The effect of mild cold exposure on UCP3 mRNA expression and UCP3 protein content in humans

OBJECTIVE: In rodents, adaptive thermogenesis in response to cold exposure and high-fat feeding is accomplished by the activation of the brown adipose tissue specific mitochondrial uncoupling protein, UCP1. The recently discovered human uncoupling protein 3 is a possible candidate for adaptive thermogenesis in humans. In the present study we examined the effect of mild cold exposure on the mRNA and protein expression of UCP3. SUBJECTS: Ten healthy male volunteers (age 24.4 +/- 1.6 y; height 1.83 +/- 0.02 m; weight 77.3 +/- 3.0 kg; percentage body fat 19 +/- 2). DESIGN: Subjects stayed twice in the respiration chamber for 60 h (20.00-8.00 h); once at 22 degrees C (72 degrees F), and once at 16 degrees C (61 degrees F). After leaving the respiration chamber, muscle biopsies were taken and RT-competitive-PCR and Western blotting was used to measure UCP3 mRNA and protein expression respectively. RESULTS: Twenty-four-hour energy expenditure was significantly increased at 16 degrees C compared to 22 degrees C (P<0.05). At 16 degrees C, UCP3T (4.6 +/- 1.0 vs 7.7 +/- 1.5 amol/microg RNA, P=0.07), UCP3L (2.0 +/- 0.5 vs 3.5 +/- 0.9 amol/microg RNA, P=0.1) and UCP3S (2.6 +/- 0.6 vs 4.2 +/- 0.7 amol/microg RNA, P=0.07) mRNA expression tended to be lower compared with at 22 degrees C, whereas UCP3 protein content was, on average, not different. However, the individual differences in UCP3 protein content (16-22 degrees C) correlated positively with the differences in 24 h energy expenditure (r=0.86, P<0.05). CONCLUSION: The present study suggests that UCP3 protein content is related to energy metabolism in humans and might help in the metabolic adaptation to cold exposure. However, the down-regulation of UCP3 mRNA with mild cold exposure suggests that prolonged cold exposure will lead to lower UCP3 protein content. What the function of such down-regulation of UCP3 could be is presently unknown.     

Expression of insulin target genes in skeletal muscle and adipose tissue in adult patients with growth hormone deficiency: effect of one year recombinant human growth hormone therapy.

Our aim was to investigate the effects of one year recombinant human growth hormone (rhGH) therapy on the regulation by insulin of gene expression in muscle and adipose tissue in adults with secondary GH deficiency (GHD). Six GHD subjects without upper-body obesity were submitted to a 3-h euglycemic hyperinsulinemic clamp before and after one year of rhGH therapy. Muscle and abdominal subcutaneous adipose tissue biopsies were taken before and at the end of each clamp. The mRNA levels of insulin receptor, p85 alpha-phosphatidylinositol-3 kinase (p85 alpha PI-3K), insulin dependent glucose transporter (Glut4), hexokinase II, glycogen synthase, lipoprotein lipase (LPL) in muscle and in adipose tissue, hormone sensitive lipase and peroxisome proliferator-activated receptor gamma (PPAR gamma) in adipose tissue were quantified by RT-competitive PCR. One year treatment with rhGH (1.25 IU/day) increased plasma IGF-I concentrations (54+/-7 vs 154+/-11 ng/ml, P<0.01) but did not affect insulin-stimulated glucose disposal rate measured during the hyperinsulinemic clamp (74+/-9 vs 85+/-5 micromol/kg free fat mass/min). Insulin significantly increased p85 alpha PI-3K, hexokinase II and Glut4 mRNA levels in muscle both before and after rhGH treatment. One year of GH therapy increased LPL mRNA levels in muscle (38+/-2 vs 70+/-7 amol/microg total RNA, P<0.05) and in adipose tissue (2490+/-260 vs 4860+/-880 amol/microg total RNA, P<0.05), but did not change the expression of the other mRNAs. We conclude from this study that GH therapy did not alter whole body insulin sensitivity and the response of gene expression to insulin in skeletal muscle of adult GHD patients, but it did increase LPL expression in muscle and adipose tissue. This result could be related to the documented beneficial effect of GH therapy on lipid metabolism.     

