Insulin and contraction directly stimulate UCP2 and UCP3 mRNA expression in rat skeletal muscle in vitro

To study the regulation of the mitochondrial uncoupling protein 2 and 3 (UCP2 and UCP3), we studied the effect of insulin and muscle contraction on UCP mRNA expression in rat skeletal muscle in vitro. Insulin dose-dependently increased skeletal muscle UCP2 and UCP3 mRNA expression in m. extensor digitorum longus (EDL) with maximal stimulation obtained at around 0.6-6 nM. The concentration of insulin giving half-maximal stimulation was 60 pM for the UCP2 and 48 pM for the UCP3 mRNA expression. The effect of insulin was maximal after 2 h and the effect was sustained during the whole study period (6 h). The insulin-induced increase in UCP mRNA was independent of the glucose uptake (as UCP mRNA was stimulated even in incubations without glucose). In addition, electrically induced contractions (in vitro) increased UCP2 and UCP3 mRNA expression 60-120 min after a single bout of contraction (for 10 min). Both the increment of UCP2 and UCP3 mRNA were sustained throughout the study period (4 h) (153 +/- 62 and 216 +/- 71% above basal, P < 0.05 respectively). Finally, 5-aminoimidazole-4-carboxamid-ribosid (AICAR), an activator of the AMP-activated protein kinase (AMPK), that is activated during exercise, was able to mimic the increase in UCP2 and UCP3 mRNA expression. In conclusion, UCP2 and UCP3 mRNA expression in skeletal muscle are stimulated rapidly by insulin and contraction in vitro, thus the stimulation is direct and not caused by changes in other hormones or metabolites. Even a brief bout of contraction induces an increase in UCP2 and UCP3 expression, an effect that could be mimicked by activation of the AMP-activated protein kinase by AICAR.     

Abundant and constant expression of uncoupling protein 2 in the liver of red sea bream Pagrus major

Four overlapping cDNA fragments encoding a partial sequence for uncoupling protein 2 (UCP2) were amplified by PCR using degenerate primers from the liver of a marine teleost fish, red sea bream (Pagrus major). The partial sequence was 674 bp long, encoding 224 amino acids. The deduced amino acid sequence from the cDNA partial sequence contained the signature motifs for mitochondrial transporter protein and revealed positional identity higher than 72.8% with UCP2 from mammals. The fish UCP2 gene was highly expressed in the liver but almost undetectable in the visceral mesenteric adipose tissue. Using beta-actin as control, the UCP2 mRNA level was determined to be at least 20-fold higher in the liver than in the visceral mesenteric adipose tissues. Neither 48 h starvation nor high lipid diet had any significant effect on liver UCP2 gene expression, indicating that the abundant UCP2 gene expression was stable and might have some basic function in a fish liver that always contains high lipid content. The striking contrast of UCP2 gene expression in the two fish fat-depot organs is consistent with their large differences in oxidative capacity. We suggest that the fish liver may adapt to a constantly high fat deposit by maintaining high UCP2 expression to constrain reactive oxygen species (ROS) production and protect hepatocytes from apoptosis.     

Full expression of uncoupling protein gene requires the concurrence of norepinephrine and triiodothyronine

We have examined the uncoupling (UCP) protein gene expression in euthyroid and hypothyroid rats. UCP mRNA levels were estimated by northern blot analysis of total RNA from brown adipose tissue (BAT). Stimuli were endogenous (cold) and exogenous norepinephrine (NE), isoproterenol, T3, and T4. While the euthyroid rats UCP mRNA levels increase 2- to 3-fold by 2 h after NE or overnight cold exposure, these stimuli and isoproterenol are ineffective in hypothyroid rats. One single dose of T4, equal to the daily production rate, brings about a normal response in hypothyroid rats exposed to cold overnight. Hypothyroid rats recover their responsiveness to NE approximately 4 h after a receptor saturating dose of T3. On the other hand, such a dose of T3 induces a 3- to 4-fold increase in UCP mRNA levels in hypothyroid rats without the need of exogenous NE, and this response is not reduced by raising ambient temperature to thermoneutrality (28 C). However, the following evidence indicates that T3 requires adrenergic input to stimulate the accumulation of UCP mRNA: first, euthyroid animals maintained at 28 C do not respond to such a treatment. Second, when T3 was injected to hypothyroid rats with unilaterally denervated BAT, only the intact side responded to T3 with an elevation of the UCP mRNA levels, but both sides remained responsive to T3 + NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of hepatic lipase gene expression and activity by fenofibrate.

The influence of the hypolipidemic drug, fenofibrate, on hepatic lipase (HL) gene expression and activity was investigated in the rat. Fenofibrate treatment provoked a dose-dependent decrease in HL mRNA levels. At a dose of 0.5% (w/w), HL mRNA levels were reduced to nearly 50% the levels in untreated controls. This decrease was parallelled by a comparable reduction in liver HL activity. The decrease in HL mRNA levels was already observed after 1 day of fenofibrate treatment. Whole liver perfusion experiments showed that the heparin-releasable HL activity in fenofibrate-treated livers dropped to 10% the activity in control livers. In conclusion, treatment with fenofibrate decreases HL gene expression, leading to a lowered activity of endothelium bound HL in fenofibrate-treated livers.     

Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction

Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.     

Thyroid-hormone effects on putative biochemical pathways involved in UCP3 activation in rat skeletal muscle mitochondria

In vitro, uncoupling protein 3 (UCP3)-mediated uncoupling requires cofactors [e.g., superoxides, coenzyme Q (CoQ) and fatty acids (FA)] or their derivatives, but it is not yet clear whether or how such activators interact with each other under given physiological or pathophysiological conditions. Since triiodothyronine (T3) stimulates lipid metabolism, UCP3 expression and mitochondrial uncoupling, we examined its effects on some biochemical pathways that may underlie UCP3-mediated uncoupling. T3-treated rats (Hyper) showed increased mitochondrial lipid-oxidation rates, increased expression and activity of enzymes involved in lipid handling and increased mitochondrial superoxide production and CoQ levels. Despite the higher mitochondrial superoxide production in Hyper, euthyroid and hyperthyroid mitochondria showed no differences in proton-conductance when FA were chelated by bovine serum albumin. However, mitochondria from Hyper showed a palmitoyl-carnitine-induced and GDP-inhibited increased proton-conductance in the presence of carboxyatractylate. We suggest that T3 stimulates the UCP3 activity in vivo by affecting the complex network of biochemical pathways underlying the UCP3 activation.     

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 beta3-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 beta3-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.     

UCP3 gene expression does not correlate with muscle oxidation rates in troglitazone-treated Zucker fatty rats

Uncoupling protein-3 (UCP3), a mitochondrial carrier protein predominantly expressed in muscle, has been suggested to release stored energy as heat. The insulin-sensitizing thiazolidinediones enhance glucose disposal in skeletal muscle and have been reported to increase the expression of uncoupling proteins in various experimental systems. We therefore studied the effect of troglitazone treatment on UCP3 gene expression in muscles from lean and obese Zucker rats. In comparison with obese littermates, basal UCP3 mRNA levels in lean Zucker rats tended to be higher in white and red gastrocnemius muscles, but were lower in soleus (P<0.001) muscle and heart (P<0.01). In lean rats, troglitazone significantly increased UCP3 gene expression in white and red gastrocnemius and heart muscles (all P<0.01). In contrast, the drug reduced UCP3 mRNA expression in red gastrocnemius and soleus muscles of obese littermates (all P<0.001). The troglitazone-dependent decrease in UCP3 gene expression was accompanied by an increased weight gain in obese rats, while no such effect was observed in lean rats. In obese rats, improvement of insulin resistance by troglitazone was associated with increased rates of basal and insulin-stimulated CO(2) production from glucose measured in soleus muscle. These studies demonstrate that effects of troglitazone on UCP3 gene expression depend on the phenotype of Zucker rats and that troglitazone-induced metabolic improvements are not related to increased uncoupling resulting from upregulation of UCP3 mRNA expression in muscle.     

Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats

We investigated the effects of aging and denervation on the gene expression of uncoupling proteins (UCPs) in slow-twitch soleus and fast-twitch gastrocnemius muscles. In a comparison between the control limbs of 6- and 24-month-old rats, the mRNA levels of UCP3, heart-type fatty acid binding protein (HFABP), and glucose transporter-4 (GLUT4) were considerably lower in the gastrocnemius muscles of the older rats, whereas no significant differences in the mRNA levels of those genes as well as UCP2 and cytochrome oxidase subunit IV (COX-IV) were observed in the soleus muscles of young and old rats. The UCP3 and COX-IV protein levels were also reduced considerably in the aged gastrocnemius muscles with atrophy. Denervation of the sciatic nerve caused an increase in UCP3 mRNA levels in both muscles, but the regulation of other genes contrasted between the two types of skeletal muscles. In spite of the increased mRNA level, a remarkable reduction in UCP3 protein was found in the denervated gastrocnemius muscles. These results indicate that the effects of aging and denervation on the gene expression of UCPs, HFABP, GLUT4, and COX-IV are different between the muscle types. The reduction in the mitochondrial UCP3 and COX proteins in aged fast-twitch muscles may have a negative effect on energy metabolism and thermogenesis in old animals.     

Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin, hyperphagia, increased tissue lipid content, and TNF-alpha

Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-alpha in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-alpha has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-alpha.     

Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP

A new cellular model for the study of brown adipocyte development and differentiation in vitro is presented. Preadipocytes isolated from brown adipose tissue (BAT) of the djungarian dwarf hamster Phodopus sungorus are able to proliferate and differentiate in vitro into true brown adipocytes able to express the BAT marker protein the uncoupling protein (UCP). Whereas basal UCP expression is very low, its mRNA levels as well as the UCP detected by immunoblotting are highly increased by beta-adrenergic stimulation. The novel, atypical beta-adrenergic compound D7114 (ICI Pharmaceuticals, Macclesfield, Cheshire, England) was found to increase the number of adipocytes as well as UCP mRNA and UCP content of mitochondria, indicating the involvement of an atypical or beta 3 receptor. Insulin was found to play an important role in brown adipocyte differentiation and mitochondrial development, whereas T3 seemed to be implicated more directly in UCP expression. In a defined, serum-free medium a synergistic stimulatory action of insulin and T3 on UCP expression was found, which seems to involve a pathway different from that of beta-adrenergic UCP stimulation.     

Effects of soy protein and isoflavone on hepatic fatty acid synthesis and oxidation and mRNA expression of uncoupling proteins and peroxisome proliferator-activated receptor gamma in adipose tissues of rats

Soy protein rich in isoflavones profoundly affects lipid metabolism in experimental animals. To distinguish the roles of the protein and isoflavone components of a soy protein preparation in regulating lipid metabolism, we compared the effects of diets containing methanol-washed soy protein low in isoflavone supplemented with a 0-, 0.5- and 4-g/kg isoflavone preparation on hepatic fatty acid metabolism and adipose tissue gene expression in rats. Diets containing soy protein irrespective of the isoflavone levels decreased the activities and mRNA expression of enzymes involved in hepatic fatty acid synthesis to similar levels. Methanol-washed soy protein compared to casein increased the mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, and supplementing the soy protein diet with isoflavone further increased this parameter dose-dependently. However, methanol-washed soy protein compared to casein was totally ineffective in altering the activities and mRNA levels of enzymes involved in fatty acid oxidation. Supplementation of soy protein diets with isoflavone slightly increased these parameters. The mRNA level of uncoupling protein (UCP) 1 in brown adipose tissue was significantly increased and mRNA levels of UCP2 and 3, and PPARgamma2 tended to be higher in rats fed methanol-washed soy protein not supplemented with isoflavone than in the animals fed casein. Adding isoflavone to the soy protein diets dose-dependently increased these parameters. These results suggested that the protein rather than isoflavone component is primarily responsible for the physiological activity of soy protein rich in isoflavones in reducing hepatic lipogenesis. However, isoflavones may have a role in regulating heptic fatty acid oxidation and adipose tissue gene expression.     

Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.     

Regulation of uncoupling protein 2 expression by long-chain fatty acids and hormones in bovine mammary epithelial cells

Although mammary epithelial cells are known to synthesize and accumulate triacylglycerol (TAG) in order to produce milk lipid in the cytosol, lipid and energy metabolism is still not fully understood. In this study, we assessed the effects of long-chain fatty acid (LCFA) on the accumulation of cytosolic TAG and uncoupling protein (UCP) 2 in cloned bovine mammary epithelial cells (bMEC). LCFAs significantly raised the expression of UCP2 mRNA and the accumulation of TAG. We observed the rapid elevation in UCP2 shown at 6 h after LCFA treatment. Insulin (5-50 ng/ml) or dexamethasone (500 nM) significantly suppressed the expression of UCP2 mRNA. These results suggest that UCP2 play an important role of lipid and energy metabolism in mammary epithelial cells.     

Up-regulation of liver uncoupling protein-2 mRNA by either fish oil feeding or fibrate administration in mice

Fish oil feeding showed less obesity in rodents, relative to other dietary oils. N-3 fatty acids rich in fish oil and fibrate compounds are peroxisome proliferator-activated receptor alpha (PPARalpha) ligands that stimulate beta-oxidation of fatty acids in liver and are used for treatment of hypertriglycemic patients. Since UCP-2, a member of an uncoupling protein family, has been shown to express in hepatocytes, the effects of these agents on the expression of UCP2 mRNA were investigated. C57BL/6J mice were divided into three groups; the first group was given a high-carbohydrate diet, and the other two groups were given a high-fat diet (60% of total energy) as safflower oil or fish oil for 5 months. Safflower oil diet fed mice developed obesity, but those fed fish oil diet did not. Therefore, the effects of fish oil feeding on the expression of UCP1, UCP2 and UCP3 in liver, skeletal muscle (gastrocnemius), white adipose tissue (WAT) and brown adipose tissue (BAT) were assessed by Northern blotting. Compared with safflower oil feeding, fish oil feeding up-regulated liver UCP2, BAT UCP2 and skeletal muscle UCP3 mRNA, while down-regulated WAT UCP2 and BAT UCP3 mRNA. Among these alterations, 5-fold up-regulation of liver UCP2 mRNA, relative to carbohydrate feeding, was noteworthy. Fenofibrate administration (about 500 mg/kg BW/d) for 2 wks also induced liver UCP2 expression by 9-fold. These data indicated that fish oil feeding and fibrate administration each up-regulated UCP2 mRNA expression in liver possibly via PPARalpha and hence each has the potential of increasing energy expenditure for prevention of obesity.