Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity,weight loss,and regulation by insulin and glucocorticoids.

The peroxisome proliferator activated receptor (PPAR gamma) plays a key role in adipogenesis and adipocyte gene expression and is the receptor for the thiazolidinedione class of insulin-sensitizing drugs. The tissue expression and potential for regulation of human PPAR gamma gene expression in vivo are unknown. We have cloned a partial human PPAR gamma cDNA, and established an RNase protection assay that permits simultaneous measurements of both PPAR gamma1 and PPAR gamma2 splice variants. Both gamma1 and gamma2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma1 was detected at lower levels in liver and heart, whereas both gamma1 and gamma2 mRNAs were expressed at low levels in skeletal muscle. To examine the hypothesis that obesity is associated with abnormal adipose tissue expression of PPAR gamma, we quantitated PPARgamma mRNA splice variants in subcutaneous adipose tissue of 14 lean and 24 obese subjects. Adipose expression of PPARgamma 2 mRNA was increased in human obesity (14.25 attomol PPAR gamma2/18S in obese females vs 9.9 in lean, P = 0.003). This increase was observed in both male and females. In contrast, no differences were observed in PPAR gamma1/18S mRNA expression. There was a strong positive correlation (r = 0.70, P < 0.001) between the ratio of PPAR gamma2/gamma1 and the body mass index of these patients. We also observed sexually dimorphic expression with increased expression of both PPAR gamma1 and PPAR gamma2 mRNAs in the subcutaneous adipose tissue of women compared with men. To determine the effect of weight loss on PPAR gamma mRNA expression, seven additional obese subjects were fed a low calorie diet (800 Kcal) until 10% weight loss was achieved. Mean expression of adipose PPAR gamma2 mRNA fell 25% (P = 0.0250 after a 10% reduction in body weight), but then increased to pretreatment levels after 4 wk of weight maintenance. Nutritional regulation of PPAR gamma1 was not seen. In vitro experiments revealed a synergistic effect of insulin and corticosteroids to induce PPAR gamma expression in isolated human adipocytes in culture. We conclude that: (a) human PPAR gamma mRNA expression is most abundant in adipose tissue, but lower level expression of both splice variants is seen in skeletal muscle; to an extent that is unlikely to be due to adipose contamination. (b) RNA derived from adipose tissue of obese humans has increased expression of PPAR gamma 2 mRNA, as well as an increased ratio of PPAR gamma2/gamma1 splice variants that is proportional to the BMI; (c) a low calorie diet specifically down-regulates the expression of PPAR gamma2 mRNA in adipose tissue of obese humans; (d) insulin and corticosteroids synergistically induce PPAR gamma mRNA after in vitro exposure to isolated human adipocytes; and (e) the in vivo modulation of PPAR gamma2 mRNA levels is an additional level of regulation for the control of adipocyte development and function, and could provide a molecular mechanism for alterations in adipocyte number and function in obesity.     

Subcutaneous adipose tissue expression of tumour necrosis factor-alpha is not associated with whole body insulin resistance in obese nondiabetic or in type-2 diabetic subjects

BACKGROUND: An association with subcutaneous adipose tissue TNFalpha expression and insulin resistance has been suggested in obesity/type-2 diabetes, but this has not been examined directly. In the first part of the study we investigated whether this association is present in 7 lean, 10 obese nondiabetic and 9 type-2 diabetic men. In the second part of the study we examined the relationship between adipose tissue TNFalpha mRNA levels and BMI in 81 nondiabetic subjects spanning a wide range of BMIs. METHODS: Subcutaneous adipose tissue TNFalpha mRNA levels and insulin sensitivity were determined with quantitative RT-competitive PCR and hyperinsulinaemic clamp, respectively. RESULTS: Subcutaneous adipose tissue TNFalpha mRNA levels were similar in 7 lean and 10 obese nondiabetic and 9 type-2 diabetic men (P = 0.68), and did not change in response to 240-min hyperinsulinaemia. TNFalpha mRNA levels and insulin sensitivity were not correlated. Unexpectedly, no correlation between TNFalpha mRNA and BMI was found. The relationship between adipose tissue TNFalpha mRNA and BMI was examined further in 31 male and 50 female nondiabetic subjects. The subcutaneous adipose tissue TNFalpha mRNA level correlated with BMI in all subjects (rS = 0.32, P < 0.01), and in a subgroup analysis in men (rS = 0.55, P < 0.01) but not in women (rS = - 0.08). The correlation in men was dependent on a fourfold higher TNFalpha mRNA level in 5 morbidly obese men while there was no difference in TNFalpha mRNA levels in lean or obese men. CONCLUSIONS: Subcutaneous adipose tissue TNFalpha expression does not correlate with insulin sensitivity in nondiabetic or type-2 diabetic men; is not regulated by acute hyperinsulinaemia; and is increased only in morbidly obese men.     

