Patrick Muzzin

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.

Ghrelin action in the brain controls adipocyte metabolism

Many homeostatic processes, including appetite and food intake, are controlled by neuroendocrine circuits involving the CNS. The CNS also directly regulates adipocyte metabolism, as we have shown here by examining central action of the orexigenic hormone ghrelin. Chronic central ghrelin infusion resulted in increases in the glucose utilization rate of white and brown adipose tissue without affecting skeletal muscle. In white adipocytes, mRNA expression of various fat storage-promoting enzymes such as lipoprotein lipase, acetyl-CoA carboxylase alpha, fatty acid synthase, and stearoyl-CoA desaturase-1 was markedly increased, while that of the rate-limiting step in fat oxidation, carnitine palmitoyl transferase-1alpha, was decreased. In brown adipocytes, central ghrelin infusion resulted in lowered expression of the thermogenesis-related mitochondrial uncoupling proteins 1 and 3. These ghrelin effects were dose dependent, occurred independently from ghrelin-induced hyperphagia, and seemed to be mediated by the sympathetic nervous system. Additionally, the expression of some fat storage enzymes was decreased in ghrelin-deficient mice, which led us to conclude that central ghrelin is of physiological relevance in the control of cell metabolism in adipose tissue. These results unravel the existence of what we believe to be a new CNS-based neuroendocrine circuit regulating metabolic homeostasis of adipose tissue.

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.

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.

Modulation of obese gene expression in rat brown and white adipose tissues

The ob gene mRNA expression in rat brown adipose tissue (BAT) and epididymal white adipose tissue (WAT) was measured on Northern blots hybridized with a rat ob gene probe. The level of ob gene mRNA in BAT was about 40% of that in WAT. Fasting (36 h) or semi‐starvation (10 days) decreased the ob gene mRNA level in both tissues by 62–68%, and cold exposure at 6°C (24 h) decreased it in BAT (−84%)_but not in WAT. Acute administration of the β 3‐adrenergic agonist Ro 16‐8714 decreased the ob gene mRNA level in BAT (−51%) and WAT (−28%) of lean Zucker rats and only in BAT (−74%) of obese falfa rats. This study demonstrates that, in the rat, the ob gene is not only expressed in WAT but also in BAT, and suggests that in these two tissues, the modulation of the ob gene expression might be more closely associated with known alterations in cell lipid content than with changes in sympathetic activity.

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)

Expression of uncoupling protein‐3 and mitochondrial activity in the transition from hypothyroid to hyperthyroid state in rat skeletal muscle

We sought a correlation between rat skeletal muscle triiodothyronine (T3)‐mediated regulation of uncoupling protein‐3 (UCP3) expression and mitochondrial activity. UCP3 mRNA expression increased strongly during the hypothyroid‐hyperthyroid transition. The rank order of mitochondrial State 3 and State 4 respiration rates was hypothyroid

β1/β2/β3-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting

Catecholamines are viewed as major stimulants of diet‐ and cold‐induced thermogenesis and of fasting‐induced lipolysis, through the β‐adrenoceptors (β1/β2/β3). To test this hypothesis, we generated β1/β2/β3‐adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold‐intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein‐1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β‐adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.

Role of glucocorticoids in the physiopathology of excessive fat deposition and insulin resistance

