Thermogenic Responses in Brown Fat Cells Are Fully UCP1-dependent
2000; Elsevier BV; Volume: 275; Issue: 33 Linguagem: Inglês
10.1074/jbc.m000547200
ISSN1083-351X
AutoresA. Matthias, Kerstin B. E. Ohlson, J. Magnus Fredriksson, Anders Jacobsson, Jan Nedergaard, Barbara Cannon,
Tópico(s)Exercise and Physiological Responses
ResumoTo examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated.Ucp1(−/−) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust (≈10-fold increase in thermogenesis);Ucp1(−/−) cells only responded ≈3% of this. Ucp1(−/−) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect inUcp1(−/−) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells. To examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated.Ucp1(−/−) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust (≈10-fold increase in thermogenesis);Ucp1(−/−) cells only responded ≈3% of this. Ucp1(−/−) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect inUcp1(−/−) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells. uncoupling protein carbonyl cyanidep-trifluoromethoxyphenylhydrazone The thermogenic capacity of brown adipocytes is unsurpassed in mammalian tissues; after the addition of the physiological stimulator norepinephrine, brown adipocytes can chronically increase their metabolism 10-fold (1Prusiner S.B. Cannon B. Lindberg O. Eur. J. Biochem. 1968; 6: 15-22Crossref PubMed Scopus (99) Google Scholar, 2Reed N. Fain J.N. J. Biol. Chem. 1968; 243: 2843-2848Abstract Full Text PDF PubMed Google Scholar) and produce heat at a rate of about 3 nanowatts/cell, corresponding to about 300 watts/kilogram of tissue (3Nedergaard J. Cannon B. Lindberg O. Nature. 1977; 267: 518-520Crossref PubMed Scopus (82) Google Scholar, 4Nedergaard J. Lindberg O. Int. Rev. Cytol. 1982; 74: 187-286Crossref PubMed Scopus (154) Google Scholar). The biochemical mechanism behind this remarkable metabolic achievement has attracted scientific interest since the heat-producing capacity of brown adipose tissue was first established (5Smith R.E. Physiologist. 1961; 4: 113Google Scholar). It is today generally accepted that the heat-producing ability of brown adipocytes is fully or partly a consequence of the presence in the mitochondria of these cells of the functionally protonophoric protein uncoupling protein-1 (UCP1)1 (thermogenin) (for reviews, see Refs. 6Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1335) Google Scholar, 7Nedergaard J. Cannon B. Ernster L. New Comprehensive Biochemistry. 23. Elsevier, Amsterdam1992: 385-420Google Scholar, 8Garlid K.D. Jaburek M. Jezek P. FEBS Lett. 1998; 438: 10-14Crossref PubMed Scopus (132) Google Scholar, 9Klingenberg M. Huang S.G. Biochim. Biophys. Acta. 1999; 1415: 271-296Crossref PubMed Scopus (313) Google Scholar).However, it is first with the development of UCP1-ablated mice in the laboratory of L. P. Kozak (10Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1067) Google Scholar) that it has become possible to approach some basic questions in the cellular physiology of brown adipocytes. These questions include whether the UCP1 mechanism is the only thermogenic mechanism of significance within the brown adipocytes and whether the mere presence of UCP1 within the brown adipocytes in itself conveys a state of semi-uncoupling to the mitochondria within the cells (as has been observed when UCP1 has been ectopically expressed (11Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1550) Google Scholar, 12Gimeno R.E. Dembski M. Weng X. Deng N. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Woolf E.A. Tartaglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar, 13Hagen T. Zhang C.-Y. Slieker L.J. Chung W.K. Leibel R.L. Lowell B.B. FEBS Lett. 1999; 454: 201-206Crossref PubMed Scopus (47) Google Scholar, 14Zhang C.-Y. Hagen T. Mootha V.K. Slieker L.J. Lowell B.B. FEBS Lett. 1999; 449: 129-134Crossref PubMed Scopus (102) Google Scholar)). Also the question of the nature of the intracellular physiological activator of UCP1 has become timely, because it has been observed that the presence of UCP1 in isolated brown fat mitochondria does not seem to increase their sensitivity to the de-energizing action of fatty acids (15Matthias A. