T3-L1 line) albeit at a much lower level than in adipose tissue (7, 22). During differentiation of 3T3-L1 preadipocytes into adipocytes, the level of obese mRNA increases with kinetics similar to those of other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein a (C/EBPa) (7). In view of the role of C/EBPa as transcriptional activator of adipocyte genes (reviewed in refs. 23 and 24), the present investigation was undertaken to determine whether C/EBPa regulates transcription of the obese gene. EXPERIMENTAL PROCEDURES Isolation and Analysis of ob Genomic Clones. Two genomic DNA libraries were used to isolate the ob promoter. The A FixII mouse (BALB/cJ) genomic DNA library (a gift from D. Nathans; Johns Hopkins University School of Medicine) was screened with the leptin cDNA fragment isolated previously Abbreviations: C/EBPa, CCAAT/enhancer binding protein a; EMSA, electrophoretic mobility-shift analysis.