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Adaptive induction of thermogenesis in brown adipose tissue (BAT) is essential for the survival of mammals after birth. We show here that G protein-coupled receptor protein 120 (GPR120) expression is dramatically induced after birth in mouse BAT. GPR120 expression in neonatal BAT is the highest among GPR120-expressing tissues in the mouse at any developmental stage tested. The induction of GPR120 in neonatal BAT is caused by postnatal thermal stress rather than by the initiation of suckling. GPR120-null neonates were found to be relatively intolerant to cold: close to one-third did not survive at 21°C, but all such pups survived at 25°C. Heat production in BAT was significantly impaired in GPR120-null pups. Deficiency in GPR120 did not modify brown adipocyte morphology or the anatomical architecture of BAT, as assessed by electron microscopy, but instead impaired the expression of uncoupling protein-1 and the fatty acid oxidation capacity of neonatal BAT. Moreover, GPR120 deficiency impaired fibroblast growth factor 21 (FGF21) gene expression in BAT and reduced plasma FGF21 levels. These results indicate that GPR120 is essential for neonatal adaptive thermogenesis.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The thermogenic activity of brown adipose tissue (BAT) and browning of white adipose tissue are important components of energy expenditure. Here we show that GPR120, a receptor for polyunsaturated fatty acids, promotes brown fat activation. Using RNA-seq to analyse mouse BAT transcriptome, we find that the gene encoding GPR120 is induced by thermogenic activation. We further show that GPR120 activation induces BAT activity and promotes the browning of white fat in mice, whereas GRP120-null mice show impaired cold-induced browning. Omega-3 polyunsaturated fatty acids induce brown and beige adipocyte differentiation and thermogenic activation, and these effects require GPR120. GPR120 activation induces the release of fibroblast growth factor-21 (FGF21) by brown and beige adipocytes, and increases blood FGF21 levels. The effects of GPR120 activation on BAT activation and browning are impaired in FGF21-null mice and cells. Thus, the lipid sensor GPR120 activates brown fat via a mechanism that involves induction of FGF21.

Pref-1 (pre-adipocyte factor-1) is known to play a central role in regulating white adipocyte differentiation, but the role of Pref-1 in BAT (brown adipose tissue) has not been analysed. In the present study we found that Pref-1 expression is high in fetal BAT and declines progressively after birth. However, Pref-1-null mice showed unaltered fetal development of BAT, but exhibited signs of over-activation of BAT thermogenesis in the post-natal period. In C/EBP (CCAAT/enhancer-binding protein) α-null mice, a rodent model of impaired fetal BAT differentiation, Pref-1 was dramatically overexpressed, in association with reduced expression of the Ucp1 (uncoupling protein 1) gene, a BAT-specific marker of thermogenic differentiation. In brown adipocyte cell culture models, Pref-1 was mostly expressed in pre-adipocytes and declined with brown adipocyte differentiation. The transcription factor C/EBPδ activated the Pref-1 gene transcription in brown adipocytes, through binding to the proximal promoter region. Accordingly, siRNA (small interfering RNA)-induced C/EBPδ knockdown led to reduced Pref-1 gene expression. This effect is consistent with the observed overexpression of C/EBPδ in C/EBPα-null BAT and high expression of C/EBPδ in brown pre-adipocytes. Dexamethasone treatment of brown pre-adipocytes suppressed Pref-1 down-regulation occurring throughout the brown adipocyte differentiation process, increased the expression of C/EBPδ and strongly impaired expression of the thermogenic markers UCP1 and PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-α]. However, it did not alter normal fat accumulation or expression of non-BAT-specific genes. Collectively, these results specifically implicate Pref-1 in controlling the thermogenic gene expression program in BAT, and identify C/EBPδ as a novel transcriptional regulator of Pref-1 gene expression that may be related to the specific role of glucocorticoids in BAT differentiation.

Retinol binding protein-4 (RBP4) is a serum protein involved in the transport of vitamin A. It is known to be produced by the liver and white adipose tissue. RBP4 release by white fat has been proposed to induce insulin resistance. We analyzed the regulation and production of RBP4 in brown adipose tissue. RBP4 gene expression is induced in brown fat from mice exposed to cold or treated with peroxisome proliferator-activated receptor (PPAR) agonists. In brown adipocytes in culture, norepinephrine, cAMP, and activators of PPARγ and PPARα induced RBP4 gene expression and RBP4 protein release. The induction of RBP4 gene expression by norepinephrine required intact PPAR-dependent pathways, as evidenced by impaired response of the RBP4 gene expression to norepinephrine in PPARα-null brown adipocytes or in the presence of inhibitors of PPARγ and PPARα. PPARγ and norepinephrine can also induce the RBP4 gene in white adipocytes, and overexpression of PPARα confers regulation by this PPAR subtype to white adipocytes. The RBP4 gene promoter transcription is activated by cAMP, PPARα, and PPARγ. This is mediated by a PPAR-responsive element capable of binding PPARα and PPARγ and required also for activation by cAMP. The induction of the RBP4 gene expression by norepinephrine in brown adipocytes is protein synthesis dependent and requires PPARγ-coactivator-1-α, which acts as a norepinephine-induced coactivator of PPAR on the RBP4 gene. We conclude that PPARγ- and PPARα-mediated signaling controls RBP4 gene expression and releases in brown adipose tissue, and thermogenic activation induces RBP4 gene expression in brown fat through mechanisms involving PPARγ-coactivator-1-α coactivation of PPAR signaling.

