Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity

Background

Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals.

Methodology and Findings

To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples.

Conclusions

ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study.     

Pathophysiology and Pathogenesis of Visceral Fat Obesity

Based on the analysis of fat distribution by computed tomography (CT) scans, the classification scheme for obesity should include visceral fat obesity in which fat accumulation is predominant in the intra-abdominal cavity. Obese subjects with visceral fat accumulation more frequently demonstrate impairment of glucose and lipid metabolism than those with subcutaneous fat accumulation. We have shown that visceral fat obesity is present in almost 90% of obese patients with ischemic heart disease. Even in non-obese subjects, visceral fat accumulation is correlated with glucose intolerance, hyperlipidemia and hypertension. Forty percent of non-obese subjects with coronary artery disease (CAD) had increased visceral fat. In non-obese subjects, visceral fat area assessed by abdominal CT at the level of the umbilicus correlates with metabolic risk factors, whereas in obese subjects the visceral fat area to subcutaneous fat area ratio provides a more significant correlation. From clinical and basic investigations, aging, sex hormones, excess intake of sucrose and lack of physical exercise have been suggested to be determinants for visceral fat accumulation. Since intra-abdominal fat (mesenteric and omentum fat) has been shown to have high activities of both lipogenesis and lipolysis, its accumulation can induce high levels of free fatty acids, a product of lipolysis, in portal circulation which go into the liver. Excess free fatty acids may cause the enhancement of lipid synthesis and gluconeo genesis as well as insulin resistance, resulting in hyperlipidemia, glucose intolerance and hypertension and finally atherosclerosis. Thus we propose a disease entity, visceral fat syndrome, which may increase susceptibility to atherosclerosis due to multiple risk factors induced by visceral fat accumulation.     

Differences in plasminogen activator inhibitor 1 in subcutaneous versus omental adipose tissue in non-obese and obese subjects.

Human adipose tissue can produce plasminogen activator inhibitor-1 (PAI-1). It has been suggested that high levels of PAI-1 are of importance in enhanced cardiovascular disease observed among obese subjects, especially abdominally obese individuals. In the present study, we investigated the level of mRNA and production of PAI-1 in adipose tissue from two adipose tissue depots (omental vs. subcutaneous). Adipose tissue from both depots was obtained from obese (mean BMI, 46.9 kg/m 2) and non-obese (mean BMI, 23.9 kg/m 2) women. PAI-1 mRNA was measured both in fresh adipose tissue obtained immediately after surgery and after the adipose tissue (fragments) had been incubated for up to 72 h. In immediately frozen adipose tissue, PAI-1 mRNA expression was similar in omental and subcutaneous adipose tissue. No differences between obese and non-obese women were found. However, when adipose tissue fragments were cultured, PAI-1 mRNA and PAI-1 production were significantly higher in omental than in subcutaneous adipose tissue (p < 0.05). In the culture system, the production of PAI-1 in obese subjects was higher than in non-obese subjects in both subcutaneous (p < 0.05) and in omental adipose tissue (p = 0.19). In order to test whether these regional differences observed after incubation of the adipose tissue were due to differences in local accumulation of cytokines that may stimulate PAI-1 by a paracrine or autocrine manner, we investigated the expression of transforming growth factor beta1 (TGF-beta1) mRNA and tumor necrosis factor alpha (TNF-alpha) mRNA and protein. No differences between the two fat depots were found. In conclusion, no differences in PAI-1 expression between omental and subcutaneous adipose tissue were observed in biopsies frozen immediately after removal, but after incubation of adipose tissue (which somehow stimulates PAI-1 production), higher levels of PAI-1 were found in omental adipose tissue than in subcutaneous adipose tissue. Finally, PAI-1 production in adipose tissue from obese women was higher in non-obese women after incubation for 72 h.     

Differences in lipogenesis and lipolysis in obese and non-obese adult human adipocytes

It has been proposed that differences in adipocyte function and/or metabolism between obese and lean individuals may manifest themselves in functional adipose tissue abnormalities that lead to metabolic disorders in obesity. We studied lipogenesis and lipolysis of omental adipocytes from obese (OB) and non-obese (NOB) humans. The specific activity of the lipogenic marker enzyme G3PDH was 50% lower in total adipocytes of OB compared to that of NOB subjects. Omental adipocytes from OB subjects also had lower basal lipolytic levels, and a lower lipolytic response to beta-adrenergic stimulus. Cholesterol depletion of adipocyte plasma membrane using methyl b-cyclodextrin caused a lipolytic effect on adipocytes of both groups together, but when obese and lean subjects were analyzed separately, the response was significant only in the obese. We present evidence of a different lipogenic and lipolytic profile in obese individuals' omental adipocytes, and propose a relevant role of plasma membrane cholesterol, where the impact of its removal in OB and NOB adipocyte lipolysis differs.     

