The FTO Obesity Gene. Genotyping and Gene Expression Analysis in Morbidly Obese Patients

BACKGROUND: Obesity has emerged as one of the most serious public health concerns in the twenty-first century. the fat mass and obesity associated gene (FTO) has been found to contribute to the risk of obesity in humans. Our aims in this study were to investigate the association of rs9939609 single nucleotide polymorphism (SNP) of the FTO gene with different obesity-related parameters, to assess the FTO gene expression in subcutaneous and visceral adipose tissues from morbidly obese and its correlations with other adipocytokine gene expressions. METHODS: The association between the rs9939609 FTO gene variant and obesity related parameters in 75 obese/morbidly obese adult patients and 180 subjects with body mass index (BMI) < 30 kg/m(2) (control group) was examined. Gene expression analyses: subcutaneous adipose tissue samples were obtained from 52 morbidly obese and five subjects with BMI < 30 kg/m(2). Visceral adipose tissue was also obtained from 35 morbidly obese patients. Weight, height, BMI, SBP, DBP, fasting glucose, lipid profile, proinsulin, insulin, leptin, and adiponectin (RIA) of patients were also obtained. Insulin resistance by HOMA(IR). rs9939609 of FTO genotyping using allele discrimination in real-time PCR. Genomic study of RNA extraction of adipose tissue and real-time PCR (RT-PCR) of adipocytokines and a housekeeping gene were quantified using TaqMan probes. Relative quantification was calculated using the DeltaDelta Ct formula. RESULTS: The minor-(A) allele frequency of rs9939609 FTO gene in the whole population was 0.39. A strong association between this A allele and obesity was found, even after age-sex adjustment (p = 0.013). We found higher levels of FTO mRNA in subcutaneous adipose tissue from morbidly obese than in the control group (p = 0.021). FTO gene expression was lower in visceral than in subcutaneous adipose depot. However, this finding did not reach the level of statistical significance. A negative correlation between subcutaneous FTO gene expression and serum triglyceride levels and a positive correlation with leptin, perilipin, and visfatin gene expressions was found. In the visceral adipose tissue, these positive correlations were statistically significant only for perilipin. CONCLUSIONS: Our results show: (1) A strong association between rs9939609 SNP of the FTO gene variant and obesity in Spanish morbidly obese adult patients; (2) positive correlations between FTO mRNA and leptin, perilipin, and visfatin gene expressions in subcutaneous adipose tissue; (3) FTO and perilipin gene expressions were positively correlated in visceral fat depot. Overall these results may suggest a role of FTO in the regulation of lipolysis as well as in total body fat rather in fat distribution patterns.     

Omentin plasma levels and gene expression are decreased in obesity

Central obesity and the accumulation of visceral fat are risk factors for the development of type 2 diabetes and cardiovascular disease. Omentin is a protein expressed and secreted from visceral but not subcutaneous adipose tissue that increases insulin sensitivity in human adipocytes. To determine the impact of obesity-dependent insulin resistance on the regulation of two omentin isoforms, gene expression and plasma levels were measured in lean, overweight, and obese subjects. Omentin 1 was shown to be the major circulating isoform in human plasma. Lean subjects had significantly higher plasma omentin 1 levels than obese and overweight subjects. In addition, higher plasma omentin 1 levels were detected in women compared with men. Plasma omentin 1 levels were inversely correlated with BMI, waist circumference, leptin levels, and insulin resistance as measured by homeostasis model assessment and positively correlated with adiponectin and HDL levels. Both omentin 1 and omentin 2 gene expression were decreased with obesity and were highly correlated with each other in visceral adipose tissue. In summary, decreased omentin levels are associated with increasing obesity and insulin resistance. Therefore, omentin levels may be predictive of the metabolic consequences or co-morbidities associated with obesity.     