Sirt3 (silent mating type information regulation 2, homolog 3), a member of the sirtuin family of protein deacetylases with multiple actions on metabolism and gene expression is expressed in association with brown adipocyte differentiation. Using Sirt3-null brown adipocytes, we determined that Sirt3 is required for an appropriate responsiveness of cells to noradrenergic, cAMP-mediated activation of the expression of brown adipose tissue thermogenic genes. The transcriptional coactivator Pgc-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) induced Sirt3 gene expression in white adipocytes and embryonic fibroblasts as part of its overall induction of a brown adipose tissue-specific pattern of gene expression. In cells lacking Sirt3, Pgc-1α failed to fully induce the expression of brown fat-specific thermogenic genes. Pgc-1α activates Sirt3 gene transcription through coactivation of the orphan nuclear receptor Err (estrogen-related receptor)-α, which bound the proximal Sirt3 gene promoter region. Errα knockdown assays indicated that Errα is required for full induction of Sirt3 gene expression in response to Pgc-1α. The present results indicate that Pgc-1α controls Sirt3 gene expression and this action is an essential component of the overall mechanisms by which Pgc-1α induces the full acquisition of a brown adipocyte differentiated phenotype.

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 β-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.

Plasma FGF21 levels and hepatic FGF21 gene expression increase dramatically after birth in mice. This induction is initiated by suckling, requires lipid intake, is impaired in PPARalpha null neonates, and is mimicked by treatment with the PPARalpha activator, Wy14,643. Neonates exhibit reduced FGF21 expression in response to fasting, in contrast to the upregulation occurring in adults. Changes in FGF21 expression due to suckling or nutritional manipulations were associated with circulating free fatty acid and ketone body levels. We mimicked the FGF21 postnatal rise by injecting FGF21 into fasting neonates, and found that this enhanced the expression of genes involved in thermogenesis within brown fat, and increased body temperature. Brown adipocytes treated with FGF21 exhibited increased expression of thermogenic genes, higher total and uncoupled respiration, and enhanced glucose oxidation. We propose that the induction of FGF21 production by the liver mediates direct activation of brown fat thermogenesis during the fetal-to-neonatal transition., (2010 Elsevier Inc. All rights reserved.)

C/EBPbeta (CCAAT/enhancer-binding protein beta) is a transcriptional regulator of the UCP1 (uncoupling protein-1) gene, the specific marker gene of brown adipocytes that is responsible for their thermogenic capacity. To investigate the role of C/EBPbeta in brown fat, we studied the C/EBPbeta-null mice. When placed in the cold, C/EBPbeta(-/-) mice did not maintain body temperature. This cold-sensitive phenotype occurred, although UCP1 and PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator-1alpha) gene expression was unaltered in brown fat of C/EBPbeta(-/-) mice. The UCP1 gene promoter was repressed by the truncated inhibitory C/EBPbeta isoform LIP (liver-enriched transcriptional inhibitory protein, the truncated inhibitory C/EBPbeta isoform). Since C/EBPbeta-null mice lack both C/EBPbeta isoforms, active LAP (liver-enriched transcriptional activatory protein, the active C/EBPbeta isoform) and LIP, the absence of LIP may have a stronger effect than the absence of LAP upon UCP1 gene expression. Gene expression for UCP2 and UCP3 was not impaired in all tissues analysed. In primary brown adipocytes from C/EBPbeta(-/-) mice, induction of gene expression by noradrenaline was preserved. In contrast, the expression of genes related to lipid storage was impaired, as was the amount of triacylglycerol mobilized after acute cold exposure in brown fat from C/EBPbeta(-/-) mice. LPL (lipoprotein lipase) activity was also impaired in brown fat, but not in other tissues of C/EBPbeta(-/-) mice. LPL protein levels were also diminished, but this effect was independent of changes in LPL mRNA, suggesting that C/EBPbeta is involved in the post-transcriptional regulation of LPL gene expression in brown fat. In summary, defective thermoregulation owing to the lack of C/EBPbeta is associated with the reduced capacity to supply fatty acids as fuels to sustain brown fat thermogenesis.

