- Email: [email protected]
Adaptive thermogenesis: Turning on the heat
Dispatch
R517
Adaptive thermogenesis: Turning on the heat Bradford B. Lowell
Brown adipocytes play important roles in the regulation of fat storage and body temperature, by virtue of their ability to uncouple mitochondrial fuel oxidation and ATP synthesis. The discovery of a tissue-specific transcriptional coactivator provides new insights into the regulation of thermogenesis by brown adipocytes. Address: Beth Israel Deaconess Medical Center and Harvard Medical School, Division of Endocrinology, Department of Medicine, 330 Brookline Avenue, Boston, Massachusetts 02215, USA. E-mail: [email protected] Current Biology 1998, 8:R517–R520 http://biomednet.com/elecref/09609822008R0517 © Current Biology Ltd ISSN 0960-9822
Brown adipocytes are highly specialized cells with a marked capacity for regulated thermogenesis. This feature is largely due to their abundant mitochondria, which uniquely possess the ability to uncouple fuel oxidation from ATP production, thus generating heat. Thermogenesis by adipocytes is a regulated, adaptive phenomenon — it is dramatically promoted by various agents, such as β-adrenergic receptor agonists, thyroid hormone, retinoic acid and ligands for peroxisome proliferator-activated receptor γ (PPARγ). The signaling pathways for these agents, however, also operate in other cell types — most notably non-thermogenic white adipocytes — and the downstream transcriptional mechanism that allows brown adipocytes to switch on their thermogenic machinery has until recently been unknown. But now Puigserver et al. [1] have reported the identification of an important link in the pathway that induces thermogenesis in brown adipocytes — a tissue-specific nuclear receptor coactivator known as PPAR gamma coactivator-1 (PGC-1). Regulated heat production plays an important part in the general control of the body’s energy budget. Triglyceride is an efficient means of storing energy, and its accumulation in white adipose tissue permits survival during starvation. Excessive fat storage, as in obesity, however, adversely affects health by increasing the risk of non-insulin-dependent diabetes mellitus and cardiovascular disease. In order to maintain proper balance, organisms must regulate fat storage. The brain plays a central role in controlling this process; it receives afferent inputs conveying information about nutritional status, integrates these signals and then modulates food intake and a component of energy expenditure that is referred to as adaptive thermogenesis. In rodents, brown adipose tissue makes an important contribution to adaptive thermogenesis [2]. This highly
specialized tissue (Figure 1) is distinguished by its intense sympathetic innervation, extensive blood supply, abundant mitochondria and unique expression of uncoupling protein-1 (UCP1). UCP1 is an inner mitochondria membrane carrier with proton transport activity which dissipates the proton electrochemical potential gradient, uncoupling fuel oxidation from conversion of ADP to ATP [3]. Consequently, stimulated brown adipocytes consume fuels, with the major by-product being the generation of heat. Recently, two proteins related to UCP1 were identified, UCP2 [4] and UCP3 [5], which are approximately 57% identical in amino-acid sequence to UCP1. UCP2 is widely expressed, whereas UCP3 is found preferentially in skeletal muscle. Whether these UCP1 homologs also act as mitochondrial uncoupling proteins is presently under investigation. Figure 1
Electron micrograph of brown fat from a cold-exposed mouse. Note the abundant mitochondria with densely packed cristae (micrograph kindly provided by Saverio Cinti).
