Sonia Samec

Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression.

Brown adipose tissue (BAT) and skeletal muscle are important sites of nonshivering thermogenesis. The uncoupling protein‐1 (UCP1) is the main effector of nonshivering thermogenesis in BAT and the recently described ubiquitous UCP2 [1] has been implicated in energy balance. In an attempt to better understand the biochemical events underlying nonshivering thermogenesis in muscle, we screened a human skeletal muscle cDNA library and isolated three clones: UCP2, UCP3L and UCP3S. The novel UCP3 was 57% and 73% identical to human UCP1 and UCP2, respectively, highly skeletal muscle‐specific and its expression was unaffected by cold acclimation. This new member of the UCP family is a candidate protein for the modulation of the respiratory control in skeletal muscle.

Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of thermogenesis or regulators of lipids as fuel substrate?

The mRNA expressions of UCP2 and UCP3, two newly described genes with high sequence homology to the uncoupling protein UCP1 in brown adipose tissue (BAT), were examined in two skeletal muscles (gastrocnemius and soleus) as well as in interscapular BAT (IBAT) of the rat in response to food deprivation and controlled refeeding. In IBAT (a tissue highly dependent on lipids for thermogenesis), the pattern of mRNA expression of UCP2 and UCP3 closely follows that of UCP1: it was markedly down‐regulated during food deprivation (when this tissues thermogenesis and lipid fuel requirements are decreased) and restored to control levels by day 5 of refeeding. By contrast, in the gastrocnemius muscle (a mixed fiber type muscle with a high capacity to shift between glucose and lipids as fuel substrate), mRNA expression of both UCP2 and UCP3 mRNA was found to be markedly up‐regulated during food deprivation (when this tissues thermogenesis is also decreased but its lipid fuel utilization is increased). The expressions were subsequently found to be markedly down‐regulated upon transition to refeeding, with mRNA levels remaining below control levels on days 3, 5, and 10 of refeeding (period of enhanced efficiency of body fat deposition). In the soleus muscle (an oxidative type muscle with higher dependency on lipids than the gastrocnemius, and hence with a lower capacity to shift between lipids and glucose as fuel substrate), UCP homologues were also found to be up‐regulated during food deprivation, but changes in their mRNA expression contrast with those in the gastrocnemius muscle both in their much lower magnitude of response to food deprivation and in their more rapid restoration to control levels during refeeding. Up‐regulation of UCP2 and UCP3 gene expressions in skeletal muscle during food deprivation was found to persist at thermoneutrality (i.e., under conditions of reduced thermoregulatory thermogenesis). Together, these tissue‐dependent differential mRNA expressions of the UCP homologues in IBAT, gastrocnemius, and soleus muscles during food deprivation and refeeding are much more consistent with a role for UCP2 and UCP3 in the regulation of lipids as fuel substrate rather than as mediators of regulatory thermogenesis.—Samec, S., Seydoux, J., Dulloo, A. G. Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of thermogenesis or regulators of lipids as fuel substrate? FASEB J. 12, 715–724 (1998)

Uncoupling Protein-3 Expression in Rodent Skeletal Muscle Is Modulated by Food Intake but Not by Changes in Environmental Temperature

A new member of the uncoupling protein (UCP) family called UCP3 has recently been cloned and shown to be highly expressed in skeletal muscle of rodents and humans. In the present study, UCP3 was overexpressed in C2C12myoblasts where it acts as an uncoupling protein. Changes in UCP3 mRNA expression were examined in rodent muscles under conditions known to modulate thermogenesis in brown adipose tissue. In skeletal muscle, UCP3 expression did not change in response to 48 h of cold exposure (6 °C), whereas it was decreased by 81% or increased 5.6-fold by 1 week of 50% food restriction or fasting, respectively. It was also decreased by 36% in soleus muscle of obese (fa/fa) as compared with lean Zucker rats. The unexpected rise of UCP3 mRNA level induced by fasting did not change in vitro muscle basal heat production rate but decreased by 31% the capacity to produce heat in response to the uncoupler carbonylcyanidep-trifluoromethoxyphenylhydrazone. This decrease may reflect underlying uncoupling by UCP3. Up-regulation of UCP3 mRNA after a 24-h fast was still observed in mice exposed at thermoneutrality. These results show that the increase in UCP3 expression induced by fasting is associated with the maintenance of thermogenesis measured in muscle in vitro and is not modulated by environmental temperature. The notion that UCP3 expression is modulated by food intake is of importance to better understand the pathophysiology of obesity in humans.

UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged.

The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5‐fold and 4‐fold, respectively, and UCP3 protein levels by 2‐fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.

Tissue-dependent upregulation of rat uncoupling protein-2 expression in response to fasting or cold.

The control of uncoupling protein‐2 (UCP2) mRNA expression in rat brown adipose tissue (BAT), heart and skeletal muscles was examined. Cold exposure (48 h) increased UCP2 mRNA in BAT, heart and soleus muscle by 2.4‐, 4.3‐ and 2.6‐fold, respectively. Fasting (48 h) had no effect on UCP2 mRNA expression neither in BAT nor in heart, but markedly increased it in skeletal muscles. While the upregulation of UCP2 mRNA in response to cold exposure is in line with a putative uncoupling role for this protein in thermoregulatory thermogenesis, the unexpected upregulation of UCP2 in skeletal muscles in response to fasting seems inconsistent with its role as an uncoupling protein involved in dietary regulation of thermogenesis.

Uncoupling proteins: their roles in adaptive thermogenesis and substrate metabolism reconsidered

During the past few years, there have been two major developments, if not revolutions, in the field of energy balance and weight regulation. The first at the molecular level, which was catalysed by developments in DNA screening technology together with the mapping of the human genome, has been the tremendous advances made in the identification of molecules that play a role in the control of food intake and metabolic rate. The second, at the systemic level, which centered upon the use of modern technologies or more robust analytical techniques for assessing human energy expenditure in response to starvation and overfeeding, has been the publication of several papers providing strong evidence that adaptive thermogenesis plays a much more important role in the regulation of body weight and body composition than previously thought. Within these same few years, several new members of the mitochondrial carrier protein family have been identified in a variety of tissues and organs. All apparently possess uncoupling properties in genetically-modified systems, with two of them (uncoupling protein (UCP) 2 and UCP3) being expressed in adipose tissues and skeletal muscles, which are generally recognised as important sites for variations in thermogenesis and/or in substrate oxidation. Considered as breakthrough discoveries, the cloning of these genes has generated considerable optimism for rapid advances in our molecular understanding of adaptive thermogenesis, and for the identification of new targets for pharmacological management of obesity and cachexia. The present paper traces first, from a historical perspective, the landmark events in the field of thermogenesis that led to the identification of these genes encoding candidate UCP, and then addresses the controversies and on-going debate about their physiological importance in adaptive thermogenesis, in lipid oxidation or in oxidative stress. The general conclusion is that UCP2 and UCP3 may have distinct primary functions, with UCP3 implicated in regulating the flux of lipid substrates across the mitochondria and UCP2 in the control of mitochondrial generation of reactive oxygen species. The distinct functions of these two UCP1 homologues have been incorporated in a conceptual model to illustrate how UCP2 and UCP3 may act in concert in the overall regulation of lipid oxidation concomitant to the prevention of lipid-induced oxidative damage.

Effect of endurance training on mRNA expression of uncoupling proteins 1, 2, and 3 in the rat.

Endurance exercise training has been shown to decrease diet‐induced thermogenesis (DIT) in rats and humans. In rodents, most thermogenesis is thought to occur in brown adipose tissue via activation of the uncoupling protein‐1 (UCP1) and in skeletal muscle. Since the level of UCP1 mRNA in rat BAT was reported to be unmodified by exercise training, the newly described uncoupling proteins UCP2 and UCP3 could be responsible for the decreased DIT in trained rats. UCP3 mRNA levels in endurancetrained rats were found to be reduced by 76% and 59% in tibialis anterior and soleus muscles, respectively. UCP2 mRNA levels were also decreased in tibialis anterior and in heart by 54% and 41%, respectively. Neither white adipose tissue UCP2 nor brown adipose tissue UCP1, UCP2, and UCP3 mRNA levels were modified. The results of this study show that a need for a higher metabolic efficiency is associated with decreased mRNA expression of the uncoupling proteins in skeletal and heart muscles, which would decrease energy dissipation in these tissues. The down‐regulation of UCP3 and UCP2 expressions might also contribute to the rapid weight gain known to occur when exercise training ceased.—Boss, O., Samec, S., Desplanches, D., Mayet, M.‐H., Seydoux, J., Muzzin, P., Giacobino, J.‐P. Effect of endurance training on mRNA expression of uncoupling proteins 1, 2, and 3 in the rat. FASEB J. 12, 339–335 (1998)

Targeted gene disruption reveals a leptin-independent role for the mouse beta3-adrenoceptor in the regulation of body composition.