Lithium impairs the appearance of the characteristic morphology of brown adipocytes and downregulates the expression of marker genes of brown adipocyte differentiation. These effects are dose-dependent and are more pronounced when exposure of preadipocytes to lithium is initiated at early stages of differentiation. Although lithium reduces the expression of genes common to both white and brown adipocytes [fatty acid binding protein aP2 (aP2/FABP) or peroxisome proliferating activated receptor gamma], genes expressed differentially in brown adipocytes, i.e., uncoupling protein 1, PPAR gamma coactivator-1alpha, and peroxisome proliferating activated receptor alpha, are particularly sensitive to lithium treatment-dependent downregulation. Brown adipocytes appear as preferential targets of the inhibitory action of lithium on adipocyte differentiation.

Objective: Human adipose depots contain remnant brown adipocytes interspersed among white adipocytes, and disturbances of brown with respect to white adipocyte biology have been implicated in highly active antiretroviral therapy (HAART)-induced lipomatosis. Brown adipocytes express the uncoupling protein-1 (UCP1) and contain a large number of mitochondria, potential targets of HAART toxicity. The aim of this study was to evaluate the effects of reverse transcriptase inhibitors (RTIs) on primary brown adipocytes differentiated in culture., Design and Methods: We analysed the effects of RTIs, nucleoside analogues (NRTIs: stavudine, zidovudine, didanosine and lamivudine) and non-nucleoside analogues (NNRTIs: nevirapine and efavirenz), on differentiation, mitochondrial biogenesis and gene expression in brown adipocytes., Results: None of the NRTIs altered brown adipocyte differentiation whereas NNTRIs had differing effects. Efavirenz blocked lipid deposition and expression of adipose marker genes but nevirapine induced lipid accumulation and adipose gene expression, promoted mitochondrial biogenesis and increased UCP1. Stavudine, zidovudine and didanosine reduced mitochondrial DNA (mtDNA) content. However, mitochondrial genome expression was only impaired in didanosine-treated adipocytes. Stavudine, but not zidovudine, induced expression of the mitochondrial transcription factors and this may explain compensatory mechanisms for the depletion of mtDNA by up-regulating mtDNA transcription. Stavudine caused a specific induction of UCP1 gene expression through direct interaction with a retinoic acid-dependent pathway., Conclusions: Specific disturbances in brown adipocytes in adipose depots may contribute to HAART-induced lipomatosis. Mitochondrial depletion does not appear to be the only mechanism explaining adverse effects in brown adipocytes because there is evidence of compensatory mechanisms that maintain mtDNA expression, and the expression of the UCP1 gene is specifically altered.

The differentiation of brown adipocytes during late fetal development or in cell culture is associated with enhanced mitochondrial biogenesis and increased gene expression for components of the respiratory chain/oxidative phosphorylation system. We have shown that this is due to a rise in mitochondrial DNA abundance and the corresponding increase in mitochondrial genome transcripts and gene products, as well as to the coordinate induction of nuclear-encoded genes for mitochondrial proteins. We studied how the expression of key components of the transcriptional regulation of mitochondrial biogenesis is regulated during this process. Changes in the expression of nuclear respiratory factor-2/GA-binding protein a and peroxisome proliferator-activated-receptor gamma coactivator-1 (increase) were opposite to those of nuclear respiratory factor-1 and Sp1 (decrease) during the developmental and differentiation-dependent induction of mitochondrial biogenesis in brown fat. These results indicate that the relative roles of transcription factors and coactivators in mediating mitochondrial biogenesis 'in vivo' are highly specific according to the cell type and stimulus that mediate the mitochondriogenic process.

Brown fat differentiation in mice is fully achieved in fetuses at term and entails the acquisition of not only adipogenic but also thermogenic and oxidative mitochondrial capacities. The present study of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein alpha (C/EBPalpha-null mice) demonstrates that C/EBPalpha is essential for all of these processes. Developing brown fat from C/EBPalpha-null mice showed a lack of uncoupling protein-1 expression, impaired adipogenesis, and reduced size and number of mitochondria per cell when compared with wild-type mice. Furthermore, immature mitochondrial morphology was found in brown fat, but not in liver or heart, from C/EBPalpha-null mice. Concordantly, expression of both nuclear and mitochondrial genome-encoded genes for mitochondrial proteins was reduced in C/EBPalpha-null brown fat, although expression of mitochondrial rRNA and mitochondrial DNA content were unaltered. Expression of nuclear respiratory factor-2, thyroid hormone nuclear receptors, and peroxisome proliferator-activated receptor gamma coactivator-1, was delayed in C/EBPalpha-null brown fat. Iodothyronine 5'-deiodinase activity and thyroid hormone content were also reduced in brown fat from C/EBPalpha-null mice, indicating for the first time a crucial role for C/EBPalpha in controlling thyroid status in developing brown fat, which may contribute to impaired mitochondrial biogenesis and cell differentiation. When survival of C/EBPalpha-null mice was achieved by transgenically expressing C/EBPalpha only in the liver, a substantial recovery in brown fat differentiation was found by day 7 of postnatal age, which is associated with a compensatory overexpression of C/EBPdelta and C/EBPbeta.

