FGF21 is a novel metabolic regulator involved in the control of glucose homeostasis, insulin sensitivity, and ketogenesis. The liver has been considered the main site of production and release of FGF21 into the blood. Here, we show that, after thermogenic activation, brown adipose tissue becomes a source of systemic FGF21. This is due to a powerful cAMP-mediated pathway of regulation of FGF21 gene transcription. Norepinephrine, acting via ␤-adrenergic, cAMP-mediated, mechanisms and subsequent activation of protein kinase A and p38 MAPK, induces FGF21 gene transcription and also FGF21 release in brown adipocytes. ATF2 binding to the FGF21 gene promoter mediates cAMP-dependent induction of FGF21 gene transcription. FGF21 release by brown fat in vivo was assessed directly by analyzing arteriovenous differences in FGF21 concentration across interscapular brown fat, in combination with blood flow to brown adipose tissue and assessment of FGF21 half-life. This analysis demonstrates that exposure of rats to cold induced a marked release of FGF21 by brown fat in vivo, in association with a reduction in systemic FGF21 half-life. The present findings lead to the recognition of a novel pathway of regulation the FGF21 gene and an endocrine role of brown fat, as a source of FGF21 that may be especially relevant in conditions of activation of thermogenic activity.Fibroblast growth factor 21 (FGF21) is a metabolic regulator involved in the control of glucose homeostasis, insulin sensitivity, and ketogenesis (1-4). Treatment with FGF21 corrects metabolic disturbances such as hyperglycemia and insulin resistance in rodent models of obesity and diabetes (1, 5-7). It also has been reported that FGF21 exerts autocrine and paracrine actions on livers that promote ketogenesis (2-4). Two recent studies in FGF21 gene-ablated mice have demonstrated that FGF21 is required for the physiological response of mice to fasting and to ketogenic diets (8, 9), although a third study did not confirm these observations (10). Moreover, FGF21 favors glucose utilization in white adipose tissue (WAT), 2 and there are conflicting data on to whether FGF21 activates or does not activate lipolysis in white fat (3, 10, 11). Recently, FGF21 has been reported to promote thermogenic activity in neonatal brown adipose tissue (BAT) and in isolated brown adipocytes (12); there are indications that FGF21 may also promote BAT thermogenic activation in adult mice (1, 6, 7). The liver is considered the main site of production and release of FGF21 into the blood. Expression of the FGF21 gene in the liver is under the control of PPAR␣, and fatty acid availability, acting via PPAR␣, seems to be the main determinant of hepatic FGF21 gene expression and release (2, 3,12,13). Extra-hepatic tissues, including white and brown adipose tissues and skeletal muscle, also express the FGF21 gene (14), and PPAR␥ activation has been reported to induce FGF21 gene expression in white adipocytes (14,15). On the basis of cell culture studies, muscle cells have been proposed to be capabl...

The beneficial effects of brown adipose tissue (BAT) are attributed to its capacity to oxidize metabolites and produce heat, but recent data suggest that secretory properties of BAT may also be involved. Here, we identify the chemokine CXCL14 (C-X-C motif chemokine ligand-14) as a novel regulatory factor secreted by BAT in response to thermogenic activation. We found that the CXCL14 released by brown adipocytes recruited alternatively activated (M2) macrophages. Cxcl14-null mice exposed to cold showed impaired BAT activity and low recruitment of macrophages, mainly of the M2 phenotype, into BAT. CXCL14 promoted the browning of white fat and ameliorated glucose/insulin homeostasis in high-fat-diet-induced obese mice. Impairment of type 2 cytokine signaling, as seen in Stat6-null mice, blunts the action of CXCL14, promoting adipose tissue browning. We propose that active BAT is a source of CXCL14, which concertedly promotes adaptive thermogenesis via M2 macrophage recruitment, BAT activation, and the browning of white fat.

