UCP2 mRNA Levels Research Articles

Synergistic Activation of UCP-3 Expression in Cultured Fetal Rat Brown Adipocytes by PPARα and PPARγ Ligands

Rat brown adipocytes express mRNAs for Uncoupling Proteins (UCP) 1, 2 and 3 and the Peroxisome Proliferator Activated Receptors (PPAR) α and γ. We have examined the effects of selective PPARα or -γ activation on changes in UCP-1 and UCP-3 mRNA levels in cultured fetal rat brown adipocytes (FBA). Rosiglitazone (1.0 μM), a selective PPARγ agonist, elicited 5- and 3-fold increases in UCP-1 and UCP-3, respectively. The PPARα ligand, Wy14643 (10.0 μM) increased UCP-3 tenfold, but decreased UCP-1. A synergistic effect on UCP-3 expression (30-fold increase; P < 0.05) was observed when FBA were exposed to a combination of Wy14643 (10.0 μM) and rosiglitazone (10.0 μM). Thus, activation of PPARγ increases UCP-1 and UCP-3 levels which are differentially regulated by PPARα. A synergistic interaction occurs between PPARα and PPARγ in the regulation of UCP-3 in FBA, probably via co-activator recruitment, suppression of co-repressor proteins or through a direct interaction at the level of the PPRE.

Assessment of mitochondrial energy coupling in vivo by 13C/31P NMR.

The recently cloned uncoupling protein homolog UCP3 is expressed primarily in muscle and therefore may play a significant role in the regulation of energy expenditure and body weight. However, investigation into the regulation of uncoupling protein has been hampered by the inability to assess its activity in vivo. In this report, we demonstrate the use of a noninvasive NMR technique to assess mitochondrial energy uncoupling in skeletal muscle of awake rats by combining (13)C NMR to measure rates of mitochondrial substrate oxidation with (31)P NMR to assess unidirectional ATP synthesis flux. These combined (31)P/(13)C NMR measurements were performed in control, 10-day triiodo-l-thyronine (T(3))-treated (model of increased UCP3 expression), and acute 2,4-dinitrophenol (DNP)-treated (protonophore and mitochondrial uncoupler) rats. UCP3 mRNA and protein levels increased 8.1-fold (+/- 1.1) and 2.8-fold (+/- 0.8), respectively, in the T(3)-treated vs. control rat gastrocnemius muscle. (13)C NMR measurements of tricarboxylic acid cycle flux as an index of mitochondrial substrate oxidation were 61 +/- 21, 148 +/- 25, and 310 +/- 48 nmol/g per min in the control, T(3), and DNP groups, respectively. (31)P NMR saturation transfer measurements of unidirectional ATP synthesis flux were 83 +/- 14, 84 +/- 14, and 73 +/- 7 nmol/g per s in the control, T(3), and DNP groups, respectively. Together, these flux measurements, when normalized to the control group, suggest that acute administration of DNP (mitochondrial uncoupler) and chronic administration of T(3) decrease energy coupling by approximately 80% and approximately 60%, respectively, and that the latter treatment correlates with an increase in UCP3 mRNA and protein expression. This NMR approach could prove useful for exploring the regulation of uncoupling protein activity in vivo and elucidating its role in energy metabolism and obesity.

Uncoupling protein-2 and -3 messenger ribonucleic acids in adipose tissue and skeletal muscle of healthy males: variability, factors affecting expression, and relation to measures of metabolic rate.