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 1999; 274: 28150-28160Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), although fatty acids have generally been believed to be the activators of UCP1 (6Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1335) Google Scholar, 7Nedergaard J. Cannon B. Ernster L. New Comprehensive Biochemistry. 23. Elsevier, Amsterdam1992: 385-420Google Scholar, 8Garlid K.D. Jaburek M. Jezek P. FEBS Lett. 1998; 438: 10-14Crossref PubMed Scopus (132) Google Scholar, 9Klingenberg M. Huang S.G. Biochim. Biophys. Acta. 1999; 1415: 271-296Crossref PubMed Scopus (313) Google Scholar). Through analysis of brown adipocytes isolated from UCP1-ablated mice, we present evidence here that no other adrenergic thermogenic mechanism exists in brown adipocytes except that associated with UCP1, and that UCP1 in its unstimulated state, when entopically expressed and under physiological control, does not induce a state of partial “uncoupling” to the mitochondria, at least not observable at the present degree of resolution. We also suggest that the activator of UCP1 is most likely not the free fatty acids themselves but a metabolite thereof.Further, brown adipose tissue in the UCP1-ablated mice demonstrates very high expression levels of the UCP1 family members UCP2 and UCP3 (10Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1067) Google Scholar, 15Matthias A. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 1999; 274: 28150-28160Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 16Cannon B. Matthias A. Golozoubova V. Ohlson K.B.E. Andersson U. Jacobsson A. Nedergaard J. Ailhaud G. Guy-Grand B. Progress in Obesity Research 8. John Libbey, London1999: 13-26Google Scholar), probably the highest combined level in any mammalian tissue. It has therefore been possible to analyze theUcp1(−/−) brown adipocytes also for signs of thermogenic (or uncoupling) effects that could be associated with the very high entopic expression of the genes for these novel uncoupling proteins, as suggested (17Jaburek M. Varecha M. Gimeno R.E. Dembski M. Jezek P. Zhang M. Burn P. Tartaglia L.A. Garlid K.D. J. Biol. Chem. 1999; 274: 26003-26007Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). We found, however, that this high entopic expression was not associated with any observable signs of mitochondrial uncoupling or thermogenesis, in contrast to what is observed when these proteins have been ectopically expressed. Thus, UCP2 or UCP3 do not substitute for UCP1 as adrenergically stimulated thermogenic proteins in brown adipocytes, even when the cellular activation mechanism for thermogenesis is intact. We therefore conclude that UCP1 distinguishes itself from the other (probably more ancient (18Stuart J.A. Harper J.A. Brindle K.M. Brand M.D. Biochim. Biophys. Acta. 1999; 1413: 50-54Crossref PubMed Scopus (64) Google Scholar)) uncoupling protein family members by being the only one that can convey to a mammalian cell a thermogenic response to adrenergic stimulation.DISCUSSIONIn the present investigation, we have demonstrated that the absence of UCP1 led to a complete loss of thermogenic capacity of isolated brown fat cells, both when they were stimulated by addition of the physiological activator norepinephrine and when thermogenesis was induced by fatty acid addition. Besides demonstrating the essential role of UCP1 for nonshivering thermogenesis in brown fat cells, these experiments also provide new information on the basal and stimulated activity of UCP1 when entopically expressed and information concerning the nature of the intracellular physiological activator of UCP1. They also indicate that UCP2 and UCP3 do not substitute for UCP1 as thermogenic proteins in these cells.No UCP1-independent Adrenergic Thermogenic Process Exists in Brown AdipocytesFrom previous studies, particularly in isolated brown fat mitochondria (reviewed e.g. in Refs. 6Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1335) Google Scholar, 7Nedergaard J. Cannon B. Ernster L. New Comprehensive Biochemistry. 