The mitochondrial uncoupling protein (UCP) is responsible for the thermogenic function of brown fat, and it is a molecular marker of the brown adipocyte cell type. Retinoic acid (RA) increased UCP mRNA levels severalfold in brown adipocytes differentiated in culture. This induction was independent of adrenergic pathways or protein synthesis. RA stimulated ucp gene expression regardless of the stage of brown adipocyte differentiation. In transient transfection experiments RA induced the expression of chloramphenicol acetyltransferase vectors driven by 4.5 kilobases of the 5'-noncoding region of the rat ucp gene, and co-transfection of expression vectors for RA receptors enhanced the action of RA. Retinoic acid receptor alpha was more effective than retinoid X receptor in promoting RA action, whereas a mixture of the two was the most effective. The RA-responsive region in the ucp gene was located at -2469/-2318 and contains three motifs (between -2357 and -2330) of the consensus half-sites characteristic of retinoic acid response elements. This 27-base pair sequence specifically binds purified retinoic acid receptor alpha as well as related proteins from brown fat nuclei. In conclusion, a novel potential regulatory pathway of brown fat development and thermogenic function has been recognized by identifying RA as a transcriptional activator of the ucp gene.

The recently identified uncoupling protein-3 (UCP-3) gene, predicted to encode a new member of the family of uncoupling proteins, is preferentially expressed in skeletal muscle and has been related to phenotypes of obesity and type 2 diabetes. We have established that during mouse ontogeny, the expression of the UCP-3 gene is switched on in skeletal muscle just after birth. The induction of UCP-3 gene expression is dependent on the initiation of suckling and particularly on lipid intake. Treatment of newborn mice with activators of peroxisome proliferator-activated receptors (PPARs), such as clofibrate, bezafibrate, or (4-chloro-6-(2,3-xylidine)-pirimidinylthio)acetic acid (WY 14,643), mimics the action of food intake on UCP-3 gene expression. The specific ligand of PPAR-alpha WY 14,643 induces UCP-3 gene expression in a time- and dose-dependent manner, whereas the thiazolidinedione BRL 49653, specific for PPAR-gamma, has no effect. These treatments act without altering circulating free fatty acids. During development, skeletal muscle expresses constitutive levels of PPAR-delta mRNA, whereas expression of the PPAR-gamma gene is undetectable. PPAR-alpha gene expression is developmentally regulated in muscle as it is first expressed at birth, just before UCP-3 gene induction occurs. The induction of UCP-3 gene expression by WY 14,643 is impaired in skeletal muscle of premature neonates, which do not express PPAR-alpha. It is proposed that the UCP-3 gene is predominantly regulated in neonatal muscle by PPAR-alpha activation.

Overexpression of adenine nucleotide translocase-1 (ANT1) is known to induce apoptosis (Bauer, M. K., Schubert, A., Rocks, O., and Grimm, S. (1999) J. Cell Biol. 147, 1493-1501), but the mechanisms involved remain unclear. In this study we show that ANT1 overexpression results in a recruitment of the IB␣-NF-B complex into mitochondria, with a coincident decrease in nuclear NF-B DNA binding activity. In this situation, NF-B transcriptionally regulated genes with antiapoptotic activity, such as Bcl-XL, MnSOD2, and c-IAP2, are down-regulated, and consequently, cells are sensitized to apoptosis. Accordingly, co-expression of p65 partially interferes with the proapoptotic effect of ANT1 overexpression. Despite the high identity of the two isoforms, overexpression of ANT2 does not exert an apoptotic effect; this lack of apoptotic activity is correlated with the absence of mitochondrial IB␣-NF-B recruitment or changes in NF-B activity. Thus, we propose that the mitochondrial recruitment of NF-B observed following ANT1 overexpression has an important role in ANT1 proapoptotic activity.Apoptosis is a form of cell death that plays a role in development, tissue homeostasis, and disease (1). The induction of apoptosis is governed by an elaborate array of checks and balances in the cell. Studies of apoptosis induction in "in vitro" systems have demonstrated that mitochondria are required for the apoptosis stimulated by a variety of different factors (2).The ANT 1 protein is localized in the inner mitochondrial membrane and exchanges cytosolic ADP for mitochondrial ATP (3). Three isoforms (ANT1, ANT2, and ANT3) with tissuespecific expression patterns have been described in humans (4). ANT interacts with several proteins of the outer mitochondrial membrane (peripheral benzodiazepine receptor, porin/VDAC, and Bax) as well as the matrix (cyclophilin D) to form the permeability transition pore (PTP) (5). The PTP appears to be an important regulator of the apoptotic process. Opening of the pore leads to a loss of mitochondrial transmembrane potential, ⌬⌿ m , which can ultimately culminate in matrix swelling and outer membrane rupture, allowing the release of apoptogenic proteins such as cytochrome c, apoptosis-inducing factor, and procaspases (6, 7). Proteins of the bcl-2 family essentially control the release of cytochrome c. Antiapoptotic members of the family (Bcl-2 and Bcl-XL) prevent cytochrome release, whereas the proapoptotic members Bax and Bak exert the opposite effect (8). Bax has been shown to interact with ANT to induce PTP opening and cytochrome c release (9). Several pharmacological compounds interfere with PTP. For instance, cyclosporin A, through its binding to cyclophilin D, prevents PTP opening, and bongkrekic acid and atractyloside are, respectively, a blocker and an inducer of apoptosis via binding of two different conformational states of ANT (10). In addition, alongside their modulation of pore formation by ANT, Bcl-2 and Bax also have been reported to influence ANT ADP/ATP antiporter activity (11). Although ...