The phytol derivatives phytanic acid and pristanic acid may activate nuclear hormone receptors and influence gene expression and cell differentiation. Phytanic acid induces brown adipocyte differentiation. It was determined that brown fat and brown adipocytes are sites of high gene expression of phytanoyl-CoA hydroxylase, the enzyme required for initiation of peroxisomal alpha-oxidation of phytanic acid. However, the effects of phytanic acid were not mediated by its alpha-oxidation product pristanic acid, which did not promote brown adipocyte differentiation or stimulate transcription of the uncoupling protein-1 gene. Moreover, acute cold exposure of mice caused a dramatic mobilization of the phytanic acid stores in brown adipose tissue thus suggesting that a high local exposure to phytanic acid in brown fat may contribute to signalling adaptive changes in the tissue in response to thermogenic activation.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis.

Thermogenic activity in brown adipose tissue (BAT) decreases during lactation; the down-regulation of the gene encoding uncoupling protein 1 (UCP1) is involved in this process. Our studies show that UCP2 mRNA expression does not change during the breeding cycle in mice. In contrast, UCP3 mRNA is down-regulated in lactation but it recovers after weaning, in parallel with UCP1 mRNA. This leads to a decrease in the content of UCP3 in BAT mitochondria during lactation. Lowering the energy-sparing necessities of lactating dams by decreasing litter size or feeding with a high-fat diet prevented the down-regulation of UCP1 mRNA and UCP3 mRNA. In most cases this resulted in a less marked decrease in UCP1 and UCP3 protein in BAT mitochondria owing to lactation. Fasting for 24 h caused a different response in UCP1 and UCP3 mRNA expression: it decreased UCP1 mRNA levels but had no effect on UCP3 mRNA abundance in virgin mice; it even increased UCP3 mRNA expression in lactating dams. These changes did not lead to modifications in UCP1 or UCP3 protein abundance. Whereas acute treatment with peroxisome-proliferator-activated receptor (PPAR)alpha and PPARgamma agonists increased UCP1 mRNA levels only in lactating dams, UCP3 mRNA expression was induced by both kinds of PPAR activator in lactating dams and by PPARalpha agonists in virgin mice. It is concluded that modifications of UCP2 mRNA levels are not part of the physiological adaptations taking place in BAT during lactation. In contrast, the down-regulation of UCP3 mRNA expression and mitochondrial UCP3 content is consistent with a role for the gene encoding UCP3 in the decrease in metabolic fuel oxidation and thermogenesis in BAT during lactation.

High expression of the peroxisome proliferator-activated receptor alpha (PPARalpha) differentiates brown fat from white, and is related to its high capacity of lipid oxidation. We analyzed the effects of PPARalpha activation on expression of the brown fat-specific uncoupling protein-1 (ucp-1) gene. Activators of PPARalpha increased UCP-1 mRNA levels severalfold both in primary brown adipocytes and in brown fat in vivo. Transient transfection assays indicated that the (-4551)UCP1-CAT construct, containing the 5'-regulatory region of the rat ucp-1 gene, was activated by PPARalpha co-transfection in a dose-dependent manner and this activation was potentiated by Wy 14,643 and retinoid X receptor alpha. The coactivators CBP and PPARgamma-coactivator-1 (PGC-1), which is highly expressed in brown fat, also enhanced the PPARalpha-dependent regulation of the ucp-1 gene. Deletion and point-mutation mapping analysis indicated that the PPARalpha-responsive element was located in the upstream enhancer region of the ucp-1 gene. This -2485/-2458 element bound PPARalpha and PPARgamma from brown fat nuclei. Moreover, this element behaved as a promiscuous responsive site to either PPARalpha or PPARgamma activation, and we propose that it mediates ucp-1 gene up-regulation associated with adipogenic differentiation (via PPARgamma) or in coordination with gene expression for the fatty acid oxidation machinery required for active thermogenesis (via PPARalpha).