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As UCP1 is expressed exclusively in brown adipocytes, intense interest has focused on transcription factors that regulate UCP1 production. The hope has been that the identification of such factors will shed important new light on the pathway of adaptive thermogenesis. Indeed, these efforts have identified a complex 220 base-pair enhancer, located approximately 2.4 kilobases upstream of the mouse [6] and rat UCP1 genes [7], which mediates brownadipocyte-specific expression of UCP1, as well as induction by β-adrenergic receptor agonists (via the second messenger cAMP), thyroid hormone [8,9], retinoic acid [10] and ligands for PPARγ [11]. As none of these signaling pathways is unique to brown adipocytes, however, the basis for development of the brown-adipocyte-specific thermogenic phenotype has been an enigma. There is particular interest in PPARγ, a ligand-activated transcription factor related to the steroid hormone receptors, which has recently been identified as a regulator of UCP1 gene expression and brown fat development. PPARγ is expressed at highest levels in white and brown adipose tissue. In white adipose tissue, PPARγ plays a critical role, along with the transcription factor C/EBPα, in promoting the differentiation of white adipocytes [12]. Furthermore, PPARγ is activated by thiazolidinediones, synthetic ligands which potently increase insulin responsiveness in animals — including humans — with noninsulin-dependent diabetes mellitus [13]. The physiologic mechanism responsible for this important therapeutic effect is presently unknown, but it may be related to effects on brown fat development and function. Over ten years ago, before the discovery of PPARγ, it was noted that thiazolidinediones stimulate brown fat thermogenesis [14,15]. More recently, it was shown that thiazolidinediones induce terminal differentiation of brown adipocyte cell lines [16]. Importantly, it was found that PPARγ binds to the UCP1 enhancer, from which it can activate transcription when coexpressed in cultured brown preadipocytes with its obligate heterodimerizing partner, the retinoid X receptor (RXR) [11]. Interestingly, transactivation of the UCP1 enhancer does not occur in nonadipocyte fibroblastic cell lines, indicating that additional factors must be present for brown-adipocyte-specific UCP1 expression. This is consistent with the observation that white adipocytes, while expressing significant amounts of PPARγ, lack UCP1 and other characteristic features of thermogenic brown adipocytes. PPARγ, like other nuclear receptors, stimulate transcription via interactions with coactivators, such as steroid receptor coactivator-1 (SRC-1) [17,18]. For the most part, these coactivators are widely expressed and consequently have not been thought to be important determinants of tissue-specific responses. With this in mind, it is of significance that Puigserver et al. [1] have cloned PGC-1, a coactivator with
relative tissue specificity. PGC-1 is expressed in brown fat, heart, muscle, kidney and brain, but not in many other tissues, most notably, white fat. Importantly, PGC-1 mRNA levels are dramatically upregulated in brown fat and skeletal muscle following acute cold exposure, which is a potent stimulator of sympathetic nervous outflow to brown fat. Numerous previous studies have shown that cold exposure markedly increases UCP1 gene expression, mitochondrial biogenesis, hyperplasia and thermogenesis in brown fat [2]. PGC-1 is a novel protein which shares little homology with previously identified coactivators. Its gene was cloned using the yeast two-hybrid assay with part of mouse PPARγ as bait. In cell-free protein–protein interaction assays, PGC-1 associates strongly with PPARγ and also the thyroid hormone, retinoic acid and estrogen receptors, but only weakly or not at all with the RXR. The binding of PGC-1 to PPARγ has two unusual features. First, the interaction is not ligand dependent — binding occurs with or with out the addition of PPARγ ligands. In contrast, modest to strong ligand dependence was found for the binding of PGC-1 to the thyroid hormone, retinoic acid and estrogen receptors. Second, PGC-1 binds to the DNA-binding and hinge domains of PPARγ, not the classical AF-2 domain which interacts with other coactivators such as SRC-1 [17,18]. As noted by Puigserver et al. [1], this raises the interesting possibility that PPARγ interacts simultaneously with PGC-1 and other coactivators to generate a larger, synergistically active macromolecular complex (Figure 2). Figure 2 Thiazolidinediones; prostaglandin J2; fatty acids; other ligands? 9-cis retinoic acid SRC-1 RXR PPARγ PGC-1 PPARγ response element
Transcriptional Transcription machinery TATA box
UCP1 Current Biology
A possible mechanism by which PGC-1 might increase the transcriptional activation potency of PPARγ. PPARγ and RXR form heterodimers on a PPARγ response element in the UCP1 enhancer. PGC-1 contacts the DNA-binding and hinge domains of PPARγ, while other coactivators, such as SRC-1 and possibly CBP, bind to its AF-2 domain. These coactivators increase ligand dependent gene transcription. The ligand for RXR is 9-cis retinoic acid; PPARγ ligands include the synthetic, antidiabetic thiazolidinediones, as well as various natural ligands, including 15-deoxy-prostaglandin J2 and certain free fatty acids.
