The hereditary breast and ovarian cancer gene, BRCA1, encodes a large polypeptide that contains the cysteine-rich RING motif, a zinc-binding domain found in a variety of regulatory proteins. Here we describe a novel protein that interacts in vivo with the N-terminal region of BRCA1. This BRCA1-associated RING domain (BARD1) protein contains an N-terminal RING motif, three tandem ankyrin repeats, and a C-terminal sequence with significant homology to the phylogenetically conserved BRCT domains that lie near the C terminus of BRCA1. The BARD1/BRCA1 interaction is disrupted by BRCA1 missense mutations that segregate with breast cancer susceptibility, indicating that BARD1 may be involved in mediating tumour suppression by BRCA1.

Conventional treatment of obesity reduces fat in mature adipocytes but leaves them with lipogenic enzymes capable of rapid resynthesis of fat, a likely factor in treatment failure. Adenovirus-induced hyperleptinemia in normal rats results in rapid nonketotic fat loss that persists after hyperleptinemia disappears, whereas pair-fed controls regain their weight in 2 weeks. We report here that the hyperleptinemia depletes adipocyte fat while profoundly down-regulating lipogenic enzymes and their transcription factor, peroxisome proliferator-activated receptor (PPAR)␥ in epididymal fat; enzymes of fatty acid oxidation and their transcription factor, PPAR␣, normally low in adipocytes, are up-regulated, as are uncoupling proteins 1 and 2. This transformation of adipocytes from cells that store triglycerides to fatty acid-oxidizing cells is accompanied by loss of the adipocyte markers, adipocyte fatty acid-binding protein 2, tumor necrosis factor ␣, and leptin, and by the appearance of the preadipocyte marker Pref-1. These findings suggest a strategy for the treatment of obesity by alteration of the adipocyte phenotype.

Overaccumulation of lipids in nonadipose tissues of obese rodents may lead to lipotoxic complications such as diabetes. To assess the pathogenic role of the lipogenic transcription factor, sterol regulatory element binding protein 1 (SREBP-1), we measured its mRNA in liver and islets of obese, leptin-unresponsive fa͞fa Zucker diabetic fatty rats. Hepatic SREBP-1 mRNA was 2.4 times higher than in lean ؉͞؉ controls, primarily because of increased SREBP-1c expression. mRNA of lipogenic enzymes ranged from 2.4-to 4.6-fold higher than lean controls, and triacylglycerol (TG) content was 5.4 times higher. In pancreatic islets of fa͞fa rats, SREBP-1c was 3.4 times higher than in lean ؉͞؉ Zucker diabetic fatty rats. The increase of SREBP-1 in liver and islets of untreated fa͞fa rats was blocked by 6 weeks of troglitazone therapy, and the diabetic phenotype was prevented. Upregulation of SREBP-1 also occurred in livers of Sprague-Dawley rats with diet-induced obesity. Hyperleptinemia, induced in lean ؉͞؉ rats by adenovirus gene transfer, lowered hepatic SREBP-1c by 74% and the lipogenic enzymes from 35 to 59%. In conclusion, overnutrition increases and adenovirus-induced hyperleptinemia decreases SREBP-1c expression in liver and islets. SREBP-1 overexpression, which is prevented by troglitazone, may play a role in the ectopic lipogenesis and lipotoxicity complicating obesity in Zucker diabetic fatty rats. Insulin stimulates hepatic lipid synthesis by selectively upregulating the expression of a lipogenic transcription factor, sterol regulatory element binding protein (SREBP)-1c (1). In insulindeficient rats, SREBP-1c expression is low and is restored almost to normal within 6 h by insulin replacement (1). It is elevated in two insulin-resistant models of leptin deficiency, the ob͞ob mouse and a transgenic mouse model of generalized lipodystrophy (2). Studies in isolated hepatocytes (1, 3, 4) and adipocytes (5) indicate that insulin induces SREBP-1c gene transcription. These reports raise the possibility that increased lipogenesis secondary to leptin unresponsiveness might also be the consequence of overexpression of SREBP-1 in nonadipose tissues. If so, its prevention might protect against lipotoxicity (6), i.e., the disease consequences of overaccumulation of unoxidized lipids in liver, skeletal muscle, pancreatic islets, and myocardium postulated to cause, respectively, insulin resistance (7, 8), noninsulin-dependent diabetes mellitus (9), and heart dysfunction (10).In obese Zucker diabetic fatty (ZDF) rats, ectopic lipid overaccumulation and lipotoxicity occur early in life as the result of a loss-of-function mutation ( fa) in their leptin receptors (11,12). All of the foregoing disease consequences are evident by the age of 14 weeks, provided the rats' diet contains at least 6% fat. Prophylactic treatment with troglitazone (TGZ) of obese, prediabetic ZDF ( fa͞fa) rats prevents the increase in ectopic triacylglycerol (TG) deposition, the insulin resistance (13, 14), the noninsulin-dependent diabetes mellitus (14), and ...