The intracellular pathways and receptors mediating the effects of retinoic acid (RA) on the brown-fat-uncoupling-protein-1 gene (ucp-1) have been analysed. RA activates transcription of ucp-1 and the RA receptor (RAR) is known to be involved in this effect. However, co-transfection of an expression vector for retinoid-X receptor (RXR) increases the action of 9-cis RA but not the effects of all-trans RA on the ucp-1 promoter in brown adipocytes. Either RAR-specific ¿p-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid¿ or RXR-specific [isopropyl-(E,E)-(R,S)-11-methoxy-3,7, 11-trimethyldodeca-2,4-dienoate, or methoprene] synthetic compounds increase the expression of UCP-1 mRNA and the activity of chloramphenicol acetyltransferase expression vectors driven by the ucp-1 promoter. The RXR-mediated action of 9-cis RA requires the upstream enhancer region at -2469/-2318 in ucp-1. During brown-adipocyte differentiation RXRalpha and RXRgamma mRNA expression is induced in parallel with UCP-1 mRNA, whereas the mRNA for the three RAR subtypes, alpha, beta and gamma, decreases. Co-transfection of murine expression vectors for the different RAR and RXR subtypes indicates that RARalpha and RARbeta as well as RXRalpha are the major retinoid-receptor subtypes capable of mediating the responsiveness of ucp-1 to retinoids. It is concluded that the effects of retinoids on ucp-1 transcription involve both RAR- and RXR-dependent signalling pathways. The responsiveness of brown adipose tissue to retinoids in vivo relies on a complex combination of the capacity of RAR and RXR subtypes to mediate ucp-1 induction and their distinct expression in the differentiated brown adipocyte.

The expression of uncoupling protein-2 (UCP2) mRNA is up-regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down-regulated. 9-cis Retinoic acid causes a dose-dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all-trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9-cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.

The brown fat uncoupling protein-1 (ucp-1) gene is regulated by the sympathetic nervous system, and its transcription is stimulated by norepinephrine, mainly through cAMP-mediated pathways. Overexpression of the catalytic subunit of protein kinase A stimulated a chloramphenicol acetyltransferase expression vector driven by the 4.5-kb 5'-region of the rat ucp-1 gene. Mutant deletion analysis indicated the presence of the main cAMP-regulatory element (CRE) in the proximal region between -141 and -54. This region contains an element at -139/-122 able to confer enhancer and protein kinase A (PKA)-dependent activity to the basal thymidine kinase promoter. The potency of this element was much higher in differentiated than in nondifferentiated brown adipocytes. Gel shift analyses indicated that a complex array of proteins from brown fat nuclei bind to the -139/-122 element, among which CRE-binding protein (CREB) and Jun proteins were identified. In transfected brown adipocytes, c-Jun was a negative regulator of basal and PKA-induced transcription from the ucp-1 promoter acting through this proximal CRE region. A double-point mutation in the -139/-122 element abolished both PKA- and c-Jun-dependent regulation through this site, and overexpression of CREB blocked c-Jun repression. Thus, an opposite action of these two transcription factors on the -139/-122 CRE is proposed. c-Jun content in brown adipocytes differentiating in culture correlated negatively with both ucp-1 gene expression and the acquisition of the brown adipocyte morphology. These findings indicate that c-Jun provides a molecular mechanism to repress the basal and cAMP-mediated expression of the ucp-1 gene before the differentiation of the brown adipocyte.

The regulation of transcription of the gene for the beta subunit of the FoF1 ATP synthase (ATPsynbeta) in brown adipose tissue has been studied as a model to determine the molecular mechanisms for mitochondrial biogenesis associated with brown adipocyte differentiation. The expression of the ATPsynbeta mRNA is induced during the brown adipocyte differentiation that occurs during murine prenatal development or when brown adipocytes differentiate in culture. This induction occurs in parallel with enhanced gene expression for other nuclear and mitochondrially-encoded components of the respiratory chain/oxidative phosphorylation system (OXPHOS). Transient transfection assays indicated that the expression of the ATPsynbeta gene promoter is higher in differentiated HIB-1B brown adipocytes than in non-differentiated HIB-1B cells. A major transcriptional regulatory site was identified between nt -306 and -266 in the ATPsynbeta promoter. This element has a higher enhancer capacity in differentiated brown adipocyte HIB-1B cells than in non-differentiated cells. Electrophoretic shift analysis indicated that Sp1and nuclear respiratory factor-2/GA-binding protein (NRF2/GABP) were the main nuclear proteins present in brown adipose tissue that bind this site. Double-point mutant analysis indicated a major role for the NRF2/GABP site in the enhancer capacity of this element in brown fat cells. It is proposed that NRF2/GABP plays a pivotal role in the co-ordinated enhancement of OXPHOS gene expression associated with mitochondrial biogenesis in brown adipocyte differentiation.