Leptin Modulates Allograft Survival by Favoring a Th2 and a Regulatory Immune Profile

Leptin, an adipose‐secreted hormone, links metabolism and immunity. Our aim was to determine whether leptin affects the alloimmune response. We used an allogeneic skin transplant model as a means to analyze the allograft immune response in Lep^ob/ob and wild‐type mice. Leptin deficiency results in an increased frequency of Treg and Th2 cells and a prolonged graft survival. These effects of leptin deficiency indicate the importance of leptin and obesity in modulating the allograft immune responses. Our data suggest a possible explanation for the increased susceptibility of hyperleptinemic obese patients to acute and chronic graft rejection.     

Adiponectin expression from human adipose tissue: relation to obesity,insulin resistance,and tumor necrosis factor-alpha expression

Adiponectin is a 29-kDa adipocyte protein that has been linked to the insulin resistance of obesity and lipodystrophy. To better understand the regulation of adiponectin expression, we measured plasma adiponectin and adipose tissue adiponectin mRNA levels in nondiabetic subjects with varying degrees of obesity and insulin resistance. Plasma adiponectin and adiponectin mRNA levels were highly correlated with each other (r = 0.80, P < 0.001), and obese subjects expressed significantly lower levels of adiponectin. However, a significant sex difference in adiponectin expression was observed, especially in relatively lean subjects. When men and women with a BMI <30 kg/m(2) were compared, women had a twofold higher percent body fat, yet their plasma adiponectin levels were 65% higher (8.6 +/- 1.1 and 14.2 +/- 1.6 micro g/ml in men and women, respectively; P < 0.02). Plasma adiponectin had a strong association with insulin sensitivity index (S(I)) (r = 0.67, P < 0.0001, n = 51) that was not affected by sex, but no relation with insulin secretion. To separate the effects of obesity (BMI) from S(I), subjects who were discordant for S(I) were matched for BMI, age, and sex. Using this approach, insulin-sensitive subjects demonstrated a twofold higher plasma level of adiponectin (5.6 +/- 0.6 and 11.2 +/- 1.1 micro g/ml in insulin-resistant and insulin-sensitive subjects, respectively; P < 0.0005). Adiponectin expression was not related to plasma levels of leptin or interleukin-6. However, there was a significant inverse correlation between plasma adiponectin and tumor necrosis factor (TNF)-alpha mRNA expression (r = -0.47, P < 0.005), and subjects with the highest levels of adiponectin mRNA expression secreted the lowest levels of TNF-alpha from their adipose tissue in vitro. Thus, adiponectin expression from adipose tissue is higher in lean subjects and women, and is associated with higher degrees of insulin sensitivity and lower TNF-alpha expression.     

Fibronectin Gene Expression in Human Adipose Tissue and Its Associations with Obesity-Related Genes and Metabolic Parameters

Background

Limited data are available on the in vivo expression of fibronectin, one of the main extracellular matrix components. We investigated the expression of fibronectin in abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) and the associations of leptin, adiponectin, and vaspin gene expression with metabolic parameters in obese women.

Methods

We recruited female subjects undergoing bariatric surgery for obesity (n?=?24) and patients undergoing benign gynecological surgery as the control group (n?=?23). We measured anthropometric variables, abdominal fat distribution, metabolic parameters, serum concentrations of leptin, adiponectin, and vaspin, and depot-specific mRNA expression of fibronectin, leptin, adiponectin, and vaspin.

Results

Fibronectin expression in both VAT and SAT was significantly lower in the obese group than in the control group. Fibronectin expression in both VAT and SAT were negatively correlated with body mass index or waist circumference, with higher prominence in VAT. In multiple regression analysis, fibronectin expression in both VAT and SAT was negatively correlated with serum leptin concentration. Fibronectin expression in VAT was negatively correlated with leptin expression in VAT. Additionally, fibronectin expression in SAT was negatively correlated with leptin expression in SAT and positively correlated with adiponectin expression in VAT and SAT.

Conclusions

We found significant negative associations between depot-specific fibronectin expression in human adipose tissue and obesity indices and obesity-related biomarkers. Our results suggest that fibronectin expression may contribute to obesity and metabolic dysregulation in humans.     