NO-1886 (ibrolipim), a lipoprotein lipase activator, increases the expression of uncoupling protein 3 in skeletal muscle and suppresses fat accumulation in high-fat diet-induced obesity in rats

Although the lipoprotein lipase (LPL) activator NO-1886 shows antiobesity effects in high-fat-induced obese animals, the mechanism remains unclear. To clarify the mechanism, we studied the effects of NO-1886 on the expression of uncoupling protein (UCP) 1, UCP2, and UCP3 in rats. NO-1886 was mixed with a high-fat chow to supply a dose of 100 mg/kg to 8-month-old male Sprague-Dawley rats. The animals were fed the high-fat chow for 8 weeks. At the end of the administration period, brown adipose tissue (BAT), mesenteric fat, and soleus muscle were collected and levels of UCP1, UCP2, and UCP3 messenger RNA (mRNA) were determined. NO-1886 suppressed the body weight increase seen in the high-fat control group after the 8-week administration (585 +/- 39 vs 657 +/- 66 g, P < .05). NO-1886 also suppressed fat accumulation in visceral (46.9 +/- 10.4 vs 73.7 +/- 14.5 g, P < .01) and subcutaneous (43.1 +/- 18.1 vs 68.9 +/- 18.8 g, P < .05) tissues and increased the levels of plasma total cholesterol and high-density lipoprotein cholesterol in comparison to the high-fat control group. In contrast, NO-1886 decreased the levels of plasma triglycerides, nonesterified free fatty acid, glucose, and insulin. NO-1886 increased LPL activity in soleus muscle (0.082 +/- 0.013 vs 0.061 +/- 0.016 mumol of free fatty acid per minute per gram of tissue, P < .05). NO-1886 increased the expression of UCP3 mRNA in soleus muscle 3.14-fold (P < .01) compared with the high-fat control group without affecting the levels of UCP3 in mesenteric adipose tissue and BAT. In addition, NO-1886 did not affect the expression of UCP1 and UCP2 in BAT, mesenteric adipose tissue, and soleus muscle. In conclusion, NO-1886 increased the expression of UCP3 mRNA and LPL activity only in skeletal muscle. Therefore, a possible mechanism for NO-1886's antiobesity effects in rats may be the enhancement of LPL activity in skeletal muscle and the accompanying increase in UCP3 expression.     

Regulation of uncoupling protein-2 and -3 by growth hormone in skeletal muscle and adipose tissue in growth hormone-deficient adults

The newly described uncoupling proteins, UCP2 and UCP3, may play a role in regulating energy expenditure (EE) in humans. GH deficiency (GHD) is associated with decreased lean body mass, increased adiposity, and reduced EE, which are reversed by GH treatment. In the present study we investigated whether GH treatment for 4 months influenced the expression of UCPs in skeletal muscle and adipose tissue in 22 GHD patients who were investigated before and after GH (n = 11) or placebo (n = 11) treatment. GH treatment increased the amount of lean body mass by 4.5% (P < 0.05) and decreased body fat mass by 12% (P < 0.05), whereas no changes in these parameters were observed after placebo treatment. The level of UCP3 messenger ribonucleic acid (mRNA) increased 3-fold (P < 0.005) in skeletal muscle and almost 2-fold (P < 0.05) in adipose tissue after GH treatment, with no changes observed after placebo treatment. Skeletal muscle UCP2 mRNA was slightly (25%), but significantly (P < 0.05), decreased, whereas the level of UCP2 mRNA in adipose tissue was unaffected after GH treatment. The T4 level was positively correlated with skeletal muscle UCP2 and UCP3 expression (r = 0.518; P < 0.05 and r = 0.463; P < 0.05, respectively). Furthermore, plasma free fatty acids were positively correlated with the expression of UCP2 (r = 0.573; P < 0.01) and UCP3 (r = 0.518; P < 0.05) in skeletal muscle. The marked increase in UCP3 expression after GH treatment indicates that the UCPs might play a role in the effects of GH on EE in GHD patients. Finally, the strong association between thyroid hormone and skeletal muscle UCP and the correlation between plasma free fatty acids and UCP expression in skeletal muscle indicate that these hormones/metabolites might influence UCP expression in humans as previously demonstrated in rodents.     

Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue.