The expression of the two novel uncoupling proteins genes, UCP2 and UCP3, is differentially regulated during prenatal maturation of brown adipose tissue in the mouse. UCP2 gene is expressed early in prenatal development, when neither UCP1 nor UCP3 gene expression yet occurs. UCP3 mRNA is absent at any stage of fetal life; it appears suddenly at birth and reaches adult levels in a few hours. UCP2 mRNA increased after birth but more slowly than UCP3 and UCP1 mRNA. Short-time exposure of adult mice to cold caused a rise in UCP2 or UCP1 mRNA levels but not in that of UCP3. The postnatal rise in UCP2 gene expression appears to be a response to the thermic stress associated with birth, similarly to UCP1, whereas different biological signals may be responsible for the surge in UCP3 gene expression at birth.

The relative abundance of the mitochondrial-encoded mRNAs for cytochrome c oxidase subunit II and NADH dehydrogenase subunit I was lower in brown adipose tissue (BAT) from lactating rats than in virgin controls. This decrease was in parallel with a significant decrease in mitochondrial 16 S rRNA levels and in the relative content of mitochondrial DNA in the tissue. BAT from lactating rats showed lowered mRNA expression of the nuclear-encoded genes for the mitochondrial uncoupling protein, subunit IV of cytochrome c oxidase and the adenine nucleotide translocase isoforms ANT1 and ANT2, whereas mRNA levels for the ATP synthase beta-subunit were unchanged. However, the relative content of this last protein was lower in BAT mitochondria from lactating rats than in virgin controls. It is concluded that lactation-induced mitochondrial hypotrophy in BAT is associated with a co-ordinate decrease in the expression of the mitochondrial genome and nuclear genes for mitochondrial proteins. This decrease is caused by regulatory events acting at different levels, including pre- and post-transcriptional regulation. BAT appears to be a useful model with which to investigate the molecular mechanisms involved in the co-ordination of the expression of the mitochondrial and nuclear genomes during mitochondrial biogenesis.

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.

CCAAT/enhancer-binding protein (C/EBP) alpha mRNA and its protein products C/EBP alpha and 30 kDa C/EBP alpha are expressed in rat brown-adipose tissue. Results also demonstrate the expression of C/EBP beta mRNA and its protein products C/EBP beta and liver inhibitory protein (LIP) in the tissue. The abundance of C/EBP alpha and C/EBP beta proteins in adult brown fat is similar to that found in adult liver. However, the expression of C/EBP alpha and C/EBP beta is specifically regulated in brown fat during development. C/EBP alpha, 30 kDa C/EBP alpha, C/EBP beta and LIP content is several-fold higher in fetal brown fat than in the adult tissue, or liver at any stage of development. Peak values are attained in late fetal life, in concurrence with the onset of transcription of the uncoupling protein (UCP) gene, the molecular marker of terminal brown-adipocyte differentiation. When adult rats are exposed to a cold environment, which is a physiological stimulus of brown-adipose tissue hyperplasia and UCP gene expression, a specific rise in C/EBP beta expression with respect to C/EBP alpha, 30 kDa C/EBP alpha and LIP is observed. Present data suggest that the C/EBP family of transcription factors has an important role in the development and terminal differentiation of brown-adipose tissue.

Primary brown adipocytes differentiated in culture were transiently transfected with plasmids containing different extensions of the 5'-flanking region of the rat uncoupling protein gene placed upstream of the bacterial chloramphenicol acetyltransferase reporter gene. Co-transfection of expression vectors for CCAAT/enhancer binding protein (C/EBP) alpha and C/EBP beta trans-activated the rat uncoupling protein gene promoter due to sequences in the 5' proximal region. DNAse I footprint analysis showed the presence of two C/EBP binding sites at positions -457/-440 and -335/-318, which interact with purified C/EBP beta as well as with C/EBP proteins present in brown fat or liver nuclear extracts. Two copies of each site placed upstream of the enhancerless SV40 promoter confer C/EBP alpha and C/EBP beta responsiveness to this heterologous promoter when co-transfected into HepG2 cells. It is concluded that the UCP gene is a target for C/EBP-dependent transcriptional regulation. This suggests that the C/EBP family of transcription factors is involved in the establishment of the characteristic phenotype of the brown adipocyte.