Targeted disruption of mouse beta3-adrenoceptor was generated by homologous recombination, and validated by an acute in vivo study showing a complete lack of effect of the beta3-adrenoceptor agonist CL 316,243 on the metabolic rate of homozygous null (-/-) mice. In brown adipose tissue, beta3-adrenoceptor disruption induced a 66% decrease (P < 0.005) in beta1-adrenoceptor mRNA level, whereas leptin mRNA remained unchanged. Chronic energy balance studies in chow-fed mice showed that in -/- mice, body fat accumulation was favored (+41%, P < 0.01), with a slight increase in food intake (+6%, NS). These effects were accentuated by high fat feeding: -/- mice showed increased total body fat (+56%, P < 0.025) and food intake (+12%, P < 0.01), and a decrease in the fat-free dry mass (-10%, P < 0.05), which reflects a reduction in body protein content. Circulating leptin levels were not different in -/- and control mice regardless of diet. The significant shift to the right in the positive correlation between circulating leptin and percentage of body fat in high fat-fed -/- mice suggests that the threshold of body fat content inducing leptin secretion is higher in -/- than in control mice. Taken together, these studies demonstrate that beta3-adrenoceptor disruption creates conditions which predispose to the development of obesity.

Skeletal muscle heterogeneity in fasting-induced upregulation of genes encoding UCP2, UCP3, PPARγ and key enzymes of lipid oxidation

Abstract. The uncoupling protein homologs UCP2 and UCP3 have been proposed as candidate genes for the regulation of lipid metabolism. Within the context of this hypothesis, we have compared, from fed and fasted rats, changes in gene expression of skeletal muscle UCP2 and UCP3 with those of carnitine palmitoyltransferase I and medium-chain acyl-CoA dehydrogenase, two key enzymes regulating lipid flux across the mitochondrial β-oxidation pathway. In addition, changes in gene expression of peroxisome proliferator-activated receptor gamma, a nuclear transcription factor implicated in lipid metabolism, were also investigated. The results indicate that in response to fasting, the mRNA levels of UCP2, UCP3, carnitine palmitoyltransferase I and medium-chain acyl-CoA dehydrogenase are markedly increased, by three- to sevenfold, in the gastrocnemius and tibialis anterior (fast-twitch muscles, predominantly glycolytic or oxidative-glycolytic), but only mildly increased, by less than twofold, in the soleus (slow-twitch muscle, predominantly oxidative). Furthermore, such muscle-type dependency in fasting-induced transcriptional changes in UCP2, UCP3, carnitine palmitoyltransferase and medium-chain acyl-CoA dehydrogenase persists when the increase in circulating levels of free fatty acids during fasting is abolished by the anti-lipolytic agent nicotinic acid – with blunted responses only in the slow-twitch muscle contrasting with unabated increases in fast-twitch muscles. Independently of muscle type, however, the mRNA levels of peroxisome proliferator-activated receptor gamma are not altered during fasting. Taken together, these studies indicate a close association between fasting-induced changes in UCP2 and UCP3 gene expression with those of key regulators of lipid oxidation, and are hence consistent with the hypothesis that these UCP homologs may be involved in the regulation of lipid metabolism. Furthermore, they suggest that in response to fasting, neither the surge of free fatty acids in the circulation nor induction of the peroxisome proliferator-activated receptor gamma gene may be required for the marked upregulation of genes encoding the UCP homologs and key enzymes regulating lipid oxidation in fast-twitch muscles.

Differences in proton leak kinetics, but not in UCP3 protein content, in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from fed and fasted rats

We have investigated the effect of 24‐h fasting on basal proton leak and uncoupling protein (UCP) 3 expression at the protein level in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. In fed rats, the two mitochondrial populations displayed different proton leak, but the same protein content of UCP3. In addition, 24‐h fasting, both at 24 and 29°C, induced an increase in proton leak only in subsarcolemmal mitochondria, while UCP3 content increased in both the populations. From the present data, it appears that UCP3 does not control the basal proton leak of skeletal muscle mitochondria.