Human SMAD4 is phosphorylated at Thr9 and Ser138 by interacting with NLK

A polymorphism in the promoter region of uncoupling protein 2 gene ?866 G/A has been associated with its expression levels, the risk of obesity, and metabolic abnormalities. We aimed to investigate the associations of uncoupling protein (UCP)2 gene variants with obesity and related traits. A total of 440 subjects, 200 obese, and 240 non-obese individuals were included in this case–control study. Hormone and glucose levels were estimated using standard protocols. Genotyping of UCP-2 gene polymorphism for all subjects was performed by the PCR–RFLP polymerase chain reaction (PCR) method. Higher Systolic blood pressure, Diastolic blood pressure, Waist to hip ratio, Leptin, Insulin, and blood glucose levels were observed in obese than non-obese (P < 0.05). The distributions of genotype (0.001) and allele (0.003) were significantly different between the non-obese and the obese groups. In the obese group, subjects with the A allele showed significant high insulin levels (<0.001) in comparison with A allele non-carriers. In conclusion, our results suggest that the ?866 AA genotype and A allele of the UCP2 gene is associated with obesity and A allele associated with hyperinsulinemia in obese subjects.     

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

Background  

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals.     

Psychophysics of sweet and fat perception in obesity: problems, solutions and new perspectives

Psychophysical comparisons seem to show that obese individuals experience normal sweet and fat sensations, they like sweetness the same or less, but like fat more than the non-obese do. These psychophysical comparisons have been made using scales (visual analogue or category) that assume intensity labels (e.g. extremely) which denote the same absolute perceived intensity to all. In reality, the perceived intensities denoted by labels vary because they depend on experiences with the substances to be judged. This variation makes comparisons invalid. Valid comparisons can be made by asking the subjects to rate their sensory/hedonic experiences in contexts that are not related to the specific experiences of interest. Using this methodology, we present the evidence that the sensory and hedonic properties of sweet and fat vary with body mass index. The obese live in different orosensory and orohedonic worlds than do the non-obese; the obese experience reduced sweetness, which probably intensifies fat sensations, and the obese like both sweet and fat more than the non-obese do. Genetic variation as well as taste pathology contribute to these results. These psychophysical advances will impact experimental as well as clinical studies of obesity and other eating disorders.     

Lipolysis in Intraabdorninal Adipose Tissues of Obese Women and Men

Intraabdominal fat in humans is located in two major depots, the omental and mesenteric. We compared basal and stimulated lipolysis in adipose tissue from these two depots and the subcutaneous abdominal depot of obese women and men. Omental fat cells of women are smaller and have lower rates of basal lipolysis than in men. Basal Iipolysis rates are significantly higher in subcutaneous than intraabdominal adipose tissues of both genders. In men, the incremental lipolytic response to norepinephrine is significantly greater in both intraabdominal fat depots than in the subcutaneous fat, while in women tlie response of tlie mesenteric is lower than tlie omental. In women, but not men, responsiveness to tlie beta agonist isoproterenol is also increased in omental tissue. Thus, in women, omental and mesenteric adipose tissues show distinctly different metabolic properties which may moderate the impact of intraabdominal obesity.     

MiRNA Expression Profile of Human Subcutaneous Adipose and during Adipocyte Differentiation

Background

Potential regulators of adipogenesis include microRNAs (miRNAs), small non-coding RNAs that have been recently shown related to adiposity and differentially expressed in fat depots. However, to date no study is available, to our knowledge, regarding miRNAs expression profile during human adipogenesis. Thereby, the aim of this study was to investigate whether miRNA pattern in human fat cells and subcutaneous adipose tissue is associated to obesity and co-morbidities and whether miRNA expression profile in adipocytes is linked to adipogenesis.