Regulation of PPAR gamma gene expression by nutrition and obesity in rodents.

The orphan nuclear receptor, peroxisome proliferator-activated receptor (PPAR) gamma, is implicated in mediating expression of fat-specific genes and in activating the program of adipocyte differentiation. The potential for regulation of PPAR gamma gene expression in vivo is unknown. We cloned a partial mouse PPAR gamma cDNA and developed an RNase protection assay that permits simultaneous quantitation of mRNAs for both gamma l and gamma 2 isoforms encoded by the PPAR gamma gene. Probes for detection of adipocyte P2, the obese gene product, leptin, and 18S mRNAs were also employed. Both gamma l and gamma 2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma 1 expression was also detected at lower levels in liver, spleen, and heart; whereas, gamma l and gamma 2 mRNA were expressed at low levels in skeletal muscle. Adipose tissue levels of gamma l and gamma 2 were not altered in two murine models of obesity (gold thioglucose and ob/ob), but were modestly increased in mice with toxigene-induced brown fat ablation uncoupling protein diphtheria toxin A mice. Fasting (12-48 h) was associated with an 80% fall in PPAR gamma 2 and a 50% fall in PPAR gamma mRNA levels in adipose tissue. Western blot analysis demonstrated a marked effect of fasting to reduce PPAR gamma protein levels in adipose tissue. Similar effects of fasting on PPAR gamma mRNAs were noted in all three models of obesity. Insulin-deficient (streptozotocin) diabetes suppressed adipose tissue gamma l and gamma 2 expression by 75% in normal mice with partial restoration during insulin treatment. Levels of adipose tissue PPAR gamma 2 mRNA were increased by 50% in normal mice exposed to a high fat diet. In obese uncoupling protein diphtheria toxin A mice, high fat feeding resulted in de novo induction of PPAR gamma 2 expression in liver. We conclude (a) PPAR gamma 2 mRNA expression is most abundant in adipocytes in normal mice, but lower level expression is seen in skeletal muscle; (b) expression of adipose tissue gamma1 or gamma2 mRNAs is increased in only one of the three models of obesity; (c) PPAR gamma 1 and gamma 2 expression is downregulated by fasting and insulin-deficient diabetes; and (d) exposure of mice to a high fat diet increases adipose tissue expression of PPAR gamma (in normal mice) and induces PPAR gamma 2 mRNA expression in liver (in obese mice). These findings demonstrate in vivo modulation of PPAR gamma mRNA levels over a fourfold range and provide an additional level of regulation for the control of adipocyte development and function.     

Differential regulation of adipose tissue glucose transporters in genetic obesity (fatty rat). Selective increase in the adipose cell/muscle glucose transporter (GLUT 4) expression.

