Reduced adiposity and improved insulin sensitivity in obese mice with antisense suppression of 4E-BP2 expression

To investigate the possible role of eukaryotic initiation factor 4E-binding protein-2 (4E-BP2) in metabolism and energy homeostasis, high-fat diet-induced obese mice were treated with a 4E-BP2-specific antisense oligonucleotide (ASO) or a control 4E-BP2 ASO at a dose of 25 mg/kg body wt or with saline twice a week for 6 wk. 4E-BP2 ASO treatment reduced 4E-BP2 levels by >75% in liver and white (WAT) and brown adipose (BAT) tissues. Treatment did not change food intake but lowered body weight by approximately 7% and body fat content by approximately 18%. Treatment decreased liver triglyceride (TG) content by >50%, normalized plasma glucose and insulin levels, and reduced glucose excursion during glucose tolerance test. 4E-BP2 ASO-treated mice showed >8.5% increase in metabolic rate, >40% increase in UCP1 levels in BAT, >45% increase in beta(3)-adrenoceptor mRNA, and 40-55% decrease in mitochondrial dicarboxylate carrier, fatty acid synthase, and diacylglycerol acyltransferase 2 mRNA levels in WAT. 4E-BP2 ASO-transfected mouse hepatocytes showed an increased fatty acid oxidation rate and a decreased TG synthesis rate. In addition, 4E-BP2 ASO-treated mice demonstrated approximately 60 and 29% decreases in hepatic glucose-6-phosphatase and phosphoenolpyruvate carboxykinase mRNA, respectively, implying decreased hepatic glucose output. Furthermore, increased phosphorylation of Akt(Ser473) in both liver and fat of 4E-BP2 ASO-treated mice and increased GLUT4 levels in plasma membrane in WAT of the ASO-treated mice were observed, indicating enhanced insulin signaling and increased glucose uptake as a consequence of reduced 4E-BP2 expression. These data demonstrate for the first time that peripheral 4E-BP2 plays an important role in metabolism and energy homeostasis.     

The control of UCP1 is dissociated from that of PGC-1alpha or of mitochondriogenesis as revealed by a study using beta-less mouse brown adipocytes in culture

In rodent brown adipose tissue, the beta-adrenergic signaling is believed, by an action on PGC-1alpha, to control UCP1 expression and mitochondriogenesis. We addressed this hypothesis using beta(1)/beta(2)/beta(3)-adrenoceptor knockout (beta-less) brown adipocytes in primary culture. In these cells: (a) proliferation and differentiation into multilocular cells were normal; (b) UCP1 mRNA expression was dramatically decreased (by 93%), whereas PGC-1alpha and mtTFA mRNA expressions were not; (c) UCP1, PGC-1alpha and COX IV protein expressions were decreased by 97%, 62% and 22%, respectively. Altogether the data show a dissociation between the control of UCP1, which is mostly beta-adrenoceptor-dependent and that of PGC-1alpha and of mitochondriogenesis which are not.     

Beta 3-adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown adipocytes in white fat.

White and brown adipocytes are usually located in distinct depots; however, in response to cold, brown adipocytes appear in white fat. This response is mediated by beta-adrenoceptors but there is a controversy about the subtype(s) involved. In the present study, we exposed to cold beta 3-adrenoceptor knockout mice (beta 3KO) on a C57BL/6J genetic background and measured in white adipose tissue the density of multilocular cells and the expression of the brown adipocyte marker uncoupling protein-1 (UCP1). In brown fat of beta 3KO mice, UCP1 expression levels were normal at 24 degrees C as well as after a 10-day cold exposure. Strikingly, under both conditions, in the white fat of beta 3KO mice the levels of UCP1 mRNA and protein as well as the density of multilocular cells were decreased. These results indicate that beta 3-adrenoceptors play a major role in the appearance of brown adipocytes in white fat and suggest that the brown adipocytes present in white fat differ from those in brown fat.     

