Expression of ob Gene in Adipose Cells

Abstract

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 ...

Effects of prolonged hyperinsulinemia on serum leptin in normal human subjects.

“…Our studies cannot rule out other indirect modes of insulin action on leptin, for instance, metabolic processes requiring insulin and glucose such as lipogenesis. In animals there is strong evidence in favor of direct effects of insulin on ob gene expression (22)(23)(24)(25)(26). Dose-dependent stimulation by insulin of ob gene expression has been shown in rat adipocytes in primary culture (22), cultured 3T3-F442A and 3T3-L1 mouse cells (23,25).…”

“…In animals there is strong evidence in favor of direct effects of insulin on ob gene expression (22)(23)(24)(25)(26). Dose-dependent stimulation by insulin of ob gene expression has been shown in rat adipocytes in primary culture (22), cultured 3T3-F442A and 3T3-L1 mouse cells (23,25). Furthermore, it has been shown that raising or lowering of plasma insulin concentrations in vivo in rodents resulted in up-or down-regulation, respectively, of leptin mRNA, and that insulin produced a dosedependent rise in leptin release from cultured human fat cells (26).…”

Plasma leptin concentrations: gender differences and associations with metabolic risk factors for cardiovascular disease

“…Furthermore, our results also suggest that, in women, the association between plasma leptin and insulin concentrations is independent of adiposity, a finding which provides further support to the observation that adipose tissue leptin secretion may be upregulated by insulin. [Diabetologia (1997) 40: 1178-1184] believed to be regulated by insulin both in vivo and in vitro [3,13,22,23].…”

“…Leptin is secreted by white adipose cells and is exclusively expressed in adipose tissue (AT) [2,[9][10][11][12][13]. In this regard, numerous studies have reported a strong relationship between adiposity and plasma leptin concentrations or its AT mRNA levels [14][15][16][17][18][19][20][21].…”

Clinical implications of leptin and its potential humoral regulators in long-term low-calorie diet therapy for obese humans

“…Several studies have demonstrated that the leptin gene expression is positively regulated by insulin both in vivo (Saladin et al, 1995;Kolaczynski et al, 1996c) and in vitro (Kolaczynski et al, 1996c;Leroy et al, 1996). Plasma leptin levels decrease after fasting and increase by refeeding in humans (Kolaczynski et al, 1996a,b;Boden et al, 1996;Dubuc et al, 1998;Geldszus et al, 1996;Wadden et al, 1998) and rodents (Frederich et al, 1995), which parallels the change in plasma insulin levels (Kolaczynski et al, 1996b).…”