Interactions between an alpha2-adrenergic antagonist and a beta3-adrenergic agonist on the expression of UCP2 and UCP3 in rats

This experimental trial was devised to assess whether selective beta3-adrenergic receptor (AR) stimulation and simultaneous blockade of alpha2-AR would affect thermoregulation. With this purpose, the individual and combined administration of a beta-AR agonist, trecadrine, and an alpha2-AR antagonist, yohimbine, were evaluated. Yohimbine produced a marked decrease (p < 0.001) in body temperature one hour after administration (5 mg kg(-1), i.p.) and blocked the thermogenic effect of trecadrine (1 mg kg(-1), i.p.) when simultaneously administered. Uncoupling protein-2 expression in skeletal muscle was downregulated (p < 0.05) by trecadrine, while yohimbine had no effect. White adipose tissue UCP2 and muscle UCP3 were not modified by either trecadrine or yohimbine administration. Liver UCP2 mRNA expression was significantly decreased by yohimbine (p < 0.05). However, this downregulation does not seem to explain the reduction in temperature produced by yohimbine given the fact that trecadrine produced a similar downregulation of hepatic UCP2 (p < 0.05). The present work indicates that alpha2-AR antagonism blocks the thermogenic effects mediated by beta3-AR stimulation, contrary to our expectations, suggesting a possible interplay between both mechanisms. Moreover, these effects are not apparently explained by changes in UCP2 and UCP3.     

Bovine UCP2 and UCP3 map to BTA15

Two recently described uncoupling proteins, UCP2 and UCP3, may have important roles in determining feed conversion and/or maintenance energy requirements in livestock. Sequence was determined for the entire coding region and four of the six introns for bovine UCP3. A partial cDNA sequence was obtained for bovine UCP2. Radiation hybrid mapping was used to place UCP3 on BTA15, a chromosome previously shown to harbor a locus influencing meat tenderness. In order to place UCP3 in the existing linkage map, five single nucleotide polymorphisms (SNPs) were developed. Linkage analysis using one of five SNPs was used to place UCP3 between 53.1 and 53.5 CM. No recombination was detected between UCP3 and IDVGA10, IDVGA32, and INRA145. The sequence of PCR products from a single BAC amplified with primers specific for either UCP2 or UCP3 indicate that UCP2 and UCP3 are separated by < 200 kb.     

Reconstitution of novel mitochondrial uncoupling proteins UCP2 and UCP3

Reconstitution of novel mitochondrial uncoupling proteins, human UCP2 and UCP3, expressed in yeast, was performed to characterize fatty acid (FA)-induced H⁺ efflux in the resulted proteoliposomes. We now demonstrate for the first time that representatives of physiologically abundant long chain FAs, saturated or unsaturated, activate H⁺ translocation in UCP2- and UCP3-proteoliposomes. Efficiency of lauric, palmitic or linoleic acid was roughly the same, but oleic acid induced faster H⁺ uniport. We have confirmed that ATP and GTP inhibit such FA-induced H⁺ uniport mediated by UCP2 and UCP3. Coenzyme Q₁₀ did not further significantly activate the observed H⁺ efflux. In conclusion, careful instant reconstitution yields intact functional recombinant proteins, UCP2 and UCP3, the activity of which is comparable with UCP1.     

Beta 2-agonist treatment enhances uterine oxytocin receptor mRNA expression in pregnant rats

The objective of this study was to disclose an interaction between Beta(2)-adrenergic (Beta(2)-ARs) and oxytocin (OT) receptors (OTRs) in the late-pregnant rat uterus. We investigated the level of uterine OTR mRNA expression after the administration of Beta(2)-AR agonists fenoterol and hexoprenaline to rats from day 18 to 22 of pregnancy, and also tested the effect of fenoterol on uterine explants. Hexoprenaline induced a maximum 24% increase of OTR mRNA. Fenoterol in vivo elicited a maximum 125% increase of OTR mRNA, in vitro produced a maximum fourfold increase in OTR mRNA. In fenoterol-treated rats the maximal contractility increasing effect of OT on isolated uterine rings was significantly higher than in intact term pregnant rats, but the EC50 values were not statistically different. It was concluded that the enhanced expression of OTR mRNA induced by Beta(2)-agonists in the late-pregnant rat uterus may be a possible drawback to effective therapy of preterm uterine contractions with Beta(2)-agonists.     

Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3

Evidence for the physiological functions of UCP2 and UCP3 is critically reviewed. They do not mediate adaptive thermogenesis, but they may be significantly thermogenic under specific pharmacological conditions. There is strong evidence that the mild regulated uncoupling they cause attenuates mitochondrial ROS production, protects against cellular damage, and diminishes insulin secretion. Evidence that they export fatty acids physiologically is weak. UCP2 and UCP3 are important potential targets for treatment of aging, degenerative diseases, diabetes, and perhaps obesity.     