Dispatch
In transient transfection assays, PGC-1 greatly augments the ability of both PPARγ and the thyroid hormone receptor to activate transcription from the UCP1 promoter. Interestingly, the combined transcriptional activation potency of PGC-1 and PPARγ was increased further by the addition of cAMP analogues. This agrees with earlier observations that β-adrenergic receptor agonists, which increase intracellular cAMP levels, and thiazolidinediones synergistically increase UCP1 mRNA expression in cultured brown adipocytes [19]. Similarly, cAMP synergizes with thyroid hormone to increase UCP1 gene expression [9]. It is not known whether the effects of cAMP on UCP1 gene expression are mediated by the cAMP response element binding protein (CREB). It is possible that PGC1, by virtue of its three potential protein kinase A phosphorylation sites, directly mediates the stimulatory effects of cAMP. This attractive possibility could account for the synergistic actions of cAMP on the activation of UCP1 gene expression that is induced by thiazolidinedione or thyroid hormone. Perhaps the most compelling argument for an involvement of PGC-1 in adaptive thermogenesis comes from the effects of retrovirally-mediated, chronic overexpression of PGC-1 in cultured white fat cells (3T3-F442A cells) [1]. Normally, these cells make little or no PGC-1 and UCP1, and have few mitochondria. The induced overexpression of PGC-1 led to an increase in the levels of mRNAs for UCP1, ATP synthase and cytochrome c-oxidase subunit Cox-IV, which are encoded by the nuclear genome, and also for cytochrome c-oxidase subunit Cox-II, which is encoded by the mitochondrial genome. PGC-1-expressing cells also had increased levels of mitochondrial DNA, indicating that overall mitochondrial biogenesis had been stimulated. The mechanism by which PGC-1 increases expression of genes encoded by the mitochondrial genome, such as Cox-II, and mitochondrial biogenesis in general, remains to be determined, but it might involve regulation of genes involved in communication between nucleus and mitochondria [20]. Overall, the observed effects of ectopic PGC-1 expression are impressive, especially as the level of PGC-1 expression achieved in the cultured white adipocytes was only 6% of that observed in brown adipose tissue from cold-exposed animals. Finally, we are left with the following perplexing question: how does a coactivator like PGC-1 confer specificity? PPARγ, in addition to being expressed in brown adipocytes, is also abundantly expressed in white adipocytes, where it plays an important role in driving expression of aP2 [12], which encodes a fatty-acid-binding protein. White adipocytes must have potent PPARγ coactivators, though not PGC-1. If transcription factors and coactivators are completely modular and interchangeable, as years of domain-swap experiments have taught us, how is it that these coactivators in white adipocytes stimulate
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PPARγ-mediated transcription of aP2, but not UCP1? Puigserver et al. [1] found that ectopic overexpression of PGC-1 in white fat cells increased expression of UCP1, but not aP2. Again, if all components of the system are modular and independent, then why is it that ectopic expression of PGC-1 fails to increase aP2 expression? One possible explanation for the specificity of PGC-1 for UCP1 over aP2 is that it interacts simultaneously with a number of transcription factors assembled on the UCP1 promoter, including PPARγ and the thyroid hormone and retinoic acid receptors, to produce a cumulative potent effect on UCP1 gene transcription (Figure 2). Alternatively, PPARγ response element of UCP1, but not that of aP2, may induce specific conformational changes in PPARγ that permit interaction with PGC-1. Examples of such interactions between DNA binding sites and transcription factors have recently been reviewed [21]. Overall, the evidence is persuasive that the PGC-1 is an important missing link between signaling pathways that are not tissue-specific and induction of the brown-fatspecific thermogenic phenotype. Acknowledgements I am grateful to Anthony Hollenberg and Jeffrey Flier for critically reading this manuscript.