Whole-body insulin resistance in the absence of obesity in FVB mice with overexpression of Dgat1 in adipose tissue

Insulin resistance is often associated with obesity. We tested whether augmentation of triglyceride synthesis in adipose tissue by transgenic overexpression of the diacylglycerol aclytransferase-1 (Dgat1) gene causes obesity and/or alters insulin sensitivity. Male FVB mice expressing the aP2-Dgat1 had threefold more Dgat1 mRNA and twofold greater DGAT activity levels in adipose tissue. After 30 weeks of age, these mice had hyperglycemia, hyperinsulinemia, and glucose intolerance on a high-fat diet but were not more obese than wild-type littermates. Compared with control littermates, Dgat1 transgenic mice were both insulin and leptin resistant and had markedly elevated plasma free fatty acid levels. Adipocytes from Dgat1 transgenic mice displayed increased basal and isoproterenol-stimulated lipolysis rates and decreased gene expression for fatty acid uptake. Muscle triglyceride content was unaffected, but liver mass and triglyceride content were increased by 20 and 300%, respectively. Hepatic insulin signaling was suppressed, as evidenced by decreased phosphorylation of insulin receptor-beta (Tyr(1,131)/Tyr(1,146)) and protein kinase B (Ser473). Gene expression data suggest that the gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, were upregulated. Thus, adipose overexpression of Dgat1 gene in FVB mice leads to diet-inducible insulin resistance, which is secondary to redistribution of fat from adipose tissue to the liver in the absence of obesity.     

Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1α,25‐dihydroxyvitamin D3 synthesis in leptin‐deficient ob/ob Mice

Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25‐hydroxyvitamin D₃ 1α‐hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone‐derived fibroblast growth factor 23 (FGF‐23) as a mediator of the influence of leptin on renal 1α‐hydroxylase mRNA expression in leptin‐deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF‐23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12‐hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF‐23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Administration of FGF‐23 (5 µg i.p. at 12‐hour intervals for 2 days) to ob/ob mice suppressed renal 1α‐hydroxylase mRNA expression. The main site of FGF‐23 mRNA expression was the bone, and leptin markedly increased the FGF‐23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1α‐hydroxylase and sodium‐phosphate cotransporters (NaP_i‐IIa and NaP_i‐IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF‐23 level and renal expression of 1α‐hydroxylase mRNA were not influenced by administration of leptin to leptin receptor–deficient (db/db) mice. These results indicate that leptin directly stimulates FGF‐23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH)₂D₃ synthesis in these mice is at least partly due to elevated bone production of FGF‐23. © 2010 American Society for Bone and Mineral Research     

Gene Expression of Leptin, Resistin, and Adiponectin in the White Adipose Tissue of Obese Patients with Non-Alcoholic Fatty Liver Disease and Insulin Resistance

Background: Adipose tissue is an active endocrine organ that secretes a variety of metabolically important substances including adipokines. These factors affect insulin sensitivity and may represent a link between obesity, insulin resistance, type 2 diabetes (DM), and nonalcoholic fatty liver disease (NAFLD). This study uses real-time polymerase chain reaction (PCR) quantification of mRNAs encoding adiponectin, leptin, and resistin on snap-frozen samples of intra-abdominal adipose tissue of morbidly obese patients undergoing bariatric surgery. Methods: Morbidly obese patients undergoing bariatric surgery were studied. Patients were classified into two groups: Group A (with insulin resistance) (N=11; glucose 149.84 ± 40.56 mg/dL; serum insulin 8.28 ± 3.52 μU/mL), and Group B (without insulin resistance) (N=10; glucose 102.2 ± 8.43 mg/dL; serum insulin 3.431 ± 1.162 μU/mL). Results: Adiponectin mRNA in intra-abdominal adipose tissue and serum adiponectin levels were significantly lower in Group A compared to Group B patients (P<0.016 and P<0.03, respectively). Although serum resistin was higher in Group A than in Group B patients (P<0.005), resistin gene expression was not different between the two groups. Finally, for leptin, neither serum level nor gene expression was different between the two groups. Serum adiponectin level was the only predictor of nonalcoholic steatohepatitis (NASH) in this study (P=0.024). Conclusions: Obese patients with insulin resistance have decreased serum adiponectin and increased serum resistin. Additionally, adiponectin gene expression is also decreased in the adipose tissue of these patients. This low level of adiponectin expression may predispose patients to the progressive form of NAFLD or NASH.     