Mitochondrial adenine nucleotide translocase 1 (ANT1), but not ANT2, can dominantly induce apoptosis [Bauer et al. (1999) J. Cell Biol. 439, 258^262]. Nothing is known, however, about the apoptotic activity of ANT3. We have transfected HeLa cells with the three human ANT isoforms to compare their potential as inducers of apoptosis. Transient overexpression of ANT3 resulted, like ANT1, in apoptosis as shown by an increase in the sub-G1 fraction, annexin V staining, low v v8 8 m , and activation of caspases 9 and 3. Moreover, the apoptosis produced by ANT3 was inhibited by bongkrekic acid and by cyclosporin A. The pro-apoptotic activities of the ANT1 and ANT3 isoforms contrast with the lack of apoptotic activity of ANT2. This ¢nding may help to identify the speci¢c factors associated with the pro-apoptotic activities of ANT isoforms. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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We have performed a sequential study on the abundance of the mRNA for uncoupling protein (UCP), subunit I1 of cytochrome-c oxidase (COII) and lipoprotein lipase in brown adipose tissue during the fetal and postnatal periods. Moreover, we have determined whether these parameters can be modulated by ambient temperature in the early hours after birth, and at which point in development this sensitivity first appears.UCP gene expression in the fetal and neonatal period has particular features when compared with overall mitochondriogenesis (COII mRNA expression) or with the expression of lipoprotein lipase mRNA. There is a specific induction of UCP gene expression between days 18 and 19 of pregnancy followed by a specific increase of UCP gene expression in utero and a further increase after birth. The acquisition of the physiological apparatus capable of the response to UCP and lipoprotein lipase gene expression to the environmental temperature is not achieved until the last day of fetal development. This result suggests that mechanisms of P-adrenergic modulation of gene expression in brown fat are already established at birth.From an experiment on iopanoic acid treatment of pregnant mothers, it was concluded that iodothyronine 5'-deiodinase activity is not necessary for the expression of the mRNAs for UCP, COII and lipoprotein lipase in the fetus whereas it is necessary for the acquisition of temperature sensitivity to these parameters at birth.Brown fat is a specialized tissue of mammals responsible for facultative thermogenesis. Its physiological significance has long been recognized in newborns when the decrease in environmental temperature at birth requires an adaptative increase in heat production [l]. Brown adipose tissue possesses a unique mitochondrial protein (so called uncoupling protein, UCP) that uncouples oxidative phosphorylation from the respiratory chain. In this way, the energy of metabolic oxidation in this tissue is dissipated as heat (for a review see [2]).Noradrenaline regulates the overall thermogenic activity of brown adipose tissue [3] and specifically modulates the expression of the UCP gene at the transcriptional level [4]. However, noradrenaline is not the only factor involved in the modulation of brown fat thermogenesis. Since the discovery of a iodothyronine 5'-deiodinase activity in brown adipose tissue [5], capable of producing 3,3',5-triiodothyronine from systemic thyroxine, a strong correlation has been established between 5'-deiodinase and thermogenic activity in different situations of adaptative increase or decrease in brown fat thermogenesis [6 -81. A key role for 3,3'$triiodothyronine, generated in situ in the regulation of brown fat activity, has therefore been suggested.