To determine whether the depletion of body fat caused by adenovirus-induced hyperleptinemia is mediated via the hypothalamus, we used as a ''bioassay'' for hypothalamic leptin activity the hypothalamic expression of a leptinregulated peptide, cocaine-and amphetamine-regulated transcript (CART). The validation of this strategy was supported by the demonstration that CART mRNA was profoundly reduced in obese rats with impaired leptin action, whether because of ablation of the ventromedial hypothalamus (VMH) or a loss-of-function mutation in the leptin receptor, as in Zucker diabetic fatty rats. We compared leptin activity in normal rats made hyperleptinemic by adenovirus-leptin treatment (43 ؎ 9 ng/ml, cerebrospinal f luid leptin 100 pg/ml) with normal rats made hyperleptinemic by a 60% fat intake (19 ؎ 4 ng/ml, cerebrospinal f luid leptin 69 ؎ 22 pg/ml). CART was increased 5-fold in the former and 2-fold in the latter, yet in adenovirus-induced hyperleptinemia, body fat had disappeared, whereas in high-fat-fed rats, body fat was abundant. Treatment of the high-fat-fed rats with adenovirus-leptin further increased their hyperleptinemia to 56 ؎ 6 ng/ml without changing CART mRNA or food intake, indicating that leptin action on hypothalamus had not been increased. Nevertheless, their body fat declined 36%, suggesting that an extrahypothalamic mechanism was responsible. We conclude that in diet-induced obesity body-fat depletion by leptin requires supraphysiologic plasma concentrations that exceed the leptin-transport capacity across the blood-brain barrier.Reduction in body fat induced by the intracerebroventricular administration of leptin (1-4) has led to the widespread assumption that the actions of leptin are regulated entirely by hypothalamic factors (5-9). However, there are now reasons to suspect that some effects of leptin, at least those that occur at high plasma concentrations, are mediated via extraneural actions. First, the plasma levels of radioiodinated leptin 20 min after intracerebroventricular injection equal or exceed those observed after its i.v. injection (10), evidence that intracerebroventricular administration of the peptide could have effects on peripheral tissues. Second, leptin receptors are widely expressed throughout the body (11-13). Third, direct in vitro action of leptin has been demonstrated in several tissues, including adipocytes (14-17). Finally, transport of leptin across the blood-brain barrier is saturable (18-21), suggesting that leptin effects that occur only at plasma concentrations above the saturation level represent direct actions on peripheral tissues.A primary goal of this study was to determine whether the rapid disappearance of body fat (22) and adipocyte dedifferentiation caused by virus-induced hyperleptinemia (23) are mediated by actions of leptin on the hypothalamus or via direct peripheral actions. Three criteria for hypothalamic activity of plasma leptin were used: (i) evidence of transport across the blood-brain barrier reflected by an increase in cerebr...