Mitochondrial uncoupling protein-2 and -3 (UCP2 and UCP3) may be involved in the modulation of resting metabolic rate and energy balance. To investigate their variability, the influence of this on the variability of energy expenditure, and potential regulatory factors of the expression of the corresponding genes, we measured their messenger ribonucleic acids (mRNAs) in muscle and white adipose tissue of lean, healthy men and correlated the abundance of these mRNAs (attomoles per microg total RNA) with measures of resting metabolic rate, hormone levels (thyroid hormones, insulin, glucagon, leptin, and catecholamines), and fuels potentially involved in energy balance regulation. We also investigated whether the thiazolidinedione, troglitazone, stimulates UCP2 and UCP3 mRNA levels to follow up on the observation that this antidiabetic drug increases the levels of expression in cultured cells. We found UCP2 and UCP3 mRNA levels to be highly variable and poorly correlated with measures of energy expenditure and with most factors affecting energy balance. Only nocturnal urinary norepinephrine excretion could explain a significant fraction of the variability in both UCP2 and UCP3 expression in muscle, but not adipose tissue. Thyroid hormone and norepinephrine excretion were found to contribute to the variability of resting metabolic rate, but this could not be explained by an effect on UCP mRNAs. Troglitazone affected neither the expression of UCPs nor the hormones or the measures of metabolic rate investigated. In conclusion, our results show that the expression of UCP2 and UCP3 genes is quite variable in healthy males and that this variability does not explain that in resting energy expenditure, and suggest that sympathetic activity is an important potential regulator of the expression of these proteins in skeletal muscle. However, the data do not support the concept that regulation of the expression of these genes is the most important level of control of UCP3 and UCP2 functions, and other levels of control have to be invoked.

Lack of Obesity and Normal Response to Fasting and Thyroid Hormone in Mice Lacking Uncoupling Protein-3

Uncoupling protein-3 (UCP3) is a mitochondrial protein that can diminish the mitochondrial membrane potential. Levels of muscle Ucp3 mRNA are increased by thyroid hormone and fasting. Ucp3 has been proposed to influence metabolic efficiency and is a candidate obesity gene. We have produced a Ucp3 knockout mouse to test these hypotheses. The Ucp3 (-/-) mice had no detectable immunoreactive UCP3 by Western blotting. In mitochondria from the knockout mice, proton leak was greatly reduced in muscle, minimally reduced in brown fat, and not reduced at all in liver. These data suggest that UCP3 accounts for much of the proton leak in skeletal muscle. Despite the lack of UCP3, no consistent phenotypic abnormality was observed. The knockout mice were not obese and had normal serum insulin, triglyceride, and leptin levels, with a tendency toward reduced free fatty acids and glucose. Knockout mice showed a normal circadian rhythm in body temperature and motor activity and had normal body temperature responses to fasting, stress, thyroid hormone, and cold exposure. The base-line metabolic rate and respiratory exchange ratio were the same in knockout and control mice, as were the effects of fasting, a beta3-adrenergic agonist (CL316243), and thyroid hormone on these parameters. The phenotype of Ucp1/Ucp3 double knockout mice was indistinguishable from Ucp1 single knockout mice. These data suggest that Ucp3 is not a major determinant of metabolic rate but, rather, has other functions.

Effects of fatty acids on uncoupling protein-2 expression in the rat heart.

Fatty acids are thought to play a role in the activity of uncoupling proteins (UCP) and have been shown to regulate the expression of genes encoding proteins involved in fatty acid handling. Therefore, we investigated whether fatty acids, which are the main substrates for the heart, affect rat cardiac UCP-2 expression in vivo and in vitro. After birth, when the contribution of fatty acid oxidation to cardiac energy conversion increases, UCP-2 expression enhanced rapidly. In the adult heart, however, UCP-2 mRNA levels did not alter during conditions associated with either enhanced (fasting, diabetes) or decreased (hypertrophy) fatty acid utilization. Exposure of neonatal cardiomyocytes and embryonic rat heart-derived H9c2 cells to fatty acids (palmitic and oleic acid) for 48 h strongly induced UCP-2 expression. Stimulation of neonatal cardiomyocytes with triiodothyronine also increased UCP-2 mRNA levels, though only in the presence of fatty acids. Ligands specific to the fatty acid-activated transcription factor PPARalpha, but not to PPARgamma, acted as inducers of cardiomyocyte UCP-2 expression. It is concluded that fatty acids promote UCP-2 expression in neonatal cardiomyocytes, which might explain the rapid increase in UCP-2 mRNA in the postnatal heart. However, UCP-2 mRNA levels in the adult heart appear to be insensitive to changes in cardiac fatty acid handling under various pathological conditions.

Modulation of uncoupling protein 2 and uncoupling protein 3: regulation by denervation, leptin and retinoic acid treatment.