Ghrelin affects gastrectomy-induced decrease in UCP1 and beta3-AR mRNA expression in mice

In this study we investigated the effects of gastrectomy (Gx) and of the gastric hormone, ghrelin, on the expression of proteins in brown adipose tissue (BAT) that are thought to be involved in thermogenesis. Heat production in BAT is known to depend upon activation and increased expression of beta3-adrenergic receptors (beta3-AR) and the consequent up-regulation of uncoupling protein 1 (UCP1). Mice were subjected to Gx or sham operation. One week later they started to receive daily subcutaneous injections of either saline or ghrelin (12 nmol) for two or eight weeks. Neither Gx nor ghrelin affected daily food intake. Gx did not lower body weight gain (except during the first post-operative week) but Gx mice responded to eight weeks of ghrelin treatment with a greater body weight increase (37%, p<0.05) than saline-injected Gx mice; sham-operated mice did not respond to ghrelin. Gx resulted in a greatly reduced expression of both UCP1 and beta3-AR mRNA in BAT (50% reduction or more, p<0.01) compared to sham-operated mice. Eight weeks of ghrelin treatment raised the UCP1 as well as the beta3-AR mRNA expression in the Gx mice, whereas two weeks of ghrelin treatment decreased UCP1 and beta3-AR mRNA expression compared to Gx mice receiving saline. In fact, mRNA expression in Gx mice after treatment with ghrelin for eight weeks was similar to that in saline-treated sham-operated mice. Ghrelin did not affect UCP1 and beta3-AR mRNA in sham-operated mice neither two nor eight weeks after the operation. The results suggest 1) that signals from the stomach stimulate BAT UCP1 (and possibly thermogenesis) and 2) that ghrelin may contribute to the control of UCP1 expression.     

CL316,243 and cold stress induce heterogeneous expression of UCP1 mRNA and protein in rodent brown adipocytes.

Uncoupling protein 1 (UCP1), the mammalian thermogenic mitochondrial protein, is found only in brown adipocytes, but its expression by immunohistochemistry is not homogeneous. Here we present evidence that the non-homogeneous pattern of immunostaining for UCP1 (referred to as the "Harlequin phenomenon") is particularly evident after acute and chronic cold (4C) stimulus and after administration of a specific beta(3)-adrenoceptor agonist (CL316,243). Accordingly, mRNA in situ expression confirmed the UCP1 non-homogeneous pattern of gene activation under conditions of adrenergic stimulus. Furthermore, morphometric analysis of immunogold-stained thin sections showed that UCP1-gold particle density was different among neighboring brown adipocytes with mitochondria of the same size and density. When the adrenergic stimulus was reduced in warm-acclimated animals (28C), UCP1 protein and mRNA expression was reduced and consequently the Harlequin phenomenon was barely visible. These data suggest the existence of an alternative and controlled functional recruitment of brown adipocytes in acute adrenergically stressed animals, possibly to avoid heat and metabolic damage in thermogenically active cells. Of note, the heat shock protein heme oxygenase 1 (HO1) is heterogeneously expressed in adrenergically stimulated brown adipose tissue and, specifically, cells expressing strong immunoreactivity for UCP1 also strongly express HO1.     

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.     

Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.     

Effect of beta1- and beta2-adrenergic stimulation on energy expenditure,substrate oxidation,and UCP3 expression in humans