Links between fatty acids and expression of UCP2 and UCP3 mRNAs

Physiological and pathological states that are associated with elevated plasma fatty acids (FAs) increase uncoupling protein 2 (UCP2) mRNA in white adipose tissue and UCP3 mRNA in skeletal muscle and heart. A direct effect of unsaturated fatty acids from all classes has been shown in various cultured cells. There is evidence that FAs could induce expression of UCPs by acting as ligands for peroxisome proliferator-activated receptors, influencing the function of sterol responsive element binding protein or activating 5'-AMP-activated protein kinase. Oleic acid has been shown to stimulate the activity of the promoter regions of UCP2 and UCP3 genes and the FA responsive regions are beginning to be characterised.     

Regulation of UCP1, UCP2, and UCP3 mRNA expression in brown adipose tissue, white adipose tissue, and skeletal muscle in rats by estrogen

The effects of ovariectomy (OVX) and estrogen substitution on body weight, body composition, food intake, weight gain, and expression of uncoupling proteins (UCPs) in brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle were studied in four groups of rats: (1) Sham-operated rats (N = 8), (2) ovariectomized rats (OVX - E) (N = 8), (3) estrogen-treated OVX rats (OVX + E) (N = 8), and (4) OVX rats on energy restriction (OVX - E + D) (N = 8). OVX was associated with an increase in food intake and body weight gain during a 5-week study period compared to sham-operated rats. The estrogen-substituted rats had a significantly lower food intake and weight gain during the 5 weeks compared to the sham-operated group. However, we also included a nontreated OVX group that was allowed to eat only enough chow to match the weight gain of the sham-operated group. To match the weight gain in the two groups, the OVX group had to consume 16% less chow than the sham-operated group. In BAT, the UCP1 expression was significantly lower in estrogen-deficient rats compared to either intact rats or estrogen-substituted rats, whereas UCP2 and UCP3 mRNA expression was similar in BAT from all four groups. In WAT, both estrogen-deficient groups had significantly lower UCP2 mRNA expression compared to the control rats and estrogen-treated rats; In contrast, the UCP3 mRNA expression in WAT was similar in all four groups. Finally, in skeletal muscle the OVX group on mild energy restriction had reduced UCP3 mRNA expression compared to control, OVX, and estrogen-treated rats. In contrast, the UCP2 mRNA expression in skeletal muscle was similar in all four groups. Thus, the findings that estrogen deficiency is followed by reduced UCP1 expression in BAT and reduced UCP2 expression in WAT in association with weight gain probably caused by a decrease in energy expenditure might indicate that UCPs play a role for the estrogen-mediated changes in body weight and energy expenditure.     

Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.     

Effects of UCP2 and UCP3 Variants on the Manifestation of Overweight in Korean Children

To investigate the associations of uncoupling protein (UCP)2 and UCP3 gene variants with overweight and related traits, we genotyped UCP2−866G>A, UCP2Ala55Val, and UCP3−55C>T in 737 Korean children and 732 adults and collected data regarding anthropometric status and blood biochemistry. Information concerning the children's lifestyles and dietary habits was collected. The UCP2−866G>A and UCP3−55C>T gene variants showed significant associations with BMI level, waist circumference, and body weight in the children but not in the adults. Compared with −866GG carriers, the −866GA and AA carriers showed a strong decreasing trend in the risk for overweight (odds ratio (OR), 0.67; 95% confidence interval (CI), 0.45–1.01; P = 0.053). In comparison with UCP3−55CC carriers, children carrying −55CT and TT showed a significant reduction in the risk of overweight (OR, 0.67; 95% CI, 0.46–0.98; P = 0.039). There was also evidence of interactions between the effects of the combined UCP2−UCP3 genotype and obesity‐related metabolic traits. The greatest protective effect against overweight was seen in those with the combined genotype non‐UCP2−866GG and non‐UCP3−55CC, as compared with those carrying both UCP2−866GG and UCP3−55CC (OR, 0.60; 95% CI, 0.38–0.95; P = 0.030). In the subgroup with a low level of physical activity, UCP3−55CC carriers had higher BMI values than UCP3−55T carriers (16.6 ± 2.3 kg/m² vs. 16.1 ± 1.9 kg/m², P = 0.016). Low physical activity may aggravate the susceptibility to overweight in UCP2−866GG and UCP3−55CC carriers.     

The reactions catalysed by the mitochondrial uncoupling proteins UCP2 and UCP3

The mitochondrial uncoupling proteins UCP2 and UCP3 may be important in attenuating mitochondrial production of reactive oxygen species, in insulin signalling (UCP2), and perhaps in thermogenesis and other processes. To understand their physiological roles, it is necessary to know what reactions they are able to catalyse. We critically examine the evidence for proton transport and anion transport by UCP2 and UCP3. There is good evidence that they increase mitochondrial proton conductance when activated by superoxide, reactive oxygen species derivatives such as hydroxynonenal, and other alkenals or their analogues. However, they do not catalyse proton leak in the absence of such acute activation. They can also catalyse export of fatty acid and other anions, although the relationship of anion transport to proton transport remains controversial.     