The concept of selective leptin resistance: evidence from agouti yellow obese mice.

Leptin, a hormone secreted by adipose tissue, acts to inhibit appetite and promote metabolism, thereby reducing body weight. Leptin also increases sympathetic activity and arterial pressure. Several murine models of obesity, including agouti obese mice, exhibit resistance to the anorexic and weight-reducing effects of leptin. Hypertension in agouti mice has been attributed to hyperleptinemia. These observations pose a seeming paradox. If these mice are leptin-resistant, then how can leptin contribute to hypertension? We tested the novel hypothesis that these mice have selective leptin resistance, with preservation of the sympathoexcitatory action despite resistance to the weight-reducing actions. Leptin-induced decreases in food intake and body weight were less in agouti obese mice than in lean littermates. In contrast, leptin-induced increases in sympathetic nerve activity did not differ in obese and lean mice. These findings support the concept of selective leptin resistance, with resistance to the metabolic actions of leptin but preservation of the sympathoexcitatory actions. This finding may have potential implications for human obesity, which is associated with elevated plasma leptin and is thought to be a leptin-resistant state. If leptin resistance is selective in obese humans, then leptin could contribute to sympathetic overactivity and its adverse consequences in human obesity.     

The natriuretic peptide system in obesity-related hypertension: new pathophysiological aspects

The association between obesity and hypertension is well known but the pathophysiology of weight-related changes on blood pressure is still a matter of debate. Although obesity-related hypertension is considered to be sodium-sensitive, little attention has been given to a possible pathophysiological role of Atrial Natriuretic Peptides (ANP) and their receptors (NPr) system. Since the early phase of weight loss induced by very-low-calorie diet or fasting is followed by a significant increase in diuresis and natriuresis together with an increase in circulating ANP, we focused our attention on the possible role of adipose tissue in mediating these changes. We first demonstrated that human and rat adipose tissue contain high levels of mRNA specific for both type A (NPr-A), which is biologically active, and type C (NPr-C) which is biologically inactive, receptors. We then demonstrated in the rat that fasting exerts a tissue-specific and gene-specific suppression of NPr-C gene expression in adipose tissue that appears to be accompanied by an increased biological activity of ANP. These experimental observations were confirmed in man studying gene expression of NPr-A and NPr-C in adipose tissue obtained through subcutaneous peri-umbilical needle aspiration in obese and non-obese hypertensive patients. We found that NPr-A: NPr-C mRNA ratio was significantly lower in obese hypertensive patients as compared with non-obese hypertensives. These findings suggest that overxpression of the clearance receptor in the obese may trap more molecules of circulating ANP so reducing their biological activity at renal level. More recent results were obtained in obese hypertensive patients in whom the intravenous bolus injection of ANP (0.6 mg/kg body weight) was performed before and after four days of very-low-calorie diet which induced a weight loss accompanied by a significant reduction of BP and an increase in the urinary excretion of cGMP. The infusion of ANP after low-calorie diet was followed by an increase of ANP levels similar to that observed before diet, but plasma cGMP, diuresis and natriuresis significantly increased only after caloric restriction and the effects of ANP infusion on BP were more pronounced. Taken together our studies suggest that the abundance of NPr-C in adipose tissue may play a significant role in explaining at least part of the sodium retention characteristic of obesity associated hypertension.     

Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys

Adiponectin is an adipose-specific plasma protein whose plasma concentrations are decreased in obese subjects and type 2 diabetic patients. This protein possesses putative antiatherogenic and anti-inflammatory properties. In the current study, we have analyzed the relationship between adiponectin and insulin resistance in rhesus monkeys (Macaca mulatta), which spontaneously develop obesity and which subsequently frequently progress to overt type 2 diabetes. The plasma levels of adiponectin were decreased in obese and diabetic monkeys as in humans. Prospective longitudinal studies revealed that the plasma levels of adiponectin declined at an early phase of obesity and remained decreased after the development of type 2 diabetes. Hyperinsulinemic-euglycemic clamp studies revealed that the obese monkeys with lower plasma adiponectin showed significantly lower insulin-stimulated peripheral glucose uptake (M rate). The plasma levels of adiponectin were significantly correlated to M rate (r = 0.66, P < 0.001). Longitudinally, the plasma adiponectin decreased in parallel to the progression of insulin resistance. No clear association was found between the plasma levels of adiponectin and its mRNA levels in adipose tissue. These results suggest that reduction in circulating adiponectin may be related to the development of insulin resistance.     

Mechanism of amelioration of insulin resistance by beta3-adrenoceptor agonist AJ-9677 in the KK-Ay/Ta diabetic obese mouse model

The mechanism by which the specific beta3-adrenoceptor agonist AJ-9677 relieves insulin resistance in vivo was investigated by studying its effects in the white and brown adipose tissues of the KK-Ay/Ta diabetic obese mouse model. AJ-9677 reduced the total weight of white adipose tissues by reducing the size of the adipocytes, an effect associated with the normalization of tumor necrosis factor-alpha (TNF-alpha) and leptin expression levels. The levels of uncoupling protein (UCP)-1 mRNA in brown adipose tissue were increased threefold. AJ-9677 caused a marked increase (20- to 80-fold) in the expression of UCP-1 in white adipose tissues. The levels of UCP-2 mRNA were increased in both the white and brown adipose tissues of diabetic obese mice, and AJ-9677 further upregulated UCP-2 mRNA levels in brown adipose tissue, but reduced its levels in white adipose tissue. UCP-3 mRNA levels were not essentially changed by AJ-9677. However, AJ-9677 significantly (two- to four-fold) upregulated the GLUT4 mRNA and protein levels in white and brown adipose tissues and the gastrocnemius. The generation of small adipocytes, presumably mediated by increased expression of UCP-1 in addition to increased lipolysis in response to AJ-9677, was associated with decreased TNF-alpha and free fatty acid production and may be the mechanism of amelioration of insulin resistance in KK-Ay/Ta diabetic obese mice.     

Altered adipose and plasma sphingolipid metabolism in obesity: a potential mechanism for cardiovascular and metabolic risk

The adipose tissue has become a central focus in the pathogenesis of obesity-mediated cardiovascular and metabolic disease. Here we demonstrate that adipose sphingolipid metabolism is altered in genetically obese (ob/ob) mice. Expression of enzymes involved in ceramide generation (neutral sphingomyelinase [NSMase], acid sphingomyelinase [ASMase], and serine-palmitoyl-transferase [SPT]) and ceramide hydrolysis (ceramidase) are elevated in obese adipose tissues. Our data also suggest that hyperinsulinemia and elevated tumor necrosis factor (TNF)-alpha associated with obesity may contribute to the observed increase in adipose NSMase, ASMase, and SPT mRNA in this murine model of obesity. Liquid chromatography/mass spectroscopy revealed a decrease in total adipose sphingomyelin and ceramide levels but an increase in sphingosine in ob/ob mice compared with lean mice. In contrast to the adipose tissue, plasma levels of total sphingomyelin, ceramide, sphingosine, and sphingosine 1-phosphate (S1P) were elevated in ob/ob mice. In cultured adipocytes, ceramide, sphingosine, and S1P induced gene expression of plasminogen activator inhibitor-1, TNF-alpha, monocyte chemoattractant protein-1, interleukin-6, and keratinocyte-derived chemokine. Collectively, our results identify a novel role for sphingolipids in contributing to the prothrombotic and proinflammatory phenotype of the obese adipose tissue currently believed to play a major role in the pathogenesis of obesity-mediated cardiovascular and metabolic disease.     