In humans, beta-adrenergic stimulation increases energy and fat metabolism. In the case of beta1-adrenergic stimulation, it is fueled by an increased lipolysis. We examined the effect of beta2-adrenergic stimulation, with and without a blocker of lipolysis, on thermogenesis and substrate oxidation. Furthermore, the effect of beta1-and beta2-adrenergic stimulation on uncoupling protein 3 (UCP3) mRNA expression was studied. Nine lean males received a 3-h infusion of dobutamine (DOB, beta1) or salbutamol (SAL, beta2). Also, we combined SAL with acipimox to block lipolysis (SAL+ACI). Energy and substrate metabolism were measured continuously, blood was sampled every 30 min, and muscle biopsies were taken before and after infusion. Energy expenditure significantly increased approximately 13% in all conditions. Fat oxidation increased 47 +/- 7% in the DOB group and 19 +/- 7% in the SAL group but remained unchanged in the SAL+ACI condition. Glucose oxidation decreased 40 +/- 9% upon DOB, remained unchanged during SAL, and increased 27 +/- 11% upon SAL+ACI. Plasma free fatty acid (FFA) levels were increased by SAL (57 +/- 11%) and DOB (47 +/- 16%), whereas SAL+ACI caused about fourfold lower FFA levels compared with basal levels. No change in UCP3 was found after DOB or SAL, whereas SAL+ACI downregulated skeletal muscle UCP3 mRNA levels 38 +/- 13%. In conclusion, beta2-adrenergic stimulation directly increased energy expenditure independently of plasma FFA levels. Furthermore, this is the first study to demonstrate a downregulation of skeletal muscle UCP3 mRNA expression after the lowering of plasma FFA concentrations in humans, despite an increase in energy expenditure upon beta2-adrenergic stimulation.     

Role of the beta(3)-adrenergic receptor and/or a putative beta(4)-adrenergic receptor on the expression of uncoupling proteins and peroxisome proliferator-activated receptor-gamma coactivator-1.

Administration of beta-adrenergic receptor (beta-AR) agonists, especially beta(3)-AR agonists, is well known to increase thermogenesis in rodents and humans. In this work we studied the role of the beta(3)-AR in regulating mRNA expression of genes involved in thermogenesis, i.e., mitochondrial uncoupling proteins UCP2 and UCP3, and peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1), in mouse skeletal muscle. For this purpose, different beta(3)-AR agonists were administered acutely to both wild type mice and mice whose beta(3)-AR gene has been disrupted (beta(3)-AR KO mice). CL 316243 increased the expression of UCP2, UCP3 and PGC-1 in wild type mice only. By contrast, BRL 37344 and CGP 12177 increased the expression of UCP2 and UCP3 in both wild type and beta(3)-AR KO mice, whereas they increased the expression of PGC-1 in wild type mice only. Finally, acute (3 h) cold exposure increased the expression of UCP2 and UCP3, but not PGC-1, in skeletal muscle of both wild type and beta(3)-AR KO mice. These results show that selective stimulation of the beta(3)-AR affects the expression of UCP2, UCP3 and PGC-1 in skeletal muscle. This effect is probably indirect, as muscle does not seem to express beta(3)-AR. In addition, our data suggest that BRL 37344 and CGP 12177 act, in part, through an as yet unidentified receptor, possibly a beta(4)-AR.     

Changes in UCP mRNA expression levels in brown adipose tissue and skeletal muscle after feeding a high-energy diet and relationships with leptin, glucose and PPARgamma

Brown adipose tissue and skeletal muscle are known to be important sites for nonshivering thermogenesis. In this context, it is accepted that uncoupling proteins (UCPs) are involved in such process, but little is known about the physiological regulation of these proteins as affected by the intake of a high-energy (cafeteria) diet inducing fat deposition. In this study, the UCP messenger RNA (mRNA) expression in interscapular brown adipose tissue (iBAT) and skeletal muscle was assessed to evaluate the influence of a dietary manipulation on energy homeostasis regulation. We report a statistically significant increase in mRNA levels of iBAT UCP1 and UCP3 and a statistical marginal rise in skeletal muscle UCP3 mRNA expression after feeding a high-energy diet, whereas no changes in UCP2 expression were found in either tissue. Furthermore, significant positive associations between iBAT UCP1 and UCP3 mRNA levels with serum leptin were found. Although the expression of the beta(3) adrenoceptor (beta(3)AR) was about 50% in the lean controls compared with the obese group in iBAT, no statistically significant changes were observed concerning peroxisome proliferator-activated receptor gamma2 (PPARgamma2) mRNA levels in muscle or iBAT. We conclude that feeding a diet inducing weight and fat gain produces different outcomes on iBAT and skeletal muscle UCP mRNA expression, revealing a tissue-dependent response for the three UCPs. Results suggest that the regulation of UCP expression in both tissues under these specific dietary conditions may be related to leptin circulating levels.     