We recently reported that the leptin-induced increase in uncoupling protein 1 (UCP1) mRNA in brown adipose tissue (BAT) is prevented by the denervation of BAT. We also reported that retinoic acid (RA) increases UCP1 mRNA in BAT. To extend these finding to UCP2 and UCP3 in BAT, we examined UCP2 and UCP3 mRNA after unilateral denervation of BAT, as well as after leptin, beta(3)-adrenergic agonist, RA, and glucocorticoid administration to rats. UCP3 mRNA was 20% less in the denervated compared with the intact BAT, whereas UCP2 mRNA was unchanged with denervation. The beta(3)-adrenergic agonist, CGP-12177 (0.75 mg/kg), increased UPC3 mRNA by 40% in the innervated and by 85% in the denervated BAT. Leptin (0.9 mg/day for 3 days) increased both UCP2 and UCP3 mRNA by 30% in the innervated and, surprisingly, in the denervated BAT. RA (7.5 mg/kg) increased UCP1 mRNA but decreased UCP2 and UCP3 mRNA by 50%, whereas methylprednisolone (65 mg/kg, two doses 24 h apart) suppressed all three uncoupling proteins by greater than 60%. The present findings indicate that: sympathetic innervation is necessary to maintain basal levels of UCP3 mRNA; beta(3)-adrenergic agonist stimulation induces UCP3 mRNA; leptin induces UCP2 and UCP3 mRNA and this induction is not dependent on sympathetic innervation; RA increases UCP1 but decreases UCP2 and UCP3 mRNA; and methylprednisolone suppresses UCP1, UCP2, and UCP3 mRNA equally. These data suggest that there are distinct patterns of regulation between UCP1, UCP2, and UCP3, and there may be at least two modes by which leptin could modulate thermogenesis in BAT; first, by increasing sympathetic stimulation of BAT and induction of UCP1 mRNA and, secondly, by increasing UCP2 and UCP3 mRNA by a mechanism independent of sympathetic stimulation.

Up-regulation of muscle UCP2 gene expression by a new beta3-adrenoceptor agonist, trecadrine, in obese (cafeteria) rodents, but down-regulation in lean animals.

The anti-obesity properties of a new beta3-adrenergic agonist (Trecadrine) were examined in a diet-induced obesity model, including the effects on OB and uncoupling protein (UCP-1 and -2) gene expression. Control rats and cafeteria-fed rats were treated with placebo or Trecadrine for 35 days. Leptin and UCP (1 and 2) mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) methodology in adipose tissue and gastrocnemius muscle. Animals fed a cafeteria diet increased body weight, fat content, white adipose tissue (WAT), brown adipose tissue (BAT) weights and oxygen consumption in relation to lean controls. A rise in plasma leptin, WAT OB gene expression as well as circulating free fatty acids levels was found in obese rats as compared with lean controls. Trecadrine administration to cafeteria-fed animals decreased fat content, WAT weight, circulating leptin and fatty acids concentrations, and WAT OB gene expression, reaching comparable values to lean controls, while WAT O2 consumption was increased in these animals. Also, an increase in BAT UCP1 mRNA levels was found through a two-way analysis of variance in control and obese animals after Trecadrine administration. Gastrocnemius muscle UCP2 gene expression was reduced in lean Trecadrine-treated and diet-induced obese animals as compared to controls, while an increase was found in cafeteria-fed animals after Trecadrine administration. A negative correlation between WAT O2 consumption and UCP2 expression was found in control animals, but not in the cafeteria-fed groups, suggesting a differential response to the beta3-adrenergic compound in lean and obese animals, which is in agreement with the reported statistical interactions between obesity and Trecadrine administration found for WAT O2 consumption and muscle UCP2 expression, as well as for plasma leptin and WAT leptin expression. The new beta3-adrenergic agonist, Trecadrine, decreases fat content and increases gastrocnemius muscle UCP2 gene expression in a diet-induced obesity model. This sheds additional light on the action mechanism of compounds with affinity for beta3-adrenoceptors and other potential anti-obesity agents.

A marked upregulation of uncoupling protein 2 gene expression in adipose tissue of hyperthyroid subjects.