Leptin and reproductive dysfunction in obese men

Infertility is somewhat more prevalent in men who are obese. They are also reported to have low sperm concentration, higher fraction of spermatozoa that look morphologically abnormal, higher DNA fragmentation index and evidence of oxidative stress. The precise cause for this remains uncertain. Leptin levels in serum and percentage body fat correlate positively, and obese men therefore usually have elevated serum leptin levels. Although leptin is important for normal reproductive function, but when present in excess, leptin could seriously affect reproductive function in men. Reports on the findings of sperm parameters in obese men, particularly those who are subfertile or infertile, seem to be similar to those reported from studies on normal-weight rats treated with leptin. Collectively, the observations reported in human and experimental animal studies point to leptin as a possible link between infertility and obesity. Herein, we review some findings on sperm function in obese subfertile or infertile men and those from animal studies following leptin treatment, and discuss the possible link between leptin and reproductive dysfunction in obese men. The large amounts of leptin secreted by the adipose tissue and its higher circulating levels could indeed be responsible for the higher prevalence of infertility in obese men.     

Neuropeptide Y is a critical modulator of Leptin's regulation of cortical bone

Leptin signaling is required for normal bone homeostasis; however, loss of leptin results in differing effects on cortical and cancellous bone, as well as altered responses between the axial and appendicular regions. Local β‐adrenergic actions are responsible for the greater cancellous bone volume in leptin‐deficient (ob/ob) mice; however, the mechanism responsible for the opposing reduction in cortical bone in ob/ob mice is not known. Here we show that blocking the leptin‐deficient increase in neuropeptide Y (NPY) expression reverses the cortical bone loss in ob/ob mice. Mice null for both NPY and leptin (NPY^–/–ob/ob), display greater cortical bone mass in both long‐bones and vertebra, with NPY^–/–ob/ob mice exhibiting thicker and denser cortical bone, associated with greater endocortical and periosteal mineral apposition rate (MAR), compared to ob/ob animals. Importantly, these cortical changes occurred without significant increases in body weight, with NPY^–/–ob/ob mice showing significantly reduced adiposity compared to ob/ob controls, most likely due to the reduced respiratory exchange ratio seen in these animals. Interestingly, cancellous bone volume was not different between NPY^–/–ob/ob and ob/ob, suggesting that NPY is not influencing the adrenergic axis. Taken together, this work demonstrates the critical role of NPY signaling in the regulation of bone and energy homeostasis, and more importantly, suggests that reduced leptin levels or leptin resistance, which occurs in obesity, could potentially inhibit cortical bone formation via increased central NPY signaling. © 2013 American Society for Bone and Mineral Research.     

Role of selective leptin resistance in diet-induced obesity hypertension

Leptin is an adipocyte-derived hormone that plays a key role in the regulation of body weight through its actions on appetite and metabolism. Leptin also increases sympathetic nerve activity (SNA) and blood pressure. We tested the hypothesis that diet-induced obesity is associated with resistance to the metabolic actions of leptin but preservation of its renal SNA and arterial pressure effects, leading to hypertension. Mice were fed a high-fat diet for 10 weeks to induce moderate obesity. The decrease in food intake and body weight induced by intraperitoneal or intracerebroventricular leptin was significantly attenuated in the obese mice. Regional SNA responses to leptin were differentially altered in diet-induced obese mice. Renal SNA response to leptin was preserved, whereas lumbar and brown adipose tissue SNA responses were attenuated in obese mice. Radiotelemetric arterial pressure was approximately 10 mmHg higher in obese mice. Furthermore, the increase in arterial pressure in response to long-term (12 days) leptin treatment was preserved in obese mice. Thus, mice with diet-induced obesity exhibit circulating hyperleptinemia and resistance to the metabolic actions of leptin. However, there is preservation of the renal sympathetic and arterial pressure responses to leptin, which represent a potential mechanism for the adverse cardiovascular consequences of obesity.