References 1. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM: A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 1998, 92:829-839. 2. Himms-Hagen J: Brown adipose tissue thermogenesis and obesity. Prog Lipid Res 1989, 28:67-115. 3. Nicholls DG, Locke RM: Thermogenic mechanisms in brown fat. Physiol Rev 1984, 64:1-64. 4. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, LeviMeyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, et al.: Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 1997, 15:269-272. 5. Boss O, Samec S, Paoloni-Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P, Giacobino JP: Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression. FEBS Lett 1997, 408:39-42. 6. Kozak UC, Kopecky J, Teisinger J, Enerback S, Boyer B, Kozak LP: An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene. Mol Cell Biol 1994, 14:59-67. 7. Cassard-Doulcier AM, Gelly C, Fox N, Schrementi J, Raimbault S, Klaus S, Forest C, Bouillaud F, Ricquier D: Tissue-specific and betaadrenergic regulation of the mitochondrial uncoupling protein gene: control by cis-acting elements in the 5′′- flanking region. Mol Endocrinol 1993, 7:497-506. 8. Cassard-Doulcier AM, Larose M, Matamala JC, Champigny O, Bouillaud F, Ricquier D: In vitro interactions between nuclear proteins and uncoupling protein gene promoter reveal several putative transactivating factors including Ets1, retinoid X receptor, thyroid hormone receptor, and a CACCC box-binding protein. J Biol Chem 1994, 269:24335-24342. 9. Silva JE, Rabelo R: Regulation of the uncoupling protein gene expression. Eur J Endocrinol 1997, 136:251-264. 10. Alvarez R, de Andres J, Yubero P, Vinas O, Mampel T, Iglesias R, Giralt M, Villarroya F: A novel regulatory pathway of brown fat thermogenesis. Retinoic acid is a transcriptional activator of the mitochondrial uncoupling protein gene. J Biol Chem 1995, 270:5666-5673. 11. Sears IB, MacGinnitie MA, Kovacs LG, Graves RA: Differentiationdependent expression of the brown adipocyte uncoupling protein gene: regulation by peroxisome proliferator-activated receptor gamma. Mol Cell Biol 1996, 16:3410-3419.
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12. Tontonoz P, Hu E, Spiegelman BM: Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 1994, 79:1147-1156. 13. Saltiel AR, Olefsky JM: Thiazolidinediones in the treatment of insulin resistance and type II diabetes. Diabetes 1996, 45:1661-1669. 14. Mercer SW, Trayhurn P: Effects of ciglitazone on insulin resistance and thermogenic responsiveness to acute cold in brown adipose tissue of genetically obese (ob/ob) mice. FEBS Lett 1986, 195:12-16. 15. Rothwell NJ, Stock MJ, Tedstone AE: Effects of ciglitazone on energy balance, thermogenesis and brown fat activity in the rat. Mol Cell Endocrinol 1987, 51:253-257. 16. Tai TAC, Jennermann C, Brown KK, Oliver BB, MacGinnitie MA, Wilkison WO, Brown HR, Lehmann JM, Kliewer SA, Morris DC, Graves RA: Activation of the nuclear receptor peroxisome proliferator-activated receptor gamma promotes brown adipocyte differentiation. J Biol Chem 1996, 271:29909-29914. 17. Zhu Y, Qi C, Calandra C, Rao MS, Reddy JK: Cloning and identification of mouse steroid receptor coactivator-1 (mSRC-1), as a coactivator of peroxisome proliferator-activated receptor gamma. Gene Expr 1996, 6:185-195. 18. Glass CK, Rose DW, Rosenfeld MG: Nuclear receptor coactivators. Curr Opin Cell Biol 1997, 9:222-232. 19. Foellmi-Adams LA, Wyse BM, Herron D, Nedergaard J, Kletzien RF: Induction of uncoupling protein in brown adipose tissue. Synergy between norepinephrine and pioglitazone, an insulin-sensitizing agent. Biochem Pharmacol 1996, 52:693-701. 20. Scarpulla RC: Nuclear control of respiratory chain expression in mammalian cells. J Bioenerg Biomembr 1997, 29:109-119. 21. Lefstin JA, Yamamoto KR: Allosteric effects of DNA on transcriptional regulators. Nature 1998, 392:885-888.