23. Elsevier, Amsterdam1992: 385-420Google Scholar, 8Garlid K.D. Jaburek M. Jezek P. FEBS Lett. 1998; 438: 10-14Crossref PubMed Scopus (132) Google Scholar, 9Klingenberg M. Huang S.G. Biochim. Biophys. Acta. 1999; 1415: 271-296Crossref PubMed Scopus (313) Google Scholar), it has been inferred that activation of UCP1 is a prerequisite for thermogenesis. Provided that this axiom, that brown fat thermogenesis occurs only through the UCP1-mediated mechanism, is accepted per se, the outcome of the present experiments, i.e. that it is not possible to elicit thermogenesis in brown fat cells from the UCP1-ablated mice, may be said to be what would be expected. However, it may be pointed out that this total elimination of the thermogenic response to norepinephrine in the Ucp1(−/−) cells finally resolves experimentally the long standing principal issue of whether other mechanisms could be responsible for, or at least contribute to, the thermogenic response to norepinephrine in brown fat cells. Possible extramitochondrial thermogenic processes that have been discussed include norepinephrine-induced activation of the plasma membrane Na+/K+-ATPase (directly or indirectly because of norepinephrine-induced plasma membrane depolarization and increased Na+ influx), an ATP-utilizing substrate cycling (of fatty acids/triglyceride or glucose/glucose 6-phosphate), glycerol 3-phosphate cycling, peroxisomal fatty acid degradation, and an α1-adrenoreceptor-induced, “coupled” respiration.It is the clear outcome of the present experiments that no such additional UCP1-independent adrenergic thermogenic component exists (although auxillary effects of these processes cannot be ruled out by the present experiments). Other cellular processes clearly make only an extremely minor contribution to norepinephrine-induced thermogenesis, as compared with that of UCP1. Considering the number of metabolic processes not supposedly linked to UCP1 activation that are stimulated by norepinephrine in these cells (e.g. ion fluxes), it is indeed remarkable that there is only such a small norepinephrine-induced UCP1-independent increase in oxygen consumption.When Entopically Expressed, UCP1 Is Not Innately an Overtly Active Mitochondrial De-energizerIt is clear from the present experiments that the entopic expression of UCP1 in the mitochondria within brown fat cells does not lead to a measurable increase in basal metabolism of these cells. In other words, the large proton (equivalent)-conducting activity of UCP1 does not manifest itself unless the cells are externally stimulated, physiologically or with fatty acids. The low respiratory rate in unstimulated cells is not because of a lack of substrate, as the addition of the adequate exogenous substrate pyruvate addition is without effect and as FCCP increases the respiratory rate even in UCP1-containing cells.The lack of overt innate uncoupling activity of UCP1 in situimplies that when brown fat-derived nonshivering thermogenesis is not needed, there is no leakage through the system and thus no waste of energy. It will be noted that in this respect the behavior of UCP1 when entopically expressed is in contrast to its properties when it is ectopically expressed in yeast cells (or in HeLa cells (41Li B. Holloszy J.O. Semenkovich C.F. J. Biol. Chem. 1999; 274: 17534-17540Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar)). Indeed, when UCP1 is expressed in certain yeast strains, these strains have decreased viability and growth rate (11Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1550) Google Scholar, 12Gimeno R.E. Dembski M. Weng X. Deng N. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Woolf E.A. Tartaglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar, 42Bathgate B. Freebairn E.M. Greenland A.J. Reid G.A. Mol. Microbiol. 1992; 6: 363-370Crossref PubMed Scopus (33) Google Scholar) (although not all authors report this (43Bouillaud F. Arechaga I. Petit P.X. Raimbault S. Levi-Meyrueis C. Casteilla L. Laurent M. Rial E. Ricquier D. EMBO J. 1994; 13: 1990-1997Crossref PubMed Scopus (111) Google Scholar, 44Gonzalez-Barroso M.M. Fleury C. Arechaga I. Zaragoza P. Levi-Meyrueis C. Raimbault S. Ricquier D. Bouillaud F. Rial E. Eur. J. Biochem. 1996; 239: 445-450Crossref PubMed Scopus (56) Google Scholar)). This loss of viability, associated with a marked decrease in mitochondrial membrane potential as estimated within the yeast cells (11Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1550) Google Scholar, 12Gimeno R.E. Dembski M. Weng X. Deng N. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Woolf E.A. Tartaglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar, 13Hagen T. Zhang C.-Y. Slieker L.J. Chung W.K. Leibel R.L. Lowell B.B. FEBS Lett. 1999; 454: 201-206Crossref PubMed Scopus (47) Google Scholar, 14Zhang C.