Adipocytes from young obese Zucker rats exhibit a hyperresponsive insulin-mediated glucose transport, together with a marked increase in cytochalasin B binding as compared with lean rat adipocytes. Here, we examined in these cells the expression of two isoforms of glucose transporter, the erythroid (GLUT 1) and the adipose cell/muscle (GLUT 4) types, in rats aged 16 or 30 d, i.e., before and after the emergence of hyperinsulinemia. GLUT 1 protein and mRNA levels were identical in the two genotypes at both ages. In contrast, the levels of GLUT 4 protein in obese rat adipocytes were 2.4- and 4.5-fold those of lean littermates at 16 and 30 d of age, respectively, in perfect agreement with the genotype effect on insulin-stimulated glucose transport activity. The levels of GLUT 4 mRNA per fat pad were increased 2.3- and 6.2-fold in obese vs. lean rats 16- and 30-d-old, indicating a pretranslational level of regulation. The obese phenotype was not associated with overexpression of GLUT 4 mRNA in gastrocnemius muscle. This work indicates that the fa gene exerts a differential control on the expression of GLUT 1 and GLUT 4 in adipose tissue and provides evidence that independent of hyperinsulinemia, genotype is a major regulatory factor of GLUT 4 expression in this tissue.     

Leptin secretion from adipose tissue in women. Relationship to plasma levels and gene expression.

The role of expression and secretion of the ob gene product, leptin, for the regulation of plasma leptin levels has been investigated in vitro using abdominal subcutaneous adipose tissue of 20 obese, otherwise healthy, and 11 nonobese women. Body mass index (BMI, mean+/-SEM; kg/m2) in the two groups was 41+/-2 and 23+/-1, respectively. Fat cell volume was 815+/-55 pl in the obese and 320+/-46 pl in the nonobese group. In the obese group, plasma leptin concentrations and adipose leptin mRNA (relative to gamma actin) were increased five and two times, respectively. Moreover, adipose tissue secretion rates per gram lipid weight or per fat cell number were also increased two and seven times, respectively, in the obese group. There were strong linear correlations (r = 0.6-0.8) between plasma leptin, leptin secretion, and leptin mRNA. All of these leptin measurements correlated strongly with BMI and fat cell volume (r = 0.7- 0.9). About 60% of the variation in plasma leptin could be attributed to variations in leptin secretion rate, BMI, or fat cell volume. We conclude that elevated circulating levels of leptin in obese women above all result from accelerated secretion rates of the peptide from adipose tissue because of increased ob gene expression. However, leptin mRNA, leptin secretion, and circulating leptin levels are all more closely related to the stored amount of lipids in the fat cells of adipose tissue than they are to an arbitrary division into obese versus nonobese.     

Induction of rat uncoupling protein-2 gene treated with tumour necrosis factor alpha in vivo

BACKGROUND: The cytokine tumour necrosis factor (TNF) alpha has been reported to induce metabolic abnormalities such as anorexia and thermogenesis. To investigate functional modulators of uncoupling protein-2 (UCP2) gene expression, we examined the effects of TNF-alpha on UCP2 mRNA expression in rats. METHODS: Mature male Wistar King A (WKA) rats at 10-11 weeks of age were treated with recombinant human TNF-alpha at a dose of 0.6 nmol 100 g-1 body weight by intraperitoneal administration. RESULTS: TNF-alpha treatment induced an increase in UCP2 mRNA expression in broadly distributed tissues including brown adipose tissue (BAT), white adipose tissue (WAT) and skeletal muscle, and an elevation of ob gene mRNA expression in WAT. After the TNF-alpha treatment, an increase in plasma leptin concentration occurred in an ob gene-dependent manner, accompanied by an anorectic effect. CONCLUSION: The present results provide evidence for a new regulatory loop involving TNF-alpha and UCP2, and add novel insights into the regulatory mechanisms of energy homeostasis.     

Impaired insulin-induced sympathetic neural activation and vasodilation in skeletal muscle in obese humans.