We have determined the developmental changes in thyroid hormone receptor expression and thyroid hormone status in rat brown adipose tissue (BAT). The present study demonstrates that c-erbA alpha and -beta genes are expressed in the developing BAT. The 6.5-kilobase (kb) beta 1, 2.6-kb alpha 2, 5.5-kb alpha 1, and 6.6-kb alpha transcripts showed peak values on day 20 of fetal life. High affinity T3-binding sites were found in fetal BAT. Binding capacity was similar (18-day-old fetuses) or higher (20-day-old fetuses) than in postnatal or adult brown fat. In contrast, T3 receptors were scarce in fetal liver, and values in adult liver were similar to those in fetal BAT on day 20. The profiles of iodothyronine 5'-deiodinase, nuclear T3 content, and receptor occupancy also showed peak values in fetal BAT on day 20, but they were very low in fetal liver. Thus, BAT has already achieved complete maturation of its thyroid status on day 20 of gestational age. This highly tissue-specific developmental pattern is unique among other mammalian thyroid-sensitive tissues studied to date. Between days 18 and 20 of rat fetal development, there is a specific induction of the uncoupling protein gene expression, the main marker of differentiated adipocytes. The results suggest that thyroid hormones may be involved in the establishment of the differentiated phenotype of the brown fat cell in fetal life.

We have performed a sequential study on the abundance of the mRNA for uncoupling protein (UCP), subunit II 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 beta-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.

Lipoprotein lipase mRNA abundance in rat brown adipose tissue increases during the first 24 h of cold exposure. Lipoprotein lipase mRNA levels do not change in brown fat throughout pregnancy and lactation, whereas enzyme activity is significantly lowered. After 5 h of acute cold or noradrenaline administration there is a 2-fold increase in lipoprotein lipase mRNA abundance, whereas lipoprotein lipase activity is stimulated to more than 6-fold the basal values. It is concluded that translational and/or posttranslational mechanisms are involved in the noradrenergic modulation of lipoprotein lipase activity in brown fat.

The net uptake/release of glucose, lactate and amino acids from the bloodstream by the interscapular brown adipose tissue of control, cold-exposed and cold-acclimated rats was estimated by measurement of arteriovenous differences in their concentrations. In the control animals amino acids contributed little to the overall energetic needs of the tissue; glucose uptake was more than compensated by lactate efflux. Cold-exposure resulted in an enhancement of amino acid utilization and of glucose uptake, with high lactate efflux. There was a net glycine and proline efflux that partly compensated the positive nitrogen balance of the tissue; amino acids accounted for about one-third of the energy supplied by glucose to the tissue. Cold-acclimation resulted in a very high increase in glucose uptake, with a parallel decrease in lactate efflux and amino acid consumption. Branched-chain amino acids, however, were more actively utilized. This was related with a much higher alanine efflux, in addition to that of glycine and proline. It is suggested that most of the glucose used during cold-exposure is returned to the bloodstream as lactate under conditions of active lipid utilization, amino acids contributing their skeletons largely in anaplerotic pathways. On the other hand, cold-acclimation resulted in an important enhancement of glucose utilization, with lowered amino acid oxidation. Amino acids are thus used as metabolic substrates by the brown adipose tissue of rats under conditions of relatively scarce substrate availability, but mainly as anaplerotic substrates, in parallel to glucose. Cold-acclimation results in a shift of the main substrates used in thermogenesis from lipid to glucose, with a much lower need for amino acids.

Uncoupling-protein (UCP) mRNA expression is decreased to 15% of virgin control levels between days 10 and 15 of pregnancy, and remains at these low values during late pregnancy and lactation. Abrupt weaning of mid-lactating rats causes a slight but significant increase in UCP mRNA. Expression of mRNA for subunit II of cytochrome c oxidase (COII) decreased to half that of virgin control in late pregnancy and during lactation. Whereas COII mRNA expression is in step with the known modifications of brown-fat mitochondria content during the breeding cycle of the rat, UCP mRNA expression appears to be diminished much earlier than the mitochondrial proton-conductance-pathway activity. On the other hand, the reactivity of brown fat to increase expression of UCP and COII mRNAs in response to acute cold or noradrenaline treatment is not impaired during lactation.

Basal iodothyronine 5'-deiodinase activity is lowered in brown fat from 20-day pregnant, 5 and 15-day lactating rats when compared with virgin controls. Acute noradrenaline treatment caused a seven fold increase in 5'-deiodinase activity in brown fat from virgin control rats. Late pregnant and lactating rats showed a reduction in noradrenaline-induced 5'-deiodinase activity in brown adipose tissue and the maximum impairment was observed in 15-day lactating rats. Lowered 5'-deiodinase activity in brown fat during late pregnancy and lactation correlates with the known reduction in the thermogenic activity of the tissue during these situations and agrees with the proposal that the rate of 3,5,3'-triiodothyronine generated in situ because of thyroxine 5'-deiodination could be an essential event related to thermogenesis in brown fat. Even though the relationship between 3,5,3'-triiodothyronine generation in the tissue and the specific thermogenic mechanisms of brown fat is unknown, present results indicate a close link between the thermogenic and 5'-deiodinase activities in physiological situations when brown adipose tissue needs to adapt to a low activity, such as that of the breeding cycle.