-Y. Hagen T. Mootha V.K. Slieker L.J. Lowell B.B. FEBS Lett. 1999; 449: 129-134Crossref PubMed Scopus (102) Google Scholar), has been interpreted to indicate that in these yeast cells, UCP1 is functionally correctly inserted in the mitochondria. However, the present experiments demonstrate that this type of high innate uncoupling is not a property of UCP1 when it is entopically expressed. Rather, when UCP1 is functionally correctly inserted in its native environment, no overt innate uncoupling effect is expected. Why UCP1 behaves differently in this respect when entopically and ectopically expressed is not known. One possibility would be that brown fat cells possess an endogenous inhibitor of UCP1 activity unique to these cells; another possibility would be that the yeast expression is so high that the normal functioning of the mitochondria is disturbed.The Nature of the Intracellular Physiological ActivatorIn this investigation, the presence of UCP1 has been demonstrated to be essential not only for norepinephrine to elicit thermogenesis but also for added fatty acids to accomplish this. In this respect, the present results may initially be considered to be discrepant with our earlier observations in isolated brown fat mitochondria, where the uncoupling effect of fatty acids was demonstrated not to be UCP1-dependent (15Matthias A. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 1999; 274: 28150-28160Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). However, the mitochondrial and cellular observations may rather be analyzed together, as done below, and through this may bring further insight to a basic question in the cellular physiology of brown adipose tissue: the nature of the signaling process leading from adrenergic receptor activation to acute UCP1 activation.There are a number of suggestions in the literature as to the nature of this “intracellular physiological activator.” Some of the candidates for the activator are summarized in Fig.7.One model (Fig. 7 A) distinguishes itself from models B–D in that the state of UCP1 in the unstimulated cell is differently formulated. According to this hypothesis, it is visualized that UCP1 is innately in an uninhibited state because it is considered not to be exposed to purine nucleotides (that inhibit UCP1 activity in experiments performed with isolated brown fat mitochondria and with isolated UCP1, as reviewed in e.g. Refs. 6Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1335) Google Scholar and 7Nedergaard J. Cannon B. Ernster L. New Comprehensive Biochemistry. 23. Elsevier, Amsterdam1992: 385-420Google Scholar). In this hypothesis, the activator may be suggested to be free fatty acids (this suggestion is based on the necessity of fatty acids for UCP1 functioning in ectopic and reconstituted system; however, it will be remembered that added fatty acids are apparently not necessary for UCP1 to function in situ, i.e. in isolated brown fat mitochondria (15Matthias A. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 1999; 274: 28150-28160Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar)). Thus, in unstimulated cells, there should be so little fatty acid available (that functions in this formulation as a co-factor) that UCP1 is unfunctional even though it is uninhibited. During stimulation, lipolysis leads to increased fatty acid levels and these, in this model, are the co-factors necessary to make UCP1 functional. Based on the data presented here alone, this model cannot be refuted, but the premises for this supposition may be challenged. The inhibitory so-called GDP-binding site on UCP1 has an affinity for free purine nucleotides of about 1 μm (45Sundin U. Cannon B. Comp. Biochem. Physiol. 1980; 65B: 463-471Google Scholar, 46Rafael J. Pampel I. Wang X. Eur. J. Biochem. 1994; 223: 971-980Crossref PubMed Scopus (19) Google Scholar). The total concentration of purine di- and triphosphate nucleotides in the cytosol is probably in the order of millimolar, but the free concentrations are lower because of the presence of Mg2+-chelated forms that are unable to inhibit UCP1 (46Rafael J. Pampel I. Wang X. Eur. J. Biochem. 1994; 223: 971-980Crossref PubMed Scopus (19) Google Scholar). However, if about equimolar concentrations of Mg2+ and purine nucleotides are found in the cytosol, about 2–4% of total purine nucleotide would be in the free form (according to Ref. 47Brooks S.P.J. Storey K.B. Anal. Biochem. 