Exercise training initiated after the onset of diabetes preserves myocardial function: effects on expression of {beta}-adrenoceptors.

The present study was undertaken to assess cardiac function and characterize beta-adrenoceptor subtypes in hearts of diabetic rats that underwent exercise training (ExT) after the onset of diabetes. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin. Four weeks after induction, rats were randomly divided into two groups. One group was exercised trained for 3 wk while the other group remained sedentary. At the end of the protocol, cardiac parameters were assessed using M-mode echocardiography. A Millar catheter was also used to assess left ventricular hemodynamics with and without isoproterenol stimulation. beta-Adrenoceptors were assessed using Western blots and [(3)H]dihydroalprenolol binding. After 7 wk of diabetes, heart rate decreased by 21%, fractional shortening by 20%, ejection fraction by 9%, and basal and isoproterenol-induced dP/dt by 35%. beta(1)- and beta(2)-adrenoceptor proteins were reduced by 60% and 40%, respectively, while beta(3)-adrenoceptor protein increased by 125%. Ventricular homogenates from diabetic rats bound 52% less [(3)H]dihydroalprenolol, consistent with reductions in beta(1)- and beta(2)-adrenoceptors. Three weeks of ExT initiated 4 wk after the onset of diabetes minimized cardiac function loss. ExT also blunted loss of beta(1)-adrenoceptor expression. Interestingly, ExT did not prevent diabetes-induced reduction in beta(2)-adrenoceptor or the increase of beta(3)-adrenoceptor expression. ExT also increased [(3)H]dihydroalprenolol binding, consistent with increased beta(1)-adrenoceptor expression. These findings demonstrate for the first time that ExT initiated after the onset of diabetes blunts primarily beta(1)-adrenoceptor expression loss, providing mechanistic insights for exercise-induced improvements in cardiac function.     

Rat brown adipose tissue thermogenic features are altered during mid-pregnancy.

Brown adipose tissue (BAT) thermogenesis is inhibited during late-pregnancy and lactation in the rat. However, scarce information concerning BAT functionality during mid-pregnancy is available. The aim of this work was to investigate uncoupling proteins and leptin expression during placentation in rat BAT as well as other key parameters in the thermogenic function of the tissue. BAT mitochondrial content was found to be reduced 50% in 11 and 13 day pregnant rats as compared to nonpregnant controls, although uncoupling protein 1 (UCP1) content was not modified. Furthermore, UCP3 mRNA levels were found to be highly increased during this period. beta3-adrenergic receptor (beta3-AR) decreased expression resulted in a higher alpha2/beta3 ratio. Finally, leptin mRNA levels in BAT were found to be 3-fold up-regulated in pregnant animals. In conclusion, we show the existence of profound changes in thermogenic features in BAT during gestational days 11 and 13, pointing to the importance of this tissue during mid-pregnancy.     

Central leptin regulates the UCP1 and ob genes in brown and white adipose tissue via different beta-adrenoceptor subtypes

The three known subtypes of beta-adrenoreceptors (beta(1)-AR, beta(2)-AR, and beta(3)-AR) are differentially expressed in brown and white adipose tissue and mediate peripheral responses to central modulation of sympathetic outflow by leptin. To assess the relative roles of the beta-AR subtypes in mediating leptin's effects on adipocyte gene expression, mice with a targeted disruption of the beta(3)-adrenoreceptor gene (beta(3)-AR KO) were treated with vehicle or the beta(1)/beta(2)-AR selective antagonist, propranolol (20 microgram/g body weight/day) prior to intracerebroventricular (ICV) injections of leptin (0.1 microgram/g body weight/day). Leptin produced a 3-fold increase in UCP1 mRNA in brown adipose tissue of wild type (FVB/NJ) and beta(3)-AR KO mice. The response was unaltered by propranolol in wild type mice, but was completely blocked by this antagonist in beta(3)-AR KO mice. In contrast, ICV leptin had no effect on leptin mRNA in either epididymal or retroperitoneal white adipose tissue (WAT) from beta(3)-AR KOs. Moreover, propranolol did not block the ability of exogenous leptin to reduce leptin mRNA in either WAT depot site of wild type mice. These results demonstrate that the beta(3)-AR is required for leptin-mediated regulation of ob mRNA expression in WAT, but is interchangeable with the beta(1)/beta(2)-ARs in mediating leptin's effect on UCP1 mRNA expression in brown adipose tissue.     