Recently, a family of uncoupling protein (UCP) genes has been discovered. The role of these genes is unknown, but it has been suggested that they are involved in regulating resting metabolic rate. In this study, we hypothesised that thyroid hormone status may influence the expression of UCP2 mRNA. The adipose tissue levels of UCP2 mRNA were measured in eight female subjects before and after treatment for thyrotoxicosis. All subjects in the hyperthyroid condition had markedly enhanced plasma levels of thyroxine (62.0 +/- 6.9 vs. 17.9 +/- 1.7, p = 0.012) and triiodothyronine (37.9 +/- 6.9 vs. 5.9 +/- 0.9, p = 0.012), accelerated heart rate (94 +/- 7 vs. 69 +/- 5, p = 0.012), decreased BMI (24.5 +/- 1.9 vs. 25.1 +/- 1.9, p = 0.025) and decreased percentage body fat (32.8 +/- 4.4 vs. 37.1 +/- 4.5, p = 0.018), as compared to the euthyroid state. Using RT-competitive-PCR, the UCP2 mRNA levels were found to be 2.5-fold upregulated in hyperthyroidism (10.4 +/- 1.7 vs. 4.2 +/- 1.3 amol/microg RNA, p = 0.012). In contrast, no difference in expression levels of the reference gene 18SrRNA was seen in the hyperthyroid versus the euthyroid state (317 +/- 49 vs. 279 +/- 25 amol/microg RNA, p = 0.48) but the difference in UCP2 mRNA levels between the hyper- and euthyroid state remained when UCP2 was related to 18SrRNA (p = 0.012). In conclusion, thyrotoxicosis markedly increases the expression of UCP2 mRNA in adipose tissue, which suggests a role for thyroid hormones in the regulation of this uncoupling protein in man.

Correlation between pancreatic islet uncoupling protein-2 (UCP2) mRNA concentration and insulin status in rats.

Hypothesizing that UCP2 may influence insulin secretion by modifying the ATP/ADP ratio within pancreatic islets, we have investigated the expression of intraislet UCP2 gene in rats showing insulin oversecretion (non-diabetic Zucker fa/fa obese rats, glucose-infused Wistar rats) or insulin undersecretion (fasting and mildly diabetic rats). We found that in Zucker fa/fa obese rats, hyperinsulinemia (1222 ± 98 pmol/1 vs. 128 ± 22 pmol/1 in lean Zucker rats) was accompanied by a significant increase in UCP2 mRNA levels. In rat submitted to a 5 day infusion with glucose, hyperinsulinemia (1126 ± 101 pmol/l vs. 215 ± 25 pmol/1 in Wistar control rats), coincided with an enhanced intraislet UCP2 gene expression, whereas a 8h or a 2 day-infusion did not induce significant changes in UCP2 mRNA expression. In rats made hypoinsulinemic and mildly diabetic by the injection of a low dose of streptozotocin, and in 4-day-fasting rats (plasma insulin 28 ± 5 pmol/1) UCP2 gene expression was sharply decreased. A 3-day-fast was ineffective. The data show the existence of a time-dependent correlation between islet mRNA UCP2 and insulin that may be interpreted as an adaptative response to prolonged insulin excess.

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.

Lack of skeletal muscle uncoupling protein 2 and 3 mRNA induction during fasting in type-2 diabetic subjects.

Skeletal muscle uncoupling protein 2 and 3 (UCP-2 and UCP-3) mRNA levels are increased during calorie restriction in lean and nondiabetic obese subjects. In this work, we have investigated the effect of a 5-day hypocaloric diet (1,045 kJ/day) on UCP-2 and UCP-3 gene expression in the skeletal muscle of type-2 diabetic obese patients. Before the diet, UCP-2 and UCP-3 mRNA levels were more abundant in diabetic than in nondiabetic subjects. The long (UCP-3(L)) and short (UCP-3(S)) forms of UCP-3 transcripts were expressed at similar levels in nondiabetic subjects, but UCP-3(S) transcripts were twofold more abundant than UCP-3(L) transcripts in the muscle of diabetic patients. Calorie restriction induced a two- to threefold increase in UCP-2 and UCP-3 mRNA levels in nondiabetic patients. No change was observed in type-2 diabetic patients. Variations in plasma nonesterified fatty acid level were positively correlated with changes in skeletal muscle UCP-3(L) (r = 0.6, P < 0.05) and adipose tissue hormone-sensitive lipase (r = 0.9, P < 0.001) mRNA levels. Lack of increase in plasma nonesterified fatty acid level and in hormone-sensitive lipase upregulation in diabetic patients during the diet strengthens the hypothesis that fatty acids are associated with the upregulation of uncoupling proteins during calorie restriction.