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Adaptive thermogenesis: Turning on the heat Bradford B. Lowell
Brown adipocytes play important roles in the regulation of fat storage and body temperature, by virtue of their ability to uncouple mitochondrial fuel oxidation and ATP synthesis. The discovery of a tissue-specific transcriptional coactivator provides new insights into the regulation of thermogenesis by brown adipocytes. Address: Beth Israel Deaconess Medical Center and Harvard Medical School, Division of Endocrinology, Department of Medicine, 330 Brookline Avenue, Boston, Massachusetts 02215, USA. E-mail: [email protected] Current Biology 1998, 8:R517–R520 http://biomednet.com/elecref/09609822008R0517 © Current Biology Ltd ISSN 0960-9822
Brown adipocytes are highly specialized cells with a marked capacity for regulated thermogenesis. This feature is largely due to their abundant mitochondria, which uniquely possess the ability to uncouple fuel oxidation from ATP production, thus generating heat. Thermogenesis by adipocytes is a regulated, adaptive phenomenon — it is dramatically promoted by various agents, such as β-adrenergic receptor agonists, thyroid hormone, retinoic acid and ligands for peroxisome proliferator-activated receptor γ (PPARγ). The signaling pathways for these agents, however, also operate in other cell types — most notably non-thermogenic white adipocytes — and the downstream transcriptional mechanism that allows brown adipocytes to switch on their thermogenic machinery has until recently been unknown. But now Puigserver et al. [1] have reported the identification of an important link in the pathway that induces thermogenesis in brown adipocytes — a tissue-specific nuclear receptor coactivator known as PPAR gamma coactivator-1 (PGC-1). Regulated heat production plays an important part in the general control of the body’s energy budget. Triglyceride is an efficient means of storing energy, and its accumulation in white adipose tissue permits survival during starvation. Excessive fat storage, as in obesity, however, adversely affects health by increasing the risk of non-insulin-dependent diabetes mellitus and cardiovascular disease. In order to maintain proper balance, organisms must regulate fat storage. The brain plays a central role in controlling this process; it receives afferent inputs conveying information about nutritional status, integrates these signals and then modulates food intake and a component of energy expenditure that is referred to as adaptive thermogenesis. In rodents, brown adipose tissue makes an important contribution to adaptive thermogenesis [2]. This highly
specialized tissue (Figure 1) is distinguished by its intense sympathetic innervation, extensive blood supply, abundant mitochondria and unique expression of uncoupling protein-1 (UCP1). UCP1 is an inner mitochondria membrane carrier with proton transport activity which dissipates the proton electrochemical potential gradient, uncoupling fuel oxidation from conversion of ADP to ATP [3]. Consequently, stimulated brown adipocytes consume fuels, with the major by-product being the generation of heat. Recently, two proteins related to UCP1 were identified, UCP2 [4] and UCP3 [5], which are approximately 57% identical in amino-acid sequence to UCP1. UCP2 is widely expressed, whereas UCP3 is found preferentially in skeletal muscle. Whether these UCP1 homologs also act as mitochondrial uncoupling proteins is presently under investigation. Figure 1
Electron micrograph of brown fat from a cold-exposed mouse. Note the abundant mitochondria with densely packed cristae (micrograph kindly provided by Saverio Cinti).