Exogenous leptin enhances energy utilization in ob/ob mice by binding its hypothalamic receptor and selectively increasing peripheral fat oxidation. Leptin also increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), but the neurotransmitter that mediates this effect has not been established. The present experiments sought to determine whether leptin regulates UCP1 expression in BAT and its own expression in white adipose tissue (WAT) through the long or short forms of leptin receptor and modulation of norepinephrine release. Mice lacking dopamine beta-hydroxylase (Dbh-/-), the enzyme responsible for synthesizing norepinephrine and epinephrine from dopamine, were treated with leptin (20 microg/g body weight/day) for 3 days before they were euthanized. UCP1 messenger RNA (mRNA) and protein expression were 5-fold higher in BAT from control (Dbh+/-) compared with Dbh-/- mice. Leptin produced a 4-fold increase in UCP1 mRNA levels in Dbh+/- mice but had no effect on UCP1 expression in Dbh-/-. The beta3-adrenergic agonist, CL-316,243 increased UCP1 expression and established that BAT from both groups of mice was capable of responding to beta-adrenergic stimulation. Similarly, exogenous leptin reduced leptin mRNA in WAT from Dbh+/- but not Dbh-/- mice. In separate experiments, leptin produced comparable reductions in food intake in both Dbh+/- and Dbh-/- mice, illustrating that norepinephrine is not required for leptin's effect on food intake. Lastly, db/db mice lacking the long form of the leptin receptor failed to increase UCP1 mRNA in response to exogenous leptin but increased UCP1 mRNA in response to CL-316,243. These studies establish that norepinephrine is required for leptin to regulate its own expression in WAT and UCP1 expression in BAT and indicate that these effects are likely mediated through the centrally expressed long form of the leptin receptor.     

Understanding the role of glucocorticoids in obesity: tissue-specific alterations of corticosterone metabolism in obese Zucker rats

The role of glucocorticoids in obesity is poorly understood. Observations in obese men suggest enhanced inactivation of cortisol by 5alpha-reductase and altered reactivation of cortisone to cortisol by 11betahydroxysteroid dehydrogenase type 1 (11betaHSD1). These changes in glucocorticoid metabolism may influence corticosteroid receptor activation and feedback regulation of the hypothalamic-pituitary-adrenal axis (HPA). We have compared corticosterone metabolism in vivo and in vitro in male obese and lean Zucker rats, aged 9 weeks (n = 8/group). Steroids were measured in 72-h urine and 0900 h trunk blood samples. 5alpha-Reductase type 1 and 11betaHSD activities were assessed in dissected tissues. Obese animals were hypercorticosteronemic and excreted more total corticosterone metabolites (2264+/-623 vs. 388+/-144 ng/72 h; P = 0.003), with a greater proportion being 5alpha-reduced or 11-oxidized. 11-Dehydrocorticosterone was also elevated in plasma (73+/-9 vs. 18+/-2 nM; P = 0.001) and urine (408+/-111 vs. <28 ng/72 h; P = 0.01). In liver of obese rats, 5alpha-reductase type 1 activity was greater (20.6+/-2.7% vs. 14.1+/-1.5%; P<0.04), but 11betaHSD1 activity (maximum velocity, 3.43+/-0.56 vs. 6.57+/-1.13 nmol/min/mg protein; P = 0.01) and messenger RNA levels (0.56+/-0.08 vs. 1.03+/-0.15; P = 0.02) were lower. In contrast, in obese rats, 11betaHSD1 activity was not different in skeletal muscle and sc fat and was higher in omental fat(36.4+/-6.2 vs. 19.2+/-6.6; P = 0.01), whereas 11betaHSD2 activity was higher in kidney (16.7+/-0.6% vs. 11.3+/-1.5%; p = 0.01). We conclude that greater inactivation of glucocorticoids by 5alpha-reductase in liver and 11betaHSD2 in kidney combined with impaired reactivation of glucocorticoids by 11betaHSD1 in liver may increase the MCR of glucocorticoids and decrease local glucocorticoid concentrations at these sites. By contrast, enhanced 11betaHSD1 in omental adipose tissue may increase local glucocorticoid receptor activation and promote obesity.     