Methodology/Principal Findings

We performed a global miRNA expression microarray of 723 human and 76 viral mature miRNAs in human adipocytes during differentiation and in subcutaneous fat samples from non-obese (n = 6) and obese with (n = 9) and without (n = 13) Type-2 Diabetes Mellitus (DM-2) women. Changes in adipogenesis-related miRNAs were then validated by RT-PCR. Fifty of 799 miRNAs (6.2%) significantly differed between fat cells from lean and obese subjects. Seventy miRNAs (8.8%) were highly and significantly up or down-regulated in mature adipocytes as compared to pre-adipocytes. Otherwise, 17 of these 799 miRNAs (2.1%) were correlated with anthropometrical (BMI) and/or metabolic (fasting glucose and/or triglycerides) parameters. We identified 11 miRNAs (1.4%) significantly deregulated in subcutaneous fat from obese subjects with and without DM-2. Interestingly, most of these changes were associated with miRNAs also significantly deregulated during adipocyte differentiation.

Conclusions/Significance

The remarkable inverse miRNA profile revealed for human pre-adipocytes and mature adipocytes hints at a closely crosstalk between miRNAs and adipogenesis. Such candidates may represent biomarkers and therapeutic targets for obesity and obesity-related complications.     

Adipokines oversecreted by omental adipose tissue in human obesity

Central-omental obesity plays a causative role in the pathogenesis of the metabolic syndrome. Adipokines are involved in the pathogenesis of this syndrome. However, adipokines secreted by omental adipose tissue (OAT) are still poorly characterized in human obesity. Therefore, we searched for novel adipokines abnormally secreted by OAT in obesity and examined their relationships with some features of metabolic syndrome and the respective contribution of adipocytes vs. stromal-vascular cells. OAT from obese and nonobese men was fractionated into adipocytes and SV cells, which were then cultured. Medium was screened by medium-scale protein arrays and ELISAs. Adipokine mRNA levels were measured by real-time RT-qPCR. We detected 16 cytokines secreted by each cellular fraction of lean and obese subjects. Of the 16 cytokines, six adipokines were newly identified as secretory products of OAT, which were dysregulated in obesity: three chemokines (growth-related oncogen factor, RANTES, macrophage inflammatory protein-1beta), one interleukin (IL-7), one tissue inhibitor of metalloproteinases (TIMP-1), and one growth factor (thrombopoietin). Their secretion and expression were enhanced in obesity, with a relatively similar contribution of the two fractions. The higher proportion of macrophages and endothelial cells in obesity may contribute to this enhanced production as well as changes in intrinsic properties of hypertrophied adipocytes. Accordingly, mRNA concentrations of most of these adipokines increased during adipocyte differentiation. Eventually, expression of the investigated adipokines did correlate with several features of the metabolic syndrome. In conclusion, six adipokines were newly identified as oversecreted by OAT in obesity. These adipokines may link obesity to its cardiovascular or metabolic comorbidities.     

Circadian rhythm of blood leptin level in obese and non-obese people

Leptin, an adipose tissue hormone, has circadian variations in its secretion. Aims of this study were to show how circadian rhythm depends on fat tissue distribution in obese and non-obese subjects. The research was carried out on 70 subjects (37 men and 33 women) with an average body mass index (BMI) of 25.22 kg/m2. Concentration of leptin in blood was measured at 8.30 a.m., 12.30 p.m. and 6.30 p.m. Basal leptin level correlated strongly with all isolated regions of subcutaneous fat tissue in women and obese subjects. Circadian changes of blood leptin level in non-obese people are more significant than these changes in obese people. Differences in circadian pattern of leptin secretion between obese and non-obese subjects were probably caused by enlarged volume of subcutaneous fat tissue in obese people. Lean subjects have subcutaneous fat in physiological range which allows influence of some hormones (insulin or cortizol) or food intake on leptin secretion.     

Decreased expression and function of adipocyte hormone-sensitive lipase in subcutaneous fat cells of obese subjects.