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As UCP1 is expressed exclusively in brown adipocytes, intense interest has focused on transcription factors that regulate UCP1 production. The hope has been that the identification of such factors will shed important new light on the pathway of adaptive thermogenesis. Indeed, these efforts have identified a complex 220 base-pair enhancer, located approximately 2.4 kilobases upstream of the mouse [6] and rat UCP1 genes [7], which mediates brownadipocyte-specific expression of UCP1, as well as induction by β-adrenergic receptor agonists (via the second messenger cAMP), thyroid hormone [8,9], retinoic acid [10] and ligands for PPARγ [11]. As none of these signaling pathways is unique to brown adipocytes, however, the basis for development of the brown-adipocyte-specific thermogenic phenotype has been an enigma. There is particular interest in PPARγ, a ligand-activated transcription factor related to the steroid hormone receptors, which has recently been identified as a regulator of UCP1 gene expression and brown fat development. PPARγ is expressed at highest levels in white and brown adipose tissue. In white adipose tissue, PPARγ plays a critical role, along with the transcription factor C/EBPα, in promoting the differentiation of white adipocytes [12]. Furthermore, PPARγ is activated by thiazolidinediones, synthetic ligands which potently increase insulin responsiveness in animals — including humans — with noninsulin-dependent diabetes mellitus [13]. The physiologic mechanism responsible for this important therapeutic effect is presently unknown, but it may be related to effects on brown fat development and function. Over ten years ago, before the discovery of PPARγ, it was noted that thiazolidinediones stimulate brown fat thermogenesis [14,15]. More recently, it was shown that thiazolidinediones induce terminal differentiation of brown adipocyte cell lines [16]. Importantly, it was found that PPARγ binds to the UCP1 enhancer, from which it can activate transcription when coexpressed in cultured brown preadipocytes with its obligate heterodimerizing partner, the retinoid X receptor (RXR) [11]. Interestingly, transactivation of the UCP1 enhancer does not occur in nonadipocyte fibroblastic cell lines, indicating that additional factors must be present for brown-adipocyte-specific UCP1 expression. This is consistent with the observation that white adipocytes, while expressing significant amounts of PPARγ, lack UCP1 and other characteristic features of thermogenic brown adipocytes. PPARγ, like other nuclear receptors, stimulate transcription via interactions with coactivators, such as steroid receptor coactivator-1 (SRC-1) [17,18]. For the most part, these coactivators are widely expressed and consequently have not been thought to be important determinants of tissue-specific responses. With this in mind, it is of significance that Puigserver et al. [1] have cloned PGC-1, a coactivator with
relative tissue specificity. PGC-1 is expressed in brown fat, heart, muscle, kidney and brain, but not in many other tissues, most notably, white fat. Importantly, PGC-1 mRNA levels are dramatically upregulated in brown fat and skeletal muscle following acute cold exposure, which is a potent stimulator of sympathetic nervous outflow to brown fat. Numerous previous studies have shown that cold exposure markedly increases UCP1 gene expression, mitochondrial biogenesis, hyperplasia and thermogenesis in brown fat [2]. PGC-1 is a novel protein which shares little homology with previously identified coactivators. Its gene was cloned using the yeast two-hybrid assay with part of mouse PPARγ as bait. In cell-free protein–protein interaction assays, PGC-1 associates strongly with PPARγ and also the thyroid hormone, retinoic acid and estrogen receptors, but only weakly or not at all with the RXR. The binding of PGC-1 to PPARγ has two unusual features. First, the interaction is not ligand dependent — binding occurs with or with out the addition of PPARγ ligands. In contrast, modest to strong ligand dependence was found for the binding of PGC-1 to the thyroid hormone, retinoic acid and estrogen receptors. Second, PGC-1 binds to the DNA-binding and hinge domains of PPARγ, not the classical AF-2 domain which interacts with other coactivators such as SRC-1 [17,18]. As noted by Puigserver et al. [1], this raises the interesting possibility that PPARγ interacts simultaneously with PGC-1 and other coactivators to generate a larger, synergistically active macromolecular complex (Figure 2). Figure 2 Thiazolidinediones; prostaglandin J2; fatty acids; other ligands? 9-cis retinoic acid SRC-1 RXR PPARγ PGC-1 PPARγ response element
Transcriptional Transcription machinery TATA box
UCP1 Current Biology
A possible mechanism by which PGC-1 might increase the transcriptional activation potency of PPARγ. PPARγ and RXR form heterodimers on a PPARγ response element in the UCP1 enhancer. PGC-1 contacts the DNA-binding and hinge domains of PPARγ, while other coactivators, such as SRC-1 and possibly CBP, bind to its AF-2 domain. These coactivators increase ligand dependent gene transcription. The ligand for RXR is 9-cis retinoic acid; PPARγ ligands include the synthetic, antidiabetic thiazolidinediones, as well as various natural ligands, including 15-deoxy-prostaglandin J2 and certain free fatty acids.