Glucocorticoids are important hormones in the regulation of metabolic homeostasis. We infused normal rats with dexamethasone given intracerebroventricularly (i.c.v.) for 3 days. This resulted in hyperphagia, hyperinsulinemia, and marked insulin resistance. Similar metabolic defects were observed following i.c.v. infusion of neuropeptide Y (NPY) in normal rats. As central dexamethasone infusion enhanced NPY content in the arcuate nucleus, it suggested that its metabolic effects are mediated by NPY. Moreover, due to the lack of effects observed in vagotomized animals, activation of the parasympathetic nervous system by central dexamethasone infusion is proposed. Glucocorticoid action is known to involve prereceptor metabolism by enzymes such as 11β-HSD-1 that converts inactive into active glucocorticoids. Mice overexpressing 11β-HSD-1 in adipose tissue were shown to be obese and insulin resistant. We recently observed that adipose tissue 11β-HSD-1 mRNA expression is increased at the onset of high-fat diet-induced obesity and positively correlated with the degree of hyperglycemia. In human obesity, increased adipose tissue 11β-HSD-1 expression and activity were also reported. Resistin is a new adipose tissue-secreted hormone shown to play a role in glucose homeostasis by increasing hepatic glucose production and inhibiting muscle and adipose tissue glucose utilization. We observed increased adipose tissue resistin expression in the early phase of high-fat diet-induced obesity as well as decreased resistin expression in response to leptin. A positive correlation between glycemia and adipose tissue resistin expression further suggested a role of this hormone in the development of insulin resistance. The melanocortin system is another important player in the regulation of energy balance. Peripheral administration of a melanocortin agonist decreased food intake and body weight and favored lipid oxidation, effects that were more marked in obese than in lean rats. It is proposed that both resistin and melanocortin agonists may influence adipose tissue 11β-HSD-1, thereby decreasing or enhancing glucose metabolism.

Uncoupling protein-3 expression in skeletal muscle and free fatty acids in obesity

The newly identified mitochondrial uncoupling proteins UCP2 and UCP3, by contrast with UCP1, are highly expressed in human beings, UCP2 being ubiquitous and UCP3 specific to skeletal muscle. Because of its tissue distribution, UCP3 might have an important role in wholebody energy homoeostasis in human beings. Variations in its degree of expression might contribute to the interindividual variability in resting energy expenditure and in the energybalance dysregulation found in obesity and type 2 diabetes. We investigated whether mRNA expression of UCP2 and UCP3 in human vastus lateralis muscle was correlated with variables related to fat or glucose metabolism and with resting energy expenditure in a mixed population of obese patients and obese patients with type 2 diabetes. Nine obese, 10 h fasted patients, including three with type 2 diabetes (untreated but dietetically controlled, fasting plasma glucose of 8·1 to 10·5 mmol/L, table), volunteered, with written consent, to participate, which was accepted by the local Human Investigation Committee. We measured lean body mass with bioelectrical impedance. Plasma glucose concentrations were determined with a Beckman Glucose Analyzer II (Beckman Instruments, CA, USA), and plasma free fatty acids and insulin concentrations with commercial kits (Wako Chemicals GmbH, Germany, and Abbott, IL). Glucose uptake was measured with a euglycaemic hyperinsulinaemic clamp, and glucose and lipid oxidation rates, as well as resting energy expenditure, were determined by indirect calorimetry. The concentrations of UCP2 and UCP3 mRNA in vastus lateralis biopsy fragments were measured by northern blot. The expression of UCP2 and UCP3 mRNA normalised to the respective concentrations of 18S rRNA were compared with 12 physical, biological, and metabolic variables and analysed (Simple Pearson ProductMoment correlations). The mRNA expression of UCP3 was positively and linearly correlated with circulating free fatty acids (r=0·83; p=0·005), whereas that of UCP2 was not (r=0·40). When adjustments were made for age, percentage of fat mass, body-mass index, lean body mass and bodyweight, the correlation between UCP3 mRNA and concentrations of free fatty acids was stronger (r=0·99; p 0·05) was seen between UCP2 or UCP3 mRNA expression and age, percentage fat mass, body-mass index, lean body mass, resting energy expenditure per kg lean body mass, bodyweight, fasting plasma glucose, insulin concentrations, insulin-induced glucose uptake, glucose oxidation, and lipid oxidation. Free fatty acids might control muscle UCP3 expression. A 10-day severe calorie restriction increases muscle UCP2 and UCP3 mRNA expression, and in rodents infusion of free fatty acids increases muscle UCP3 mRNA. Our data suggest a role for muscle UCP3 in the metabolic adaptations to increases in fatty-acid supply, and, therefore, the involvement of UCP3 in a compensatory mechanism linking obesity to increased muscle thermogenesis.