Regulation of Gene Expression by Activation of the Peroxisome Proliferator-Activated Receptor γ with Rosiglitazone (BRL 49653) in Human Adipocytes

To better define the mechanism of action of the thiazolidinediones, we incubated freshly isolated human adipocytes with rosiglitazone and investigated the changes in mRNA expression of genes encoding key proteins of adipose tissue functions. Rosiglitazone (10−6 M, 4 h) increased p85αphosphatidylinositol 3-kinase (p85αPI-3K) and uncoupling protein-2 mRNA levels and decreased leptin expression. The mRNA levels of insulin receptor, IRS-1, Glut 4, lipoprotein lipase, hormone-sensitive lipase, acylation-stimulating protein, fatty acid transport protein-1, angiotensinogen, plasminogen activator inhibitor-1, and PPARγ1 and γ2 were not modified by rosiglitazone treatment. Activation of RXR, the partner of PPARγ, in the presence of rosiglitazone, increased further p85αPI-3K and UCP2 mRNA levels and produced a significant augmentation of Glut 4 expression. Because p85αPI-3K is a major component of insulin action, the induction of its expression might explain, at least in part, the insulin-sensitizing effect of the thiazolidinediones.

The Bioenergetics of Brown Fat Mitochondria from UCP1-ablated Mice: UCP1 IS NOT INVOLVED IN FATTY ACID-INDUCED DE-ENERGIZATION (“UNCOUPLING”)

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.

Skeletal muscle UCP2 and UCP3 expression in trained and untrained male subjects.

The new uncoupling proteins, UCP2 and UCP3, are thought to play a role in energy efficiency in humans. Endurance training has been suggested to have effects on resting metabolic rate and energy efficiency. We therefore determined UCP2 and UCP3 mRNA levels in skeletal muscle of trained and untrained male subjects. Using reverse transcription-polymerase chain reaction (RT-PCR), expression of UCP2, UCP3L and UCP3S mRNA were measured in muscle biopsies from the quadriceps femoris in eight trained (23.9+/-1.6 y; 70.6+/-3.1 kg; 14+/-3% body fat; maximal power output (Wmax): 5. 6+/-0.4 W/kg; mean+/-s.d.) and 10 lean, untrained (22.1+/-2.9 y; 72. 0+/-7.9 kg; 18+/-4% body fat; Wmax: 3.9+/-0.4 W/kg; mean+/-s.d.) subjects. In six of the trained subjects, UCP2 and UCP3 mRNA were measured before and after an exercise bout to exhaustion. To correct for differences in mitochondrial content, levels of UCP2 and UCP3 mRNA were expressed relative to cytochrome-b, a marker of mitochondrial content. Acute exercise had no effect on the expression of UCP3L or UCP3S, but in five out of six subjects UCP2 expression decreased after exercise, although the difference was not statistically significant (P=0.11). Trained subjects had significantly reduced mRNA levels of UCP3L (P=0.028) and UCP3S (P=0. 031). VO2max expressed per kg of fat-free mass was negatively correlated with UCP3L (r=-0.61, P=0.009) and UCP3S (r=-0.52, P=0. 028). Mechanical efficiency correlated negatively with UCP3L (r=-0. 56, P=0.019), UCP3S (r=-0.47, P=0.048) and tended to correlate with UCP2 (r=-0.46, P=0.06). The lower levels of UCP3 mRNA in trained subjects and the inverse relationship of UCP3 expression and mechanical efficiency suggest that exercise training produces an adaptive physiological response in skeletal muscle improving mechanical efficiency.