Iodothyronine 5'-deiodinase activity in rat brown adipose tissue has a characteristic pattern of developmental changes that is completely different from that of the liver. Fetal brown fat exhibits an extremely high iodothyronine 5'-deiodinase activity that is approx. 10-fold that in adult rats. Even though brown fat iodothyronine 5'-deiodinase activity falls suddenly at birth, there is a new peak in the activity around days 5-7 of life, whereas it remains very low afterwards. Just after birth, brown adipose tissue iodothyronine 5'-deiodinase activity is already capable of stimulation by noradrenaline. The postnatal peak in brown fat iodothyronine 5'-deiodinase correlates with the known increase in the thermogenic activity of the tissue in the neonatal rat, thus reinforcing the suggestion that local 3',3,5-triiodothyronine generation could be an important event related to thermogenesis in brown adipose tissue. However, the high fetal activity was only slightly related to the thermogenic activity of brown fat. Moreover, the increased iodothyronine 5'-deiodinase activity of brown adipose tissue during fetal and neonatal life suggests a substantial contribution by brown fat in the overall extrathyroidal 3',3,5-triiodothyronine production in these physiological periods.

Iodothyronine 5'-deiodinase activity appears to be a type I enzyme in bovine brown adipose tissue, on the basis of its high Km for 3,3',5'-tri-iodothyronine ('reverse T3') (in the micromolar range) and sensitivity to propylthiouracil inhibition. This enzyme activity is already detectable in perirenal adipose tissue of bovine fetuses in the second month of gestation, reaches peak values around the seventh month of fetal life, declines before birth, becomes lower after parturition and finally undetectable in the adult cow. Iodothyronine 5'-deiodinase activity is present in the pericardic, peritoneal and intermuscular adipose depots of the neonatal calf, but it is always undetectable in the subcutaneous adipose tissue. It is concluded that iodothyronine 5'-deiodinase is a specific feature of brown fat in the bovine species that is not shared by white adipose tissue. white adipose tissue. Peak values of 5'-deiodinating activity appear as an early event in the prenatal differentiation programme of bovine brown-fat cells as they occur when uncoupling-protein-gene expression first starts.

Tri-iodothyronine (T3)production by interscapular brown fat was studied by measurements of arterio-venous differences and blood flow across the tissue in rats exposed to the following situations: controls, acute cold, chronic cold and starvation. Results demonstrate that brown adipose tissue is a source of systemic T3 in the rat and that the T3 release is modulated according to the physiological situation of the animal: increased in cold exposure and inhibited in starvation.

Brown adipose tissue iodothyronine 5'-deiodinase increases progressively in fetuses from the day 17 of pregnancy on, it reaches peak values on the 20th day of gestation and declines in the last days of fetal life as well as during the first day of life. Birth of premature fetuses causes a sudden drop in the enzyme activity. Postmaturity is associated to a decrease in brown fat 5'-deiodinase similar to that found after birth in fetuses born at term. In the first hours of life brown fat iodothyronine 5'-deiodinase is essentially insensitive to the cold-stimulus. Present data indicates that, differently from adult rats, brown fat iodothyronine 5'-deiodinase activity during the perinatal period is dissociated from the thermogenic activity of the tissue. It is suggested that factors different from the action of the sympathetic nervous system may play a main role in brown fat iodothyronine 5'-deiodinase activity modulation in the fetal and neonatal life.

Brown adipose tissue iodothyronine 5'-deiodinase activity is significantly lower in 17-day pregnant rats compared with virgin controls and remains low during late pregnancy and lactation. It fully recovers with abrupt weaning, but only partially with spontaneous weaning. Even though this profile of changes is remarkably in step with the known pattern of modifications in brown fat thermogenesis during the breeding cycle, the lowered iodothyronine 5'-deiodinase activity appearing between days 15 and 17 of pregnancy occurs earlier than the reduction in brown adipose tissue thermogenesis. Brown fat 3,3',5-tri-iodothyronine content is also reduced in late pregnant, early and mid-lactating rats, most probably as a consequence of the lowered 5'-deiodination of thyroxine in situ. Acute insulin treatment increases brown fat iodothyronine 5'-deiodinase activity in virgin animals as well as in late-pregnant and lactating rats, despite the lowered basal enzyme activity levels in the latter groups. Thus an impaired response to insulin in brown fat does not appear to be a factor leading to the lowered iodothyronine 5'-deiodinase activity during late pregnancy and lactation.