Dispatch
In transient transfection assays, PGC-1 greatly augments the ability of both PPARγ and the thyroid hormone receptor to activate transcription from the UCP1 promoter. Interestingly, the combined transcriptional activation potency of PGC-1 and PPARγ was increased further by the addition of cAMP analogues. This agrees with earlier observations that β-adrenergic receptor agonists, which increase intracellular cAMP levels, and thiazolidinediones synergistically increase UCP1 mRNA expression in cultured brown adipocytes [19]. Similarly, cAMP synergizes with thyroid hormone to increase UCP1 gene expression [9]. It is not known whether the effects of cAMP on UCP1 gene expression are mediated by the cAMP response element binding protein (CREB). It is possible that PGC1, by virtue of its three potential protein kinase A phosphorylation sites, directly mediates the stimulatory effects of cAMP. This attractive possibility could account for the synergistic actions of cAMP on the activation of UCP1 gene expression that is induced by thiazolidinedione or thyroid hormone. Perhaps the most compelling argument for an involvement of PGC-1 in adaptive thermogenesis comes from the effects of retrovirally-mediated, chronic overexpression of PGC-1 in cultured white fat cells (3T3-F442A cells) [1]. Normally, these cells make little or no PGC-1 and UCP1, and have few mitochondria. The induced overexpression of PGC-1 led to an increase in the levels of mRNAs for UCP1, ATP synthase and cytochrome c-oxidase subunit Cox-IV, which are encoded by the nuclear genome, and also for cytochrome c-oxidase subunit Cox-II, which is encoded by the mitochondrial genome. PGC-1-expressing cells also had increased levels of mitochondrial DNA, indicating that overall mitochondrial biogenesis had been stimulated. The mechanism by which PGC-1 increases expression of genes encoded by the mitochondrial genome, such as Cox-II, and mitochondrial biogenesis in general, remains to be determined, but it might involve regulation of genes involved in communication between nucleus and mitochondria [20]. Overall, the observed effects of ectopic PGC-1 expression are impressive, especially as the level of PGC-1 expression achieved in the cultured white adipocytes was only 6% of that observed in brown adipose tissue from cold-exposed animals. Finally, we are left with the following perplexing question: how does a coactivator like PGC-1 confer specificity? PPARγ, in addition to being expressed in brown adipocytes, is also abundantly expressed in white adipocytes, where it plays an important role in driving expression of aP2 [12], which encodes a fatty-acid-binding protein. White adipocytes must have potent PPARγ coactivators, though not PGC-1. If transcription factors and coactivators are completely modular and interchangeable, as years of domain-swap experiments have taught us, how is it that these coactivators in white adipocytes stimulate
R519
PPARγ-mediated transcription of aP2, but not UCP1? Puigserver et al. [1] found that ectopic overexpression of PGC-1 in white fat cells increased expression of UCP1, but not aP2. Again, if all components of the system are modular and independent, then why is it that ectopic expression of PGC-1 fails to increase aP2 expression? One possible explanation for the specificity of PGC-1 for UCP1 over aP2 is that it interacts simultaneously with a number of transcription factors assembled on the UCP1 promoter, including PPARγ and the thyroid hormone and retinoic acid receptors, to produce a cumulative potent effect on UCP1 gene transcription (Figure 2). Alternatively, PPARγ response element of UCP1, but not that of aP2, may induce specific conformational changes in PPARγ that permit interaction with PGC-1. Examples of such interactions between DNA binding sites and transcription factors have recently been reviewed [21]. Overall, the evidence is persuasive that the PGC-1 is an important missing link between signaling pathways that are not tissue-specific and induction of the brown-fatspecific thermogenic phenotype. Acknowledgements I am grateful to Anthony Hollenberg and Jeffrey Flier for critically reading this manuscript.