Etomoxir, Sodium 2-[6-(4-Chlorophenoxy)hexyl]oxirane-2-carboxylate, Up-Regulates Uncoupling Protein-3 mRNA Levels in Primary Culture of Rat Preadipocytes

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of primary culture of rat preadipocytes for 24 h with 40 μM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated UCP-3 mRNA levels (3.6-fold induction), whereas changes in UCP-2 mRNA levels were not significant. As a consequence of increased UCP-3 expression, a fall in the mitochondrial membrane potential was detected by flow cytometry. Etomoxir treatment modified neither L-CPT-I (liver-type) nor PPARα mRNA levels in preadipocytes. In contrast, mRNA expression of acyl-CoA oxidase (ACO), the rate-limiting enzyme of peroxisomal fatty acid β-oxidation, whose transcription is controlled by PPARα, was significantly induced (1.3-fold induction, P = 0.015). These findings suggest that the effects of etomoxir were mediated by PPARα. Since it has been reported that the intracellular accumulation of lipids following the inhibition of CPT-I by etomoxir leads to a PPARα-mediated metabolic response that increases the expression of genes involved in alternate fatty acid oxidation pathways, these results seem to implicate UCP-3 in this protective metabolic response. It remains to be studied whether reductions in the expression of UCP-3 could compromise this response, giving rise to lipotoxic effects on cells.

Skeletal muscle UCP3 and UCP2 gene expression in response to inhibition of free fatty acid flux through mitochondrial beta-oxidation.

Studies of starvation and refeeding have implicated the genes coding for uncoupling protein-3 and -2 (UCP3, UCP2) as candidate genes in the regulation of lipids as metabolic fuels in skeletal muscle. To gain insight into the role of free fatty acid (FFA) flux in regulating the expression of these muscle UCP homologues, we recently reported that, in response to the anti-lipolytic agent nicotinic acid, utilized to reduce FFA flux at the input supply (i.e. circulating) level in fed and fasted rats, expression of the UCP3 and UCP2 genes was reduced in the soleus (predominantly slow-oxidative fibres), but not in the gastrocnemius (predominantly fast-glycolytic fibres) or tibials anterior (predominantly fast-oxidative-glycolytic fibres) muscles. In the present study, we examined UCP2 and UCP3 gene expression in these muscles from fed or fasted rats treated with etomoxir, an inhibitor of FFA flux at the output (i.e. mitochondrial oxidation) level. Fasting per se resulted in a threefold increase in serum FFA (P < 0.001) and in marked increases in the messenger ribonucleic acid (mRNA) expression of both UCP2 and UCP3 in all three muscles (P < 0.001). Treatment with etomoxir had no significant effect on serum FFA in the fed rats, but further elevated serum FFA in the fasted rats (P < 0.001). The mRNA levels of both UCP3 and UCP2 in response to etomoxir were significantly reduced in the tibialis anterior muscle in both fed and fasted states (P < 0.01), unaltered in the gastrocnemius muscle in both fed and fasted states and unaltered in the soleus muscle in the fed state, but increased in the fasted state, in parallel with the etomoxir-induced changes in serum FFA levels. Taken together, these results suggest the existence of positive feedback loops between FFA flux and muscle UCPs only in oxidative muscles--with that loop operating at the input FFA supply level for muscles with predominantly slow-oxidative fibres, and at the output FFA oxidation level for muscles with predominantly fast-oxidative-glycolytic fibres.

Uncoupling Protein-3 mRNA Levels Are Increased in White Adipose Tissue and Skeletal Muscle of Bezafibrate-Treated Rats

Fibrates are hypolipidemic drugs that are also able to improve glucose tolerance in animals and diabetic patients through an unknown mechanism. Since uncoupling proteins (UCP) seem to play an important role in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM), we examined whether treatment of rats with bezafibrate for 3, 7, or 15 days modified UCP mRNA levels. Using RT-PCR, we observed a weak ectopic expression of UCP-1 and a 2-fold increase in UCP-3 mRNA levels in white adipose tissue after 7 and 15 days of treatment. Moreover, bezafibrate administration caused a 1.7-fold induction in UCP-3 mRNA levels in skeletal muscle on day 7. Since UCP-3 mRNA levels are reduced in skeletal muscle of diabetic patients, this effect may be involved in the improvement of insulin sensitivity caused by bezafibrate in NIDDM.

New members of uncoupling protein family implicated in energy metabolism.