The sympathetic nervous system is an important regulatory mechanism of both metabolic and cardiovascular function, and altered sympathetic activity may play a role in the etiology and/or complications of obesity. In lean subjects, insulin evokes sympathetic activation and vasodilation in skeletal muscle. In obese subjects such vasodilation is impaired and, in turn, may contribute to insulin resistance. To examine the relationship between sympathetic and vasodilatory responses in skeletal muscle to hyperinsulinemia, we simultaneously measured muscle sympathetic nerve activity (MSNA) and calf blood flow at basal and during a 2-h hyperinsulinemic (6 pmol/kg per min) euglycemic clamp in eight lean and eight obese subjects. The major findings of this study are twofold: obese subjects had a 2.2 times higher fasting rate of MSNA, and euglycemic hyperinsulinemia, which more than doubled MSNA and increased calf blood flow by roughly 30% in lean subjects, had only a minor vasodilatory and sympathoexcitatory effect in obese subjects. In contrast, two non-insulin-sympathetic stimuli evoked comparably large increases in MSNA in lean and obese subjects. We conclude that insulin resistance in obese subjects is associated with increased fasting MSNA and a specific impairment of sympathetic neural responsiveness to physiological hyperinsulinemia in skeletal muscle tissue.     

Glucose transporter levels in spontaneously obese (db/db) insulin-resistant mice.

In the present study we examined mRNA and protein levels for the muscle/adipose tissue glucose transporter (GLUT-4) in various tissues of spontaneously obese mice (C57BL/KsJ, db/db) and their lean littermates (db/+). Obese (db/db) mice were studied at 5 wk of age, when they were rapidly gaining weight and were severely insulin resistant, evidenced by hyperglycemia (plasma glucose 683 +/- 60 vs. 169 +/- 4 mg/dl in db/+, P less than 0.05) and hyperinsulinemia (plasma insulin 14.9 +/- 0.53 vs. 1.52 +/- 0.08 ng/ml in db/+, P less than 0.05). The GLUT-4 mRNA was reduced in quadriceps muscle (67.5 +/- 8.5%, P = 0.02), but unaltered in adipose tissue (120 +/- 19%, NS), heart (95.7 +/- 6.1%, NS), or diaphragm (75.2 +/- 12.1%, NS) in obese (db/db) mice relative to levels in lean littermates. The GLUT-4 protein, measured by quantitative immunoblot analysis using two different GLUT-4 specific antibodies, was not different in five insulin-sensitive tissues including diaphragm, heart, red and white quadriceps muscle, and adipose tissue of obese (db/db) mice compared with tissue levels in lean littermates; these findings were consistent when measured relative to tissue DNA levels as an index of cell number. These data suggest that the marked defect in glucose utilization previously described in skeletal muscle of these young obese mice is not due to a decrease in the level of the major muscle glucose transporter. An alternate step in insulin-dependent activation of the glucose transport process is probably involved.     

Increased adipose tissue expression of tumor necrosis factor-alpha in human obesity and insulin resistance.

Obesity is frequently associated with insulin resistance and abnormal glucose homeostasis. Recent studies in animal models have indicated that TNF-alpha plays an important role in mediating the insulin resistance of obesity through its overexpression in fat tissue. However, the mechanisms linking obesity to insulin resistance and diabetes in humans remain largely unknown. In this study we examined the expression pattern of TNF-alpha mRNA in adipose tissues from 18 control and 19 obese premenopausal women by Northern blot analysis. TNF-alpha protein concentrations in plasma and in conditioned medium of explanted adipose tissue were measured by ELISA. Furthermore, the effects of weight reduction by dietary treatment of obesity on the adipose expression of TNF-alpha mRNA were also analyzed in nine premenopausal obese women, before and after a controlled weight-reduction program. These studies demonstrated that obese individuals express 2.5-fold more TNF-alpha mRNA in fat tissue relative to the lean controls (P < 0.001). Similar increases were also observed in adipose production of TNF-alpha protein but circulating TNF-alpha levels were extremely low or undetectable. A strong positive correlation was observed between TNF-alpha mRNA expression levels in fat tissue and the level of hyperinsulinemia (P < 0.001), an indirect measure of insulin resistance. Finally, body weight reduction in obese subjects which resulted in improved insulin sensitivity was also associated with a decrease in TNF-alpha mRNA expression (45%, P < 0.001) in fat tissue. These results suggest a role for the abnormal regulation of this cytokine in the pathogenesis of obesity-related insulin resistance.     

