The product of the recently cloned mouse obese (ob) gene is likely to play an important role in a loop regulating the size of the adipose tissue mass. The hormonal regulation of the ob gene could affect adiposity. To investigate this point, the effect of insulin on ob gene expression was examined in cells of the 3T3-F442A preadipocyte clonal line. ob mRNA is absent from exponentially growing, undifferentiated cells as well as from confluent preadipose cells. Terminal differentiation of preadipose to adipose cells leads to the expression of ob mRNA detected by a sensitive and quantitative ribonuclease protection assay. In adipose cells, the level of ob mRNA is sensitive to insulin in the nanomolar range of concentrations with an increase from an average of 1 copy to 5-10 copies/cell. The effect of insulin was fully reversible and takes place primarily at a transcriptional level. The ob mRNA shows a rapid turnover, with a halflife of approximately 2 h in the absence or presence of insulin. The level of secreted Ob protein is also regulated by insulin. These results indicate that the ob gene is expressed in mature fat cells only and support the possibility that insulin is an important regulator of ob gene expression.The "lipostat" or "adipostat" theory postulates that the size of body fat stores is regulated by a feedback loop (1). This hypothesis is based upon the recovery of initial body weight following lipectomy (2) and parabiosis experiments between genetically obese and wild-type mice suggesting the existence of putative factor(s) regulating food intake (3). The recently cloned ob gene from mouse, rat, and human encodes a circulating factor of 16 kDa that is secreted from adipocytes from various adipose depots (4 -8). The OB protein, named leptin, appears to act at a distant site since injections of the leptin decrease food intake and body weight in ob/ob mice and their lean counterparts (9 -12). This phenomenon implicates directly or indirectly the hypothalamus since mice with chemical lesions of the ventromedial nucleus of the hypothalamus (VMH), 1 after becoming rapidly hyperinsulinemic, express a dramatic increase in the levels of ob mRNA (5, 13). A substantial fall in ob mRNA in the epididymal fat of lean mice has been observed after fasting; this phenomenon is rapidly reversed on refeeding (13-16). The correlation between insulin level and the levels of ob mRNA and plasma leptin suggests that insulin may have direct effects on ob gene expression (15)(16)(17)(18). In this paper, we present data using cultured adipocytes that support this hypothesis. EXPERIMENTAL PROCEDURESCell Culture-Culture conditions of cells of the 3T3-F442A clonal line have been described (19). Cells were plated at 10 3 cells/cm 2 in 60-or 100-mm dishes and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. This medium was defined as standard medium. At confluence, standard medium was supplemented with 2 nM triiodothyronine (T 3 ) and 3 nM insulin, termed differentiation medium, for 10 ...

families are trans-acting nuclear factors playing a regulatory role in the differentiation of preadipocytes into adipocytes (1, 2). PPAR␥2 is predominantly expressed in adipose tissue and plays a critical role in the adipocyte differentiation process (3, 4). Expression of C/EBP␤ and C/EBP␦ genes is not restricted to adipose tissue but is induced early and transiently during the program of adipocyte differentiation. Expression of C/EBP␤ and C/EBP␦ decreases following adipocyte maturation, whereas expression of C/EBP␣ and PPAR␥2 gene is induced (5). C/EBP-binding sites have been identified in the PPAR␥2 promoter, and it has been recently shown that C/EBP proteins directly control transcription from the PPAR␥2 promoter (6). The adipogenic role of C/EBP␤ and C/EBP␦ has been previously demonstrated. Their ectopic expression in fibroblasts leads, in the presence of adipogenic hormones, to the adipocyte phenotype (7-9). Finally, the generation by homologous recombination of C/EBP␤ Ϫ/Ϫ ⅐␦ Ϫ/Ϫ mice has clearly established the essential role of these two C/EBPs for the acquisition of adipocytes both in vitro and in vivo (10). However, the extracellular factors and the intracellular signaling pathways involved in the regulation of C/EBP␤ and C/EBP␦ expression in preadipose cells are poorly defined. During the course of our investigation to identify extracellular factors that regulate early events in adipocyte differentiation, secretion of leukemia inhibitory factor (LIF) by preadipocytes was observed. LIF is known to induce differentiation of the murine myeloid leukemia cell line M1, to maintain pluripotent embryonic stem (ES) cells (11,12), and to modulate stem cell and differentiated cell type functions in vitro and in vivo (13). LIF and the related cytokines cardiotrophin (CT-1) and ciliary neurotrophic factor (CNTF) act through heterodimeric receptors comprised of LIF receptor and gp130 (14). We show in the current study that exogenous LIF stimulates terminal differentiation of Ob1771 and 3T3-F442A preadipose cells and induces adipocyte differentiation of multipotent mouse embryonic fibroblasts (MEF). Moreover, by using an antagonist of LIF receptor or genetically modified ES cells, we show that LIF receptor plays a key role during adipocyte differentiation. These results are at variance with previous reports showing an anti-adipogenic effect of LIF in 3T3-L1 preadipocytes (15,16). The differential response to LIF of 3T3-L1 cells versus other cells is discussed.The expression of C/EBP␤ and C/EBP␦ genes was rapidly induced in Ob1771 and 3T3-F442A preadipose cells after LIF addition. The role of the mitogen-activated protein kinase (MAPK) pathway in early events induced by LIF has been

The ob gene product leptin is secreted from adipose tissue. Leptin has dramatic effects on food intake and energy expenditure in rodents. Brown adipose tissue is the first form of adipose tissue to appear during development, and is present at birth in most species. The development of a leptin feedback system in early life and the relative role of the brown and white adipose tissues have not yet been revealed. We have investigated the expression of ob/leptin mRNA in brown adipose tissue around birth and with respect to feeding. Northern blotting analysis and in situ hybridization experiments demonstrated the presence of leptin mRNA in brown adipose tissue at 0, 18, and 24 h after birth. The leptin mRNA level was decreased at 8 h postpartum in fed animals and at 18 or 24 h in the absence of feeding. In addition, circulating leptin was detected in the plasma of newborn rats at 0, 10, or 24 h after birth, whereas it was not detectable in 10 h-old animals that did not suckle at their mother. The presence at birth of ob mRNA and circulating leptin, as well as the early effect of suckling on ob mRNA levels, suggests the precocious involvement of leptin in the control of food intake.

Embryonic stem cells, derived from the inner cell mass of murine blastocysts, can be maintained in a totipotent state in vitro. In appropriate conditions embryonic stem cells have been shown to differentiate in vitro into various derivatives of all three primary germ layers. We describe in this paper conditions to induce differentiation of embryonic stem cells reliably and at high efficiency into adipocytes. A prerequisite is to treat early developing embryonic stem cell-derived embryoid bodies with retinoic acid for a precise period of time. Retinoic acid could not be substituted by adipogenic hormones nor by potent activators of peroxisome proliferator-activated receptors. Treatment with retinoic acid resulted in the subsequent appearance of large clusters of mature adipocytes in embryoid body outgrowths. Lipogenic and lipolytic activities as well as high level expression of adipocyte specific genes could be detected in these cultures. Analysis of expression of potential adipogenic genes, such as peroxisome proliferator-activated receptors gamma and delta and CCAAT/enhancer binding protein beta, during differentiation of retinoic acid-treated embryoid bodies has been performed. The temporal pattern of expression of genes encoding these nuclear factors resembled that found during mouse embryogenesis. The differentiation of embryonic stem cells into adipocytes will provide an invaluable model for the characterisation of the role of genes expressed during the adipocyte development programme and for the identification of new adipogenic regulatory genes.