Decreased lipolytic effect of catecholamines in adipose tissue has repeatedly been demonstrated in obesity and may be a cause of excess accumulation of body fat. However, the mechanisms behind this lipolysis defect are unclear. The role of hormone-sensitive lipase was examined using abdominal subcutaneous adipocytes from 34 obese drug-free and otherwise healthy males or females and 14 non-obese control subjects. The enzyme catalyzes the rate-limiting step of the lipolysis pathway. The maximum lipolytic capacity of fat cells was significantly decreased in obesity when measured using either a non-selective beta-adrenergic receptor agonist (isoprenaline) or a phosphodiesterase resistant cyclic AMP analogue (dibutyryl cyclic AMP). Likewise, enzyme activity, protein expression, and mRNA of hormone-sensitive lipase were significantly decreased in adipocytes of obese subjects. The findings were not influenced by age or gender. The data suggest that a decreased expression of hormone-sensitive lipase in subcutaneous fat cells, which in turn causes decreased enzyme function and impaired lipolytic capacity of adipocytes, is present in obesity. Impaired expression of the hormone-sensitive lipase gene might at least in part explain the enzyme defect.     

Plasma proteome analysis in diet-induced obesity-prone and obesity-resistant rats

One of the major issues in the field of obesity is why some humans become obese and others resist development of obesity when exposed to high-calorie diets. Despite the same genetic background, namely obesity-prone (OP) and -resistant (OR) rats, differing responses have been demonstrated in a high fat diet-induced rodent model. The aim of the present study was to discover novel obesity-related biomarkers for susceptibility and/or resistance to obesity by proteomic analysis of OP and OR rat plasma. After feeding of high fat diet, OP rats gained approximately 25% more body weight than OR rats and were used for proteomic analysis using 2-DE combined with MALDI-TOF-MS. We categorized identified proteins into three groups by analysis of both average spot density in each group and individual spot density of six rats as a function of body weight. Consequently, category (1) included inter-α-inhibitor H4 heavy chain and fetuin B precursor, which can be used as novel plasma biomarkers for risk of obesity. Nine proteins of category (2) and (3) can also be plausible plasma markers in the study of obesity. This proteomic study is an important advancement over the previous steps needed for identification of OP and OR rats.     

Differential expression of adipose tissue proteins between obesity-susceptible and -resistant rats fed a high-fat diet

One of the major questions in the field of obesity is why some humans become obese (obesity prone, OP) and others resist the development of obesity (obesity resistant, OR) when exposed to a high-calorie diet, which has not been completely studied. Therefore, in the present study, in order to gain insight into the molecular mechanisms underlying this propensity, we have performed a comparative analysis of protein expression profiles in white adipose tissue (WAT) and brown adipose tissue (BAT) of rats fed a high-fat diet by 2-DE and MALDI-TOF-MS. Protein mapping of homogenates revealed significant alterations to a number of proteins; 60 and 70 proteins were differentially regulated in BAT and WAT, respectively. For careful interpretation of proteomic results, we categorized the identified proteins into two groups by analysis of both average spot density of pooled six rat adipose tissues and individual spot density of each adipose tissue of six rats as a function of body weight. One of the most striking findings of this study was that significant changes of Ehd1 and laminin receptor in BAT as well as antiquitin, DJ-1 protein, and paraoxonase 2 in WAT were found for the first time in obese rats. In addition, we confirmed the increased expression of some thermogenic enzymes and decreased lipogenic enzymes in adipose tissues of OR rats by immunoblot analysis. To our knowledge, this is the first proteomic study of profiling of protein modulation in OP and OR rats, thereby providing the first global evidence for different propensities to obesity between OP and OR rats.     

肥胖者脂肪组织中TNF-α表达与脂肪细胞大小的相关性分析

目的探讨肥胖者网膜脂肪和皮下脂肪两处肿瘤坏死因子-α(TNF-α)蛋白的表达与脂肪细胞大小的相关性。方法选取正常体重者16名,中心型肥胖者32名拟行外科手术患者,术中取出网膜脂肪和皮下脂肪标本,测定脂肪细胞大小,采用western blot方法测定TNF-α蛋白表达。结果肥胖者网膜脂肪和皮下脂肪两处TNF-α蛋白的水平均比正常体重对照组表达高(P<0.01),肥胖者网膜脂肪组织TNF-α蛋白表达高于皮下脂肪(P<0.05),同时研究发现肥胖者皮下脂肪细胞和网膜脂肪细胞大小均明显大于正常体重组(P<0.05),且肥胖者网膜脂肪和皮下脂肪两处脂肪组织TNF-α蛋白表达与脂肪细胞大小呈正相关(网膜:r=0.808,P<0.01;皮下:r=0.452,P<0.05)。结论肥胖者网膜脂肪和皮下脂肪细胞增大,在肥胖相关胰岛素抵抗的发生中起到了重要的作用。