1992; 201: 119-126Crossref PubMed Scopus (323) Google Scholar), corresponding to some 50–100 μm free nucleotide, i.e. a concentration widely in excess of that needed for full UCP1 inhibition. It is therefore difficult to see how UCP1 could be in an uninhibited state within the cell. It is also notable that in this hypothesis, the GDP-binding site on UCP1 is devoid of any regulatory role in thermogenesis. Thus, although the present experiments cannot rule out this hypothesis, it does not seem to fulfil other criteria for an activation process.In contrast, the basis for the hypotheses in Fig. 7, B—D,is that UCP1 activity is inhibited in the resting state because of the effect of cytosolic purine nucleotides (ATP, ADP, GTP, and GDP together); this point of view is based on the behavior of UCP1 in isolated brown fat mitochondria (as reviewed in e.g. Refs.6Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1335) Google Scholar, 7Nedergaard J. Cannon B. Ernster L. New Comprehensive Biochemistry. 23. Elsevier, Amsterdam1992: 385-420Google Scholar, 8Garlid K.D. Jaburek M. Jezek P. FEBS Lett. 1998; 438: 10-14Crossref PubMed Scopus (132) Google Scholar, 9Klingenberg M. Huang S.G. Biochim. Biophys. Acta. 1999; 1415: 271-296Crossref PubMed Scopus (313) Google Scholar). In this case, an activator must therefore somehow overcome this inhibition.According to one group of hypotheses (Fig. 7 B), norepinephrine generates an activator of UCP1 independent of its action on hormone-sensitive lipase (e.g. cytosolic alkanization). This activator then overcomes the purine nucleotide inhibition. Considering the data presented here, that stimulation by added fatty acids is UCP1-dependent, the postulation of a further, nonlipolysis-related, activator would seem unnecessarily complex.Alternatively, the intracellular physiological activator may be a product of lipolysis. This could be the released free fatty acids themselves (Fig. 7 C). This is presently the prevalent hypothesis for norepinephrine activation of thermogenesis (6Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1335) Google Scholar, 48Gonzalez-Barroso M.M. Fleury C. Bouillaud F. Nicholls D.G. Rial E. J. Biol. Chem. 1998; 273: 15528-15532Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The findings presented here do not in themselves contradict such a proposal, but this hypothesis is made less likely based on our studies of isolated brown fat mitochondria, where we were unable to distinguish a UCP1-dependent de-energization induced by free fatty acids (15Matthias A. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 1999; 274: 28150-28160Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). As the de-energization observed in the isolated brown fat mitochondria was UCP1-independent, it probably merely represented the general uncoupling effect of fatty acids observed in any mitochondrial preparation (37Wojtczak L. Schönfeld P. Biochim. Biophys. Acta. 1993; 1183: 41-57Crossref PubMed Scopus (314) Google Scholar, 38Skulachev V.P. Biochim. Biophys. Acta. 1998; 1363: 100-124Crossref PubMed Scopus (810) Google Scholar), and the effect may therefore even be considered artifactual. Therefore these mitochondrial observations make it unlikely that fatty acids are the direct activators of UCP1 within the cell; another activator would seem to be necessary. A hypothesis for an activation scheme, based on the fact that fatty acids added to the cells stimulate thermogenesis in a UCP1-dependent way (Fig.5), is presented in D. However, it may be wondered why fatty acids, that clearly function as UCP1-independent uncouplers in isolated brown fat mitochondria (15Matthias A. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 1999; 274: 28150-28160Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), are unable to uncouple in an UCP1-independent way when the same mitochondria are confined to cells. A possibility is that the high levels of fatty acid-binding proteins found in these cells (49Daikoku T. Shinohara Y. Shima A. Yamazaki N. Terada H. FEBS Lett. 1997; 410: 383-386Crossref PubMed Scopus (41) Google Scholar) do not allow cytosolic free fatty acid levels to become sufficiently high to reach the probably unphysiological levels necessary for UCP1-independent uncoupling of the mitochondria within the cells.In view of the results presented here, that fatty acid uncoupling is UCP1-dependent in cells in combination with the fact that fatty acid in themselves were apparently unable to activate UCP1 in isolated brown fat mitochondria (15Matthias A. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 1999; 274: 28150-28160Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), it would seem most plausible to propose that the activation sequence in the cells involves adrenergic stimulation of lipolysis and thus intracellular release of fatty acids. However, it may be suggested that it is not the released fatty acids themselves that activate UCP1 but rather a fatty acid metabolite (in a broad sense) (Fig. 7 D); this metabolite would be formed irrespective of whether the fatty acids are of endogenous or exogenous origin. The nature of such an activating metabolite is presently unknown. However, one downstream product of fatty acid metabolism, fatty acyl-CoA esters, can compete in isolated mitochondria with purine nucleotides bound to UCP1 and increase ion transport through the protein, i.e. activate UCP1 (50Cannon B. Sundin U. Romert L. FEBS Lett. 1977; 74: 43-46Crossref PubMed Scopus (56) Google Scholar, 51Katiyar S.S. Shrago E. Biochem. Biophys. Res. Commun. 1991; 175: 1104-1111Crossref PubMed Scopus (23) Google Scholar), although no studies in reconstituted systems have as yet confirmed these effects.UCP2 and UCP3From cDNA libraries, mRNAs coding for proteins now dubbed UCP2 (11Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1550) Google Scholar, 12Gimeno R.E. Dembski M. Weng X. Deng N. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Woolf E.A. Tartaglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar) and UCP3 (52Boss O. Samec S. Paoloni-Giacobino A. Rossier C. Dulloo A. Seydoux J. Muzzin P. Giacobino J.P. FEBS Lett. 1997; 408: 39-42Crossref PubMed Scopus (996) Google Scholar, 53Vidal-Puig A. Solanes G. Grujic D. Flier J.S. Lowell B.B. Biochem. Biophys. Res. Commun. 1997; 235: 79-82Crossref PubMed Scopus (681) Google Scholar) were recently identified. These mRNAs represent proteins more homologous to UCP1 than any other proteins presently identified. Because of this relatively close homology, an evident initial suggestion was that these proteins should also have thermogenesis/uncoupling as their function. This suggestion gained initial support from experiments in which these proteins were ectopically expressed in yeast strains (11Fleury C. Neverova M. Collins S. Raimbault S. Champigny O. Levi-Meyrueis C. Bouillaud F. Seldin M.F. Surwit R.S. Ricquier D. Warden C.H. Nat. Genet. 1997; 15: 269-272Crossref PubMed Scopus (1550) Google Scholar, 12Gimeno R.E. Dembski M. Weng X. Deng N. Shyjan A.W. Gimeno C.J. Iris F. Ellis S.J. Woolf E.A. Tartaglia L.A. Diabetes. 1997; 46: 900-906Crossref PubMed Scopus (0) Google Scholar, 13Hagen T. Zhang C.-Y. Slieker L.J. Chung W.K. Leibel R.L. Lowell B.B. FEBS Lett. 1999; 454: 201-206Crossref PubMed Scopus (47) Google Scholar, 14Zhang C.-Y. Hagen T. Mootha V.K. Slieker L.J. Lowell B.B. FEBS Lett. 1999; 449: 129-134Crossref PubMed Scopus (102) Google Scholar, 54Gong D.W. He Y. Karas M. Reitman M. J. Biol. Chem. 1997; 272: 24129-24132Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 55Liu Q. Bai C. Chen F. Wang R. MacDonald T. Gu M. 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A characteristic for these systems with ectopic expression was also that a very high degree of mitochondrial uncoupling was observed (i.e. a very much lowered mitochondrial membrane potential within the cells) and this has been understood as being the reason for the poor growth of the yeast strains.Serendipitously, the present experiments may be helpful in establishing whether these conclusions from experiments with ectopically expressed UCP2/UCP3 are also valid when UCP2/UCP3 are entopically expressed. This is because in the brown adipose tissue of the UCP1-ablated animals, high expression levels of UCP2/UCP3 are found (10Enerbäck S. Jacobsson A. Simpson E.M. Guerra C. Yamashita H. Harper M.-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1067) Google Scholar, 15Matthias A. Jacobsson A. Cannon B. Nedergaard J. J. Biol. Chem. 1999; 274: 28150-28160Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 16Cannon B. Matthias A. Golozoubova V. Ohlson K.B.E. 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