Growth hormone (GH) substitution in GH-deficient patients inhibits 11beta-hydroxysteroid dehydrogenase type 1 messenger ribonucleic acid expression in adipose tissue

CONTEXT: Local tissue activity of glucocorticoids is in part determined by the isoenzymes 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) and 11beta-HSD2, interconverting inert cortisone and active cortisol. Increased tissue activity of cortisol may play a central role in the features of GH deficiency and the metabolic syndrome. OBJECTIVE: We investigated the effects of GH treatment on adipose tissue 11beta-HSD mRNA. SUBJECTS AND METHODS: A randomized placebo-controlled double-blind study design was used. Twenty-three GH-deficient patients (16 males and seven females) were randomized to 4 months of GH treatment (2 IU/m2) (n = 11) or placebo treatment (n = 12). Adipose tissue biopsies and blood samples were obtained before and after treatment. Biopsies were obtained from the abdominal sc depot at the level of the umbilicus and do not necessarily reflect the metabolically more important visceral adipose tissue. Gene expressions were determined by real-time RT-PCR. RESULTS: GH treatment decreased 11beta-HSD1 mRNA 66% [95% confidence interval (CI), 23-107%; P < 0.01] and increased 11beta-HSD2 mRNA 167% (95% CI, 33-297%; P < 0.05) in adipose tissue. Serum IGF-I and IGF-I mRNA increased in the GH-treated group by 187% (95% CI, 122-250%; P < 0.001) and 470% (95% CI, 88-846%; P < 0.01). The change in 11beta-HSD1 mRNA expression was negatively correlated with the change in serum IGF-I (R = -0.434; P < 0.05). In contrast, the change in 11beta-HSD2 mRNA expression was positively correlated with the change in serum IGF-I (R = 0.487; P < 0.05), and even stronger with the change in IGF-I mRNA expression (R = 0.798; P < 0.0001). CONCLUSION: GH treatment is able to decrease 11beta-HSD1 mRNA and increase 11beta-HSD2 and accordingly may be able to reduce the amount of locally produced cortisol in adipose tissue.     

Expression and activity of 20alpha-hydroxysteroid dehydrogenase (AKR1C1) in abdominal subcutaneous and omental adipose tissue in women

We examined the expression and activity of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) in abdominal adipose tissue in women. This recently characterized enzyme from the aldoketoreductase 1C family is responsible for the conversion of progesterone into 20alpha-hydroxyprogesterone. Abdominal sc (SC) and omental (OM) adipose tissue biopsies were obtained from a sample of 32 women aged 47.7 +/- 5.9 yr (body mass index 27.6 +/- 5.0 kg/m(2)) undergoing abdominal hysterectomies. Body composition and body fat distribution measurements were performed before the surgery by dual-energy x-ray absorptiometry and computed tomography, respectively. The expression of 20alpha-HSD was determined by real-time RT-PCR, and its activity was measured in whole-tissue homogenates. mRNA and activity of the enzyme were detected in both the SC and OM fat depots, the two measures being significantly higher in the SC compartment. Women characterized by a visceral adipose tissue area of 100 cm(2) or greater had an increased 20alpha-HSD conversion rate in their OM adipose tissue, compared with women without visceral obesity (13.99 +/- 2.07 vs. 7.92 +/- 0.83 fmol/microg protein per 24 h, P < 0.05). Accordingly, a positive correlation was found between OM adipose tissue 20alpha-HSD activity and computed tomography-measured visceral adipose tissue area (r = 0.36, P < 0.05). Significant positive correlations were also found between OM 20alpha-HSD activity and OM adipocyte diameter (r = 0.49, P < 0.05) and OM adipose tissue LPL activity (r = 0.36, P = 0.06). In conclusion, 20alpha-HSD activity and mRNA were detected in SC and OM adipose tissue in women, and OM 20alpha-hydroxylation of progesterone was highest in women with visceral obesity. Additional studies are required to establish whether local conversion of progesterone may impact on the metabolism and function of adipocytes located within the abdominal cavity.     