Acute insulin administration does not affect plasma leptin levels in lean or obese subjects

Abstract. Whether leptin levels are related to insulin sensitivity or subject to acute regulation by insulin is not known. In 12 obese [body mass index (BMI) = 34.0 ±1.5 kg m^-2] and 12 lean (BMI = 22.2 ±0.6 kg m^-2) non-diabetic subjects, plasma leptin concentrations were measured in the fasting state and during 2 hours of euglycaemic hyperinsulinaemia (˜600 pmol L^-2). Fasting plasma leptin was significantly higher in obese (26.6 ±3.2) than in lean subjects (6.4 ±1.2 ng mL^-1, P = 0.0001), and in women (21.1 ±3.3) than in men (7.3 = 2.3 ng mL^-1, P = 0.01). In univariate analysis, fasting plasma leptin was strongly related to all anthropometric measures (body weight, fat mass, percent fat mass, waist and hip circumferences). In multiple regression, per cent adiposity, hip circumference and duration of obesity explained 90% of the variability in fasting leptin concentrations. Fasting and stimulated (OGTT) insulin levels, insulin sensitivity (22.6 ±1.9 vs 36.7 ±2.0 μmol min^-1 kg^-1 in lean and obese subjects, respectively, P < 0.0001), glucose area, and serum triglycerides were positively related to fasting plasma leptin concentrations; none of these associations, however, was statistically significant after adjusting for BMI. During the clamp, plasma leptin concentrations remained constant in both lean and obese subjects. We conclude that neither insulin levels nor sensitivity relate to leptin levels independently of fat mass, and that leptin is not subject to acute (2 hours) regulation by insulin in lean or obese humans.     

Visfatin expression in subcutaneous adipose tissue of pre-menopausal women: relation to hormones and weight reduction

Background A novel adipokine, visfatin, was found to be related to adiposity in humans and regulated by a number of hormonal signals. The aim of this study was to investigate the relationships of visfatin expression in adipose tissue with potential regulatory factors such as insulin, testosterone and tumor necrosis factor‐α (TNF‐α) and to elucidate the effect of a diet induced weight reduction on adipose tissue mRNA expression and plasma levels of visfatin. Materials and methods Biopsies of subcutaneous abdominal adipose tissue (SCAAT) and plasma samples were obtained at the beginning of the study from 47 pre‐menopausal women (age 38·7 ± 1·7 years, body mass index (BMI) 27·9 ± 1·4 kg m^?2), consisting of 15 lean, 16 overweight and 16 obese subjects. The subgroup of 32 overweight/obese women (age 42·1 ± 1·9 years, BMI 31·2 ± 0·9 kg m^?2) underwent a 12 week hypocaloric weight reducing diet and samples were obtained at the end of the diet. Biopsy samples were analysed for visfatin and TNF‐α mRNA levels and plasma was analysed for relevant metabolites and hormones. Results In the group of 47 subjects visfatin mRNA expression in SCAAT was negatively correlated with plasma free testosterone (r = –0. 363, P < 0·05) and BMI (r = –0·558, P < 0·01) and positively associated with adipose tissue TNF‐α mRNA expression (r = 0·688, P < 0·01). The diet resulted in the reduction of body weight and in the decrease of plasma insulin, free testosterone and TNF‐α levels. In the group of overweight/obese subjects visfatin mRNA in SCAAT increased after the diet and the diet induced increase was positively correlated with the magnitude of body weight loss. Conclusion Visfatin mRNA expression in SCAAT is associated with TNF‐α expression, plasma free testosterone and BMI in pre‐menopausal women. A weight reducing hypocaloric diet results in the increase of visfatin mRNA in SCAAT.     

Lactate release from the subcutaneous tissue in lean and obese men.