References 1. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM: A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 1998, 92:829-839. 2. Himms-Hagen J: Brown adipose tissue thermogenesis and obesity. Prog Lipid Res 1989, 28:67-115. 3. Nicholls DG, Locke RM: Thermogenic mechanisms in brown fat. Physiol Rev 1984, 64:1-64. 4. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, LeviMeyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, et al.: Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 1997, 15:269-272. 5. Boss O, Samec S, Paoloni-Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P, Giacobino JP: Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression. FEBS Lett 1997, 408:39-42. 6. Kozak UC, Kopecky J, Teisinger J, Enerback S, Boyer B, Kozak LP: An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene. Mol Cell Biol 1994, 14:59-67. 7. Cassard-Doulcier AM, Gelly C, Fox N, Schrementi J, Raimbault S, Klaus S, Forest C, Bouillaud F, Ricquier D: Tissue-specific and betaadrenergic regulation of the mitochondrial uncoupling protein gene: control by cis-acting elements in the 5′′- flanking region. Mol Endocrinol 1993, 7:497-506. 8. Cassard-Doulcier AM, Larose M, Matamala JC, Champigny O, Bouillaud F, Ricquier D: In vitro interactions between nuclear proteins and uncoupling protein gene promoter reveal several putative transactivating factors including Ets1, retinoid X receptor, thyroid hormone receptor, and a CACCC box-binding protein. J Biol Chem 1994, 269:24335-24342. 9. Silva JE, Rabelo R: Regulation of the uncoupling protein gene expression. Eur J Endocrinol 1997, 136:251-264. 10. Alvarez R, de Andres J, Yubero P, Vinas O, Mampel T, Iglesias R, Giralt M, Villarroya F: A novel regulatory pathway of brown fat thermogenesis. Retinoic acid is a transcriptional activator of the mitochondrial uncoupling protein gene. J Biol Chem 1995, 270:5666-5673. 11. Sears IB, MacGinnitie MA, Kovacs LG, Graves RA: Differentiationdependent expression of the brown adipocyte uncoupling protein gene: regulation by peroxisome proliferator-activated receptor gamma. Mol Cell Biol 1996, 16:3410-3419.
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12. Tontonoz P, Hu E, Spiegelman BM: Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 1994, 79:1147-1156. 13. Saltiel AR, Olefsky JM: Thiazolidinediones in the treatment of insulin resistance and type II diabetes. Diabetes 1996, 45:1661-1669. 14. Mercer SW, Trayhurn P: Effects of ciglitazone on insulin resistance and thermogenic responsiveness to acute cold in brown adipose tissue of genetically obese (ob/ob) mice. FEBS Lett 1986, 195:12-16. 15. Rothwell NJ, Stock MJ, Tedstone AE: Effects of ciglitazone on energy balance, thermogenesis and brown fat activity in the rat. Mol Cell Endocrinol 1987, 51:253-257. 16. Tai TAC, Jennermann C, Brown KK, Oliver BB, MacGinnitie MA, Wilkison WO, Brown HR, Lehmann JM, Kliewer SA, Morris DC, Graves RA: Activation of the nuclear receptor peroxisome proliferator-activated receptor gamma promotes brown adipocyte differentiation. J Biol Chem 1996, 271:29909-29914. 17. Zhu Y, Qi C, Calandra C, Rao MS, Reddy JK: Cloning and identification of mouse steroid receptor coactivator-1 (mSRC-1), as a coactivator of peroxisome proliferator-activated receptor gamma. Gene Expr 1996, 6:185-195. 18. Glass CK, Rose DW, Rosenfeld MG: Nuclear receptor coactivators. Curr Opin Cell Biol 1997, 9:222-232. 19. Foellmi-Adams LA, Wyse BM, Herron D, Nedergaard J, Kletzien RF: Induction of uncoupling protein in brown adipose tissue. Synergy between norepinephrine and pioglitazone, an insulin-sensitizing agent. Biochem Pharmacol 1996, 52:693-701. 20. Scarpulla RC: Nuclear control of respiratory chain expression in mammalian cells. J Bioenerg Biomembr 1997, 29:109-119. 21. Lefstin JA, Yamamoto KR: Allosteric effects of DNA on transcriptional regulators. Nature 1998, 392:885-888.