Severe quantitative and qualitative brown adipocyte defects are common in obesity. To investigate whether aberrant expression of tumor necrosis factor ␣ (TNF-␣) in obesity is involved in functional brown fat atrophy, we have studied genetically obese (ob͞ob) mice with targeted null mutations in the genes encoding the two TNF receptors. The absence of both TNF receptors or p55 receptor alone resulted in a significant reduction in brown adipocyte apoptosis and an increase in ␤3-adrenoreceptor and uncoupling protein-1 expression in obese mice. Increased numbers of multilocular functionally active brown adipocytes, and improved thermoregulation was also observed in obese animals lacking TNF-␣ function. These results indicate that TNF-␣ plays an important role in multiple aspects of brown adipose tissue biology and mediates the abnormalities that occur at this site in obesity. O besity in experimental models is ubiquitously associated with abnormalities in brown adipose tissue (BAT) (1, 2). In adult obese animals, the amount of thermogenically active BAT as well as the expression of ␤ 3 -adrenoceptors in brown adipocytes are substantially reduced, potentially resulting in alterations of metabolic function and thermoregulation (3-5). The molecular basis of these obesity-related changes is poorly understood. It has been demonstrated that expression of tumor necrosis factor ␣ (TNF-␣) in adipose tissue is elevated in a variety of experimental obesity models (6-9) and obese humans (10, 11). Owing to its ability to inhibit insulin receptor signaling (12-14), TNF-␣ represents a component of obesity-related insulin resistance (15). More recently, TNF-␣ has been shown to induce brown and white adipocyte apoptosis in vitro (16)(17)(18). Interestingly, in genetic models of obesity, the number of apoptotic cells in the brown fat is dramatically higher than that in control animals (16). This leads to reduction in the number of multilocular thermogenically active brown fat cells and therefore causes BAT functional atrophy, which is characterized by defective thermoregulation in both genetic and dietary obesity. In addition, because BAT is an important target for insulin action and other aspects of energy metabolism, its atrophy could further contribute to the abnormal metabolic profile in obesity. Here, we have tested whether abnormal TNF-␣ production in obesity contributes to any of these abnormalities in BAT. Materials and MethodsGeneration of ob͞ob Mice Deficient in TNF Receptors (TNFR). Obese (ob͞ob) mice deficient in TNFR were generated as described (19)(20)(21). Mice with targeted null mutations at both TNFR 1 and 2 loci (p55, respectively) were crossed with Ob͞ob mice, to produce animals heterozygous at the TNFR1, TNFR2, and ob loci (p55, and Ob͞ob). The resulting triple heterozygotes were then crossbred with each other to produce OB͞ob mice wild type at TNFR loci or with homozygous null mutations in each or both TNFR. The subsequent intercrossing of these animals produced control OB͞OB and ob͞ob mice and littermates with m...

1 In the present work, we study the eect of NO on the proliferation and dierentiation of brown fat cells in primary cultures. 2 Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until con¯uence and treated daily with the NO donating agents, S-nitroso-acetyl penicillamine (SNAP) or Snitroso-L-glutathione (GSNO). Both agents (300 mM) decreased cell proliferation approximately 8 fold on day 8. The inhibitory eect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory eect was reversible after removal of the media containing NO donors. 3 Daily treatment with nitric oxide synthase inhibitors, such as N G -nitro-L-arginine methyl ester (L-NAME, 300 mM), led to the stimulation of cell proliferation by 44+5%, n=3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth. 4 Daily treatment with both SNAP or GSNO induced signi®cant mitochondriogenesis, measured as the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L-NAME was without eect. 5 The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor-g and uncoupling protein-1, which are upregulated during dierentiation. 6 Increasing cyclic GMP in cells by 8-bromo-cyclic GMP (100 ± 1000 mM) did not reproduce the observed NO eects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO-stimulated guanylyl cyclase, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), reduced the expression of peroxisome proliferator activated receptor-g and uncoupling protein-1.

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