Lactate concentration in the subcutaneous interstitial fluid and adipose tissue blood flow (ATBF, ml/100 g.min) were simultaneously measured with the microdialysis technique combined with 133Xe clearance in the abdominal and femoral subcutaneous adipose tissue in nine lean and nine obese men. The studies were performed both in the postabsorptive state and 2 h after an oral glucose load and the results compared to the lactate levels in arterialized venous plasma. After an overnight's fast, arterial lactate was 738 +/- 49 and 894 +/- 69 microM (mean +/- SE) (P < 0.05) in the lean and obese subjects, respectively. The interstitial lactate levels were significantly higher than blood lactate in both subject groups without any regional differences. Abdominal and femoral ATBF was 3.2 +/- 0.6 vs. 2.8 +/- 0.4 and 1.7 +/- 0.3 vs. 2.4 +/- 0.4 ml/100 g.min (P < 0.05) in lean and obese subjects, respectively. Mean apparent lactate release from the abdominal vs. femoral adipose tissue in the fasting state was 10.5 +/- 3.1 vs. 8.6 +/- 2.3 and 6.0 +/- 2.3 vs. 8.5 +/- 2.3 mumol/kg.min (NS) in lean and obese subjects, respectively. Both plasma and interstitial lactate levels increased significantly after an oral glucose load in both subject groups. However, apparent lactate release increased significantly only in the lean group. It is concluded that subcutaneous adipose tissue is a significant source of whole-body lactate release in the postabsorptive state and that this is further enhanced in obese subjects due to their large adipose mass.     

Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes.

Obese human subjects have increased protein-tyrosine phosphatase (PTPase) activity in adipose tissue that can dephosphorylate and inactivate the insulin receptor kinase. To extend these findings to skeletal muscle, we measured PTPase activity in the skeletal muscle particulate fraction and cytosol from a series of lean controls, insulin-resistant obese (body mass index > 30) nondiabetic subjects, and obese individuals with non-insulin-dependent diabetes. PTPase activities in subcellular fractions from the nondiabetic obese subjects were increased to 140-170% of the level in lean controls (P < 0.05). In contrast, PTPase activity in both fractions from the obese subjects with non-insulin-dependent diabetes was significantly decreased to 39% of the level in controls (P < 0.05). By immunoblot analysis, leukocyte antigen related (LAR) and protein-tyrosine phosphatase 1B had the greatest increase (threefold) in the particulate fraction from obese, nondiabetic subjects, and immunodepletion of this fraction using an affinity-purified antibody directed at the cytoplasmic domain of leukocyte antigen related normalized the PTPase activity when compared to the activity from control subjects. These findings provide further support for negative regulation of insulin action by specific PTPases in the pathogenesis of insulin resistance in human obesity, while other regulatory mechanisms may be operative in the diabetic state.     

肥胖者网膜脂肪组织中叉头状转录因子O1、葡萄糖转运体4mRNA的表达及其与胰岛素抵抗相关性的研究

目的观察肥胖者网膜脂肪组织中叉头状转录因子O1（FOXO1）、葡萄糖转运体4（GLUT4）mRNA表达,探讨FOXO1在肥胖和胰岛素抵抗发生中的作用。方法聚集15例肥胖者和17例非肥胖者的网膜脂肪组织应用半定量反转录聚合酶链反应（RT—PCR）测定FOXO1、GLUT4mRNA表达,并测定其他临床指标,分析各指标之间的相关性及与胰岛素敏感性的关系。结果肥胖者FOXO1 mRNA的表达显著高于非肥胖对照组,（0．577±0．038VS0．359±0．023）（P〈0．01）,GLUT4mRNA的表达明显低于非肥胖对照组,（0．386±0．037VS0．646±0．034）（P〈0．01）;网膜脂肪组织FOXO1 mRNA的表达与体质量指数（BMI）、腰臀比（WHR）、空腹胰岛素（FINs）、胰岛素抵抗指数（HOMA—IR）、甘油三酯（TG）的表达呈正相关（r=0．963、0．939、0．974、0．924、0．600,均P〈0．01）,与GLUT4 mRNA的表达呈负相关（r=0．866,P〈0．01）,多元逐步回归分析示BMI、HOMA—IR、GLUT4mRNA为FOXO1mRNA的独立相关因素。结论肥胖者的网膜脂肪组织中的FOXO1表达明显增加,FOXO1可能是肥胖和胰岛素抵抗的联系者,可能是通过减少GLUT4的表达引起肥胖者胰岛素抵抗的。