1. The regulation of energy metabolism involves food intake and energy expenditure. Mitochondrial uncoupling proteins (UCP) are implicated in energy expenditure. 2. cDNA of a homologue of UCP highly expressed in rat skeletal muscle, UCP-3, is isolated and sequenced. Rat UCP-2 cDNA is also isolated and sequenced. 3. Rat UCP-3 cDNA probe hybridized two bands, a major band at 2.5 kb and a minor band at 2.8 kb in rat tissues. The mRNA was expressed at the highest level in the skeletal muscle, and moderately in the interscapular brown adipose tissue (BAT). Only weak signals were detected in the epididymal white adipose tissue (WAT) and the heart. Rat UCP-2 cDNA probe hybridized a 1.7 kb band detected widely in the whole body, especially abundant in the lung and the spleen. In contrast to the UCP-3 gene expression, the UCP-2 gene expression was expressed at substantial levels in the WAT and only at slight levels in the skeletal muscle and BAT. 4. The UCP-3 gene expression is augmented two-fold in the gastrocnemius muscle from rats fed a high-fat diet (P < 0.05). The UCP-3 mRNA levels remained unchanged in the interscapular BAT, and epididymal WAT. The levels of the UCP-2 gene expression are augmented significantly in the epididymal WAT (1.6-fold; P < 0.05), while no significant increase is observed in the gastrocnemius muscle and interscapular BAT.

Dietary vitamin A supplementation in rats: suppression of leptin and induction of UCP1 mRNA

All-trans-retinoic acid (RA), an active metabolite of vitamin A, induces the gene expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) and suppresses leptin gene expression in white adipose tissue (WAT) when given as an acute dose. These contrasting effects of RA leave in doubt the overall effect of chronic RA or vitamin A supplementation on energy homeostasis. To investigate the effects of dietary vitamin A supplementation on leptin and UCP1 gene expression, rats were fed either a normal diet (2.6 retinol/kg diet) or a vitamin A-supplemented diet (129 mg retinol/kg diet) for 8 weeks, and adiposity, serum leptin levels, leptin mRNA levels in perirenal WAT, UCP1 and UCP2 mRNA levels in BAT, and β3-adrenergic receptor mRNA levels in BAT and WAT were examined. Rats on both diets gained a similar amount of weight, but there was a small 9% decrease in the adiposity index in the vitamin A-supplemented rats. Dietary vitamin A supplementation increased UCP1 gene expression in BAT by 31%, but suppressed leptin gene expression by 44% and serum leptin levels by 65%. UCP2 and β3-adrenergic receptor gene expression in BAT and perirenal WAT were unchanged by the vitamin A diet. These data suggest that dietary vitamin A has a role in regulating energy homeostasis by enhancing UCP1 gene expression and decreasing serum leptin levels.—Kumar, M. V., G. D. Sunvold, and P. J. Scarpace. Dietary vitamin A supplementation in rats: suppression of leptin and induction of UCP1 mRNA. J. Lipid Res. 1999. 40: 824–829.

Effects of obesity and stable weight reduction on UCP2 and UCP3 gene expression in humans.

The molecular determinants of energy expenditure are presently unknown. Recently, two uncoupling protein homologues, UCP2 and UCP3, have been identified. UCP2 is expressed widely, and UCP3 is expressed abundantly in skeletal muscle. Both could be important regulators of energy balance. In this paper, we investigated whether altered UCP2 and UCP3 mRNA levels are associated with obesity or weight reduction. UCP2, UCP3 long and short mRNA levels were examined in skeletal muscle and in white adipose tissue of lean, obese, and weight-reduced individuals by RNase protection assay. Expression of UCP2, UCP3S, and UCP3L mRNA in skeletal muscle was similar in lean individuals and in individuals with obesity at stable weight. In contrast, UCP3L and UCP3S mRNAs were decreased by 38% (p<0.0059) and 48% (p<0.0047), respectively, in 20% weight-reduced patients with obesity at stable weight. In contrast, UCP2 mRNA levels were increased by 30% in skeletal muscle of 20% weight-reduced subjects with obesity. In a different set of patients, mostly lean, UCP3L mRNA in skeletal muscle was decreased by 28% (p = 0.0425) after 10% weight reduction at stable weight. Expression of UCP2 mRNA in subcutaneous adipose tissue was similar in lean individuals and in individuals with obesity, and was increased by 58% during active weight loss. Stabilization at reduced body weight in humans is associated with a decrease in UCP3 mRNA in muscle. It is possible that reduced UCP3 expression could contribute to decreased energy expenditure in weight-stable, weight-reduced individuals.