Up-regulation of uncoupling proteins by beta-adrenergic stimulation in L6 myotubes

Catecholamine-induced and beta-adrenergic receptor (beta-AR)-mediated thermogenesis in skeletal muscle is a significant component of whole-body energy expenditure. Skeletal muscle expresses uncoupling protein (UCP) 2 and UCP3, which can dissipate the transmitochondrial electrochemical gradient and thereby may be involved in regulation of energy metabolism. We investigated the effects of beta-AR stimulation on UCP2 and UCP3 expression in L6 myotubes. Stimulation of the cells with epinephrine increased the UCP3 mRNA level transiently at 6 h, and also the UCP2 mRNA level at 6-24 h. The stimulatory effects of epinephrine were also observed in the presence of carbacyclin and 9-cis retinoic acid, and mimicked by isoproterenol and salbutamol (beta2-AR agonists), but abolished by propranolol and ICI-118,551 (beta2-AR antagonists). Pharmacological and mRNA analyses revealed the existence of beta2-AR, but not beta1- and beta3-ARs, in L6 myotubes. These results suggested that catecholamines up-regulate UCP2 and UCP3 expression through direct action on the beta2-AR in skeletal muscle.     

Acute central infusion of leptin modulates fatty acid mobilization by affecting lipolysis and mRNA expression for uncoupling proteins

Chronic administration of leptin has been shown to reduce adiposity through energy intake and expenditure. The present study aims to examine how acute central infusion of leptin regulates peripheral lipid metabolism, as assessed by markers indicative of their mobilization and utilization. A bolus infusion of 1 microg/rat leptin into the third cerebroventricle increased the expression of mRNA for hormone-sensitive lipase (HSL), an indicator of lipolysis, in white adipose tissue (WAT). This was accompanied by elevation of plasma levels of glycerol, but not of free fatty acids, as compared to the saline control (P < 0.03). The same treatment with leptin decreased plasma insulin levels but did not affect the plasma glucose level (P < 0.05 for insulin). Among the major regulators of the transportation or utilization of energy substrates, leptin treatment increased expression of mRNA for uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), UCP2 in WAT, and UCP3 in quadriceps skeletal muscle, but not those for fatty acid-binding protein in WAT, carnitine phosphate transferase-1, a marker for beta oxidation of fatty acids in muscle, nor glucose transporter 4 in WAT and muscle (P < 0.01 for HSL, P < 0.05 for UCP1, and P < 0.005 for UCP2 and UCP3). These results indicate that, even in a single bolus, leptin may regulate the mobilization and/or utilization of energy substrates such as fatty acids by affecting lipolytic activity in WAT and by increasing the expression of UCPs in BAT, WAT, and muscle.     

4E-BP3 regulates eIF4E-mediated nuclear mRNA export and interacts with replication protein A2

In nucleus, eIF4E regulates the nucleus export of specific mRNA. In this study, altered 4E-BP3 (eIF4E-binding protein 3) expression resulted in profoundly affected cyclin D1 protein levels, partially due to changes in the cytoplasmic cyclin D1 mRNA levels in both U2OS and MCF7 cells, whereas altered 4E-BP1 expression did not affect eIF4E-mediated cyclin D1 mRNA export. 4E-BP3 also affected a subset of growth promoting mRNAs exported in an eIF4-dependent manner. Furthermore, 4E-BP3 interacted with dephosphorylated RPA2 (replication protein A2). The results indicated 4E-BP3 acts as an inhibitor of eIF4E-mediated mRNA export in the examined cells, and 4E-BP3 inhibition of eIF4E-mediated mRNA export is regulated by the phosphorylation state of RPA2.