糖皮质激素对哮喘大鼠Clara细胞分泌蛋白表达的影响

目的　观察吸入糖皮质激素对大鼠支气管哮喘 (简称哮喘 )模型肺组织Clara细胞分泌蛋白 (CCSP)mRNA表达的影响。方法　用卵白蛋白 (OVA)致敏、雾化建立大鼠哮喘模型 ,以逆转录 聚合酶链反应 (RT PCR)、斑点免疫印迹法 ,分别检测大鼠激发哮喘 3天后肺组织CCSPmRNA表达水平、支气管肺泡灌洗液 (BALF)CCSP蛋白浓度及雾化吸入布地奈德的影响。结果　哮喘组大鼠肺组织CCSPmRNA表达量为 0 .5 6± 0 .0 5 ,与正常对照组 (0 .6 5± 0 .0 4 )比较差异有显著性 (P <0 .0 1) ;激素治疗组CCSPmRNA表达量为 0 .6 3± 0 .0 4 ,与哮喘组 (0 .5 6± 0 .0 5 )比较差异也有显著性 (P <0 .0 5 )。哮喘组大鼠BALF中CCSP蛋白含量为 4 9± 5 ,与正常对照组 (6 0± 5 )比较 ,差异有显著性 (P <0 .0 1) ;激素治疗组CCSP蛋白含量 (5 7± 5 )与哮喘组比较 (P <0 .0 5 ) ,BALF中嗜酸细胞比例降低 ,气道炎症反应减轻。结论　CCSPmRNA表达减少导致CCSP水平下降 ,从而促进气道炎症而参与哮喘发病。雾化吸入糖皮质激素可增加CCSPmRNA表达 ,抑制哮喘气道炎症。     

Hormonal regulation of human leptin in vivo: effects of hydrocortisone and insulin

OBJECTIVE: To investigate the effects of continuous i.v. infusion of hydrocortisone or insulin on leptin secretion in humans. SUBJECTS: Six, nonfasting healthy adults (four women, two men), aged (mean +/- s.e.m.) 36.6 +/- 1.7 y; body mass index (BMI) 27.6 +/- 0.9 kg/m2. DESIGN: Randomized, placebo-controlled, cross-over study, with a 2-week 'wash-out' period. INTERVENTIONS: Intravenous infusion of hydrocortisone (3.3 microg/(kg min)), insulin (1 mU/(kg min)) or normal saline (placebo) for 24 h. MEASUREMENTS: Blood sampling every 1-2 h for measurement of glucose, insulin, cortisol and leptin; subcutaneous abdominal fat biopsy for determination of leptin mRNA expression. RESULTS: Plasma cortisol increased to 50.0 +/- 0.4 microg/dl during hydrocortisone infusion, but was unaltered during saline or insulin infusion. The plasma insulin levels were: 28.5 +/- 4.7 microU/ml (placebo), 40.8 +/- 9.2 microU/ml (hydrocortisone, P=0.214), and 243 +/- 23.0 microU/ml (insulin, P=0.0002). Peak hyperleptinemia occurred after 16h of insulin and 20h of hydrocortisone infusion; peak/baseline plasma leptin levels (ng/ml) were 18.2 +/- 4.2/15.1 +/- 3.3 (placebo, P=0.056), 42.1 +/- 7.0/16.0 +/- 3.8 (hydrocortisone, + 163%, P= 0.008) and 30.2 +/- 4.3/16.6 +/- 2.7 (insulin, +83%, P= 0.024). Adipocyte leptin mRNA increased by 350% after the hydrocortisone infusion. CONCLUSION: Hydrocortisone, a natural glucocorticoid, induces hyperleptinemia in vivo, with a potency greater than that of insulin. The interaction between glucocorticoids and leptin may be of metabolic significance in humans.     

A polymorphism in the adiponectin gene influences adiponectin expression levels in visceral fat in obese subjects

OBJECTIVE: Reduced serum adiponectin levels have been found in obesity and type 2 diabetes and variations in the adiponectin gene (APM1) have been associated with type 2 diabetes and features of the metabolic syndrome in different populations. STUDY DESIGN: Here, we investigated the expression of APM1 in adipose tissue and studied the relationship between variation in APM1 expression, the APM1 G276T polymorphism, the common PPARG Pro12Ala polymorphism and clinical features of 36 morbidly obese (body mass index (BMI) 41.5 +/- 4.9 kg/m2) nondiabetic subjects. RESULTS: APM1 mRNA expression in visceral fat was correlated with serum adiponectin levels (r = 0.54, P = 0.012). In visceral, but not in subcutaneous, adipose tissue APM1 mRNA level was 38% higher among carriers of the APM1 G276T T allele (G/T and T/T) than among carriers of the G/G genotype (0.91 +/- 0.06 for G/T and T/T carriers vs 0.66 +/- 0.09 for G/G carriers, P = 0.013). Carriers of the T allele also had significantly higher body fat percent compared to G/G carriers (65 +/- 6 vs 56 +/- 10%, P = 0.011).Conclusion:Our results indicate that genetic variation in APM1 influences the expression of the gene in visceral adipose tissue and suggest a potential role for such variation in regulation of body fat accumulation in obese subjects.