Nitric oxide was found to trigger mitochondrial biogenesis in cells as diverse as brown adipocytes and 3T3-L1, U937, and HeLa cells. This effect of nitric oxide was dependent on guanosine 3',5'-monophosphate (cGMP) and was mediated by the induction of peroxisome proliferator-activated receptor gamma coactivator 1alpha, a master regulator of mitochondrial biogenesis. Moreover, the mitochondrial biogenesis induced by exposure to cold was markedly reduced in brown adipose tissue of endothelial nitric oxide synthase null-mutant (eNOS-/-) mice, which had a reduced metabolic rate and accelerated weight gain as compared to wild-type mice. Thus, a nitric oxide-cGMP-dependent pathway controls mitochondrial biogenesis and body energy balance.

Calorie restriction extends life span in organisms ranging from yeast to mammals. Here, we report that calorie restriction for either 3 or 12 months induced endothelial nitric oxide synthase (eNOS) expression and 3',5'-cyclic guanosine monophosphate formation in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and adenosine triphosphate production, and an enhanced expression of sirtuin 1. These effects were strongly attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP

Mammalian breast adipose tissue is replaced by a milk-secreting gland during pregnancy; the reverse process takes place upon interruption of lactation. Morphological and bromodeoxyuridine studies provide indirect evidence that mouse mammary adipocytes transform into secretory epithelial cells during pregnancy and revert to adipocytes after lactation. By using the Cre-loxP recombination system we show that the mammary gland of whey acidic protein (WAP)-Cre͞R26R mice, in which secretory epithelial cells express the lacZ gene during pregnancy, contains labeled adipocytes during involution. Conversely, adipocyte P2-Cre͞R26R mice, in which adipocytes are labeled before pregnancy, contain labeled secretory epithelial cells during pregnancy. We conclude that reversible adipocyte-to-epithelium and epithelium-to-adipocyte transdifferentiation occurs in the mammary gland of adult mice during pregnancy and lactation.Cre͞LoxP recombination system ͉ X-Gal ͉ morphology

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...

We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3␤ (GSK3␤), an event inactivating this kinase. Leptin-mediated GSK3␤ phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Lepr db/db mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3␤ phosphorylation and mimicked by the GSK3␤ inhibitor SB216763. At concentrations preventing GSK3␤ phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptinmediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3␤ activity.The adipocyte-derived hormone leptin acts as satiety signal in hypothalamic nuclei to regulate energy homeostasis (1, 2). Mice lacking leptin (Lep ob/ob mice) display obesity and several associated abnormalities (1). Excluding very rare cases of humans with genetic obesity, obese human subjects have high circulating leptin levels and hypothalamic insensitivity to leptin (1).Five leptin receptors (LEPRs) 2 in the mouse have been identified, including long (LEPRb) and short isoforms (LEPRa and LEPRc-e). LEPRb, which is ineffective in Lepr db/db mice, phosphorylates Janus kinase 2 (JAK2), which in turn phosphorylates LEPRb tyrosine residues to mediate downstream signaling (3, 4). Recruitment of the signal transducer and activator of transcription-3 (STAT3) is broadly considered a molecular signature of hypothalamic leptin signaling and drives subsequent induction of genes, including that of the feedback inhibitor, suppressor of cytokine signaling-3 (SOCS3). LEPRb stimulation can also activate the extracellular signal-regulated kinase (ERK) signaling in the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI...

The intracellular localization and activity of the nitric oxide synthase (NOS) isoforms were investigated in rat brown adipocytes. Immunohistochemistry showed cytoplasmic and nuclear staining for the endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms; accordingly, anti-L-citrulline antibody, a marker of NOS activity, immunostained both the cytoplasm and the nucleus. The presence of metabolically active NOS in the nucleus was further confirmed by immunoblotting analyses of subcellular fractions of homogenates from cultured brown adipocytes and by measurements of NOS activity in the cytosol and nucleus. Sympathetic stimulation in vivo (i.e. cold exposure or L L 3 -adrenergic agonist treatment) and in vitro (i.e. noradrenaline treatment of cultured cells) significantly increased both cytosolic and nuclear eNOS and iNOS expression and activities. By contrast, the number of iNOS-positive, but not eNOS-positive, nuclei was significantly lower in the functionally impaired brown fat of genetically obese Zucker fa/fa rats. These data suggest the existence of a noradrenaline-modulated functional NOS system in the nucleus of brown adipocytes.

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.