Effects of Obesity and Stable Weight Reduction on UCP2 and UCP3 Gene Expression in Humans

VIDAL-PUIG, ANTONIO, MICHAEL ROSENBAUM, ROBERT C. CONSIDINE, RUDOLPH L. LEIBEL, G. LYNIS DOHM, AND BRADFORD B. LOWELL. Effects of obesity and stable weight reduction on UCP2 and UCP3 gene expression in humans. Obes Res. Objectives: 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. Research Methods and Procedures: 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. Results: 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. Discussion: 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.     

Up-regulation of splenic prohormone convertases PC1 and PC2 in diabetic rats.

Organisms respond to infection in a complex manner involving bidirectional interactions between the neuroendocrine and immune systems. Many of the bioactive endocrine/immune factors are synthesized in a precursor form and are expected to be activated by prohormone convertases (PCs). Since patients with both type 1 and type 2 diabetes have an increased incidence and severity of infections, we hypothesized that in a condition of hyperglycemia, these processing enzymes would be activated in an immune tissue, the spleen. To test this hypothesis, we treated rats with intraperitoneal streptozotocin (STZ) (50 mg/kg/day) daily for 5 days and measured splenic PC1 and PC2 mRNA by ribonuclease protection assay. We found that PC1 mRNA was increased 6.0+/-0.02-fold (P<0.05) and PC2 mRNA was increased 1.80+/-0.01-fold (P<0.005) in the spleen of rats that received STZ compared to rats that received vehicle. Western blot indicated that the 75-kDa form of PC1 was the only form of PC1 present in the spleen and that this form increased with STZ treatment. Immunohistochemistry revealed that PC1 was found in both the white pulp (T-lymphocytes) and red pulp (monocytes and macrophages) and that its increase in immunoreactivity occurred primarily in the white pulp. PC2 and pro-opiomelanocortin (POMC, a possible splenic substrate for PC1/PC2) immunoreactivity was found predominantly in the red pulp. STZ induced an increase in splenic PC1 and POMC, but not PC2 protein levels. We conclude that in the STZ model of diabetes, splenic PCs are induced, which could lead to an increased activation of many immune-derived hormones. We speculate that this up-regulation of prohormone converting enzymes may be related to the increased infections seen in patients with both type 1 and type 2 diabetes.     

The effect of indomethacin on uterine contractility and luteal regression in pregnant rats at term.

Treatment of pregnant rats with 1 mg indomethacin/kg twice daily i.m. beginning on Day 20 delayed the onset of parturition by about 21 hr and prolonged the duration of spontaneous parturition by 4 hr. Plasma progesterone and oestradiol levels were determined in daily samples of peripheral blood, and uterine contractions were recorded before and during parturition by means of small, chronically implanted intrauterine balloons which were connected to pressure transducers via fluid-filled catheters. Indomethacin treatment did not inhibit or suppress spontaneous or oxytocin-induced contractions, which were of the same intensity in indomethacin-treated as in control rats. Parturition was induced with oxytocin in the same proportion of treated and control rats, but its induction was not successful in treated rats until 1 day later than in control rats, but its induction was not successful in treated rats until 1 day later than in controls. The onset of parturition was always related to the plasma progesterone level, which declined at a slower rate in indomethacin-treated than in control rats, reaching baseline values approximately 1 day later in the treated animals. The appearance of 20alpha-hydroxysteroid dehydrogenase in the CL of pregnant rats normally occurs on Day 21 of gestation, but activity was not observed until about 1 (0-3) day later in the indomethacin-treated rats, indicating that luteolysis was retarded. Prostaglandin F-2alpha infusions given on Day 21 reversed the effects of indomethacin treatment on plasma progesterone, luteal 20alpha-hydroxysteroid dehydrogenase activity and the timing and duration of parturition, and reduced the high perinatal mortality associated with indomethacin treatment, suggesting that the effects of indomethacin were related to its inhibitory action on prostaglandin synthetase activity. It is concluded that, in rats, indomethacin exerts its effects on parturition through inhibition of luteal regression which was significantly retarded but not prevented, and that indomethacin does not have a direct effect on myometrial contractility.     

Glutathionylation acts as a control switch for uncoupling proteins UCP2 and UCP3

The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys(25) and Cys(259) as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes.     

Thermogenic responses in brown fat cells are fully UCP1-dependent. UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty scid-induced thermogenesis

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 ( approximately 10-fold increase in thermogenesis); Ucp1(-/-) cells only responded approximately 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 in Ucp1(-/-) 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.