Fatty acid binding protein expression in different human adipose tissue depots in relation to rates of lipolysis and insulin concentration in obese individuals

Two fatty acid binding proteins (FABPs) are expressed in adipose tissue, adipocyte lipid binding protein (ALBP) and keratinocyte lipid binding protein (KLBP). This study investigated FABP expression in visceral and subcutaneous human adipose tissue depots and associations with lipolytic differences between the depots and circulating insulin concentrations. ALBP and KLBP (protein and RNA) were quantified in subcutaneous and omental adipose tissue from obese individuals and expressed relative to actin. ALBP RNA and protein expression was significantly higher in subcutaneous compared to omental adipose tissue (both p < 0.05), whereas KLBP RNA and protein expression was no different between the two sites. There were significant inverse correlations between serum insulin concentrations and the ALBP/KLBP RNA ratio in both subcutaneous and omental adipose tissue (both p < 0.02). Basal rates of glycerol and fatty acid release measured in adipocytes isolated from subcutaneous and omental adipose tissue were significantly higher in the former (p 0.02). Therefore the relative ALBP/KLBP content of human adipose tissue is different in different adipose tissue depots and at the RNA level is related to the circulating insulin concentration, at least in obese subjects. The higher rates of basal lipolysis in adipocytes isolated from subcutaneous compared to omental adipose tissue might be related to the increased ALBP content of the former. Therefore adipose tissue FABPs are interesting candidates for investigation to further our understanding of the insulin resistance syndrome and regulation of lipolysis.     

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.     

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.     

Insulin sensitive and resistant obesity in humans: AMPK activity, oxidative stress, and depot-specific changes in gene expression in adipose tissue

We previously reported that adenosine monophosphate-activated protein kinase (AMPK) activity is lower in adipose tissue of morbidly obese individuals who are insulin resistant than in comparably obese people who are insulin sensitive. However, the number of patients and parameters studied were small. Here, we compared abdominal subcutaneous, epiploic, and omental fat from 16 morbidly obese individuals classified as insulin sensitive or insulin resistant based on the homeostatic model assessment of insulin resistance. We confirmed that AMPK activity is diminished in the insulin resistant group. A custom PCR array revealed increases in mRNA levels of a wide variety of genes associated with inflammation and decreases in PGC-1α and Nampt in omental fat of the insulin resistant group. In contrast, subcutaneous abdominal fat of the same patients showed increases in PTP-1b, VEGFa, IFNγ, PAI-1, and NOS-2 not observed in omental fat. Only angiotensinogen and CD4(+) mRNA levels were increased in both depots. Surprisingly, TNFα was only increased in epiploic fat, which otherwise showed very few changes. Protein carbonyl levels, a measure of oxidative stress, were increased in all depots. Thus, adipose tissues of markedly obese insulin resistant individuals uniformly show decreased AMPK activity and increased oxidative stress compared with insulin sensitive patients. However, most changes in gene expression appear to be depot-specific.     

A role of lipin in human obesity and insulin resistance: relation to adipocyte glucose transport and GLUT4 expression

The mouse lipin gene, Lpin1, is important for adipose tissue development and is a candidate gene for insulin resistance. Here, we investigate the adipose tissue expression levels of the human LPIN1 gene in relation to various clinical variables as well as adipocyte function. LPIN1 gene expression was induced at an early step in human preadipocyte differentiation in parallel with peroxisome proliferator-activated receptor gamma. Lipin mRNA levels were higher in fat cells than in adipose tissue segments but showed no difference between subcutaneous and omental depots. Moreover, LPIN1 expression levels were reduced in obesity, improved following weight reduction in obese subjects, and were downregulated in women with the metabolic syndrome. With respect to adipocyte function, adipose LPIN1 gene expression was strongly associated with both basal and insulin-mediated subcutaneous adipocyte glucose transport as well as mRNA levels of glucose transporter 4 (GLUT4). We show that body fat accumulation is a major regulator of human adipose LPIN1 expression and suggest a role of LPIN1 in human preadipocyte as well as mature adipocyte function.     

Expression and activity of steroid aldoketoreductases 1C in omental adipose tissue are positive correlates of adiposity in women

We examined expression and activity of steroid aldoketoreductase (AKR) 1C enzymes in adipose tissue in women. AKR1C1 (20alpha-hydroxysteroid dehydrogenase; 20alpha-HSD), AKR1C2 (3alpha-HSD-3), and AKR1C3 (17beta-HSD-5) are involved mainly in conversion of progesterone to 20alpha-hydroxyprogesterone and inactivation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol. Abdominal subcutaneous and omental adipose tissue biopsies were obtained during abdominal hysterectomies in seven women with low visceral adipose tissue (VAT) area and seven age- and total body fat mass-matched women with visceral obesity. Women with elevated VAT areas were characterized by significantly higher omental adipose tissue 20alpha-HSD and 3alpha-HSD-3 mRNA abundance compared with women with low VAT accumulations (1.4- and 1.6-fold differences, respectively; P < 0.05). Omental and subcutaneous adipose tissue 3alpha-HSD activities were significantly higher in women with high vs. low VAT areas (P < 0.05 for both comparisons). Total and visceral adiposities were positively associated with omental 20alpha-HSD mRNA level (r = 0.75, P < 0.003 for fat mass; r = 0.57, P < 0.04 for VAT area) and omental 3alpha-HSD-3 mRNA level (r = 0.68, P < 0.01 for fat mass; r = 0.74, P < 0.003 for VAT area). Enzyme activities in both depots were also positively correlated with adiposity measures. Omental adipose tissue enzyme expression and activity were positively associated with omental adipocyte size and LPL activity. In conclusion, mRNA abundance and activity of AKR1C enzymes in abdominal adipose tissue compartments are positive correlates of adiposity in women. Increased progesterone and/or dihydrotestosterone reduction in abdominal adipose tissue may impact locally on fat cell metabolism.     

Proliferation and differentiation of human adipocyte precursor cells: Differences between the preperitoneal and subcutaneous compartments

Human adipocyte precursor cells (APC) have been characterized in their proliferation and differentiation potential from subcutaneous, omental, and mesenteric depots, mostly from morbidly obese patients. Cells from the preperitoneal adipose compartment have not been characterized yet, least of all when obtained from normal weight subjects. The aim was to compare proliferation and differentiation of subcutaneous (SC) and preperitoneal (PP) APC derived from adipose tissue in healthy subjects with different body mass. SC and PP adipose tissue was obtained during surgery of inguinal hernias in five healthy non‐obese subjects and three obese otherwise healthy men. APC, obtained by collagenase digestion, were cultured. Proliferation was assayed by cell counting and differentiation by oil red O staining and flow cytometry using Nile Red staining. Proliferation of SC was higher than PP APC. Such differences between both compartments were even higher in APC obtained from obese patients. Conversely PP APC differentiated earlier in vitro compared with SC cells. These results agree with published data on fat cell proliferation. However regarding differentiation, our data show that APC from deeper depots (in this case PP) differentiate earlier than subcutaneous APC. This is different to previous studies performed in mesenteric or omental adipose tissue. J. Cell. Biochem. 111: 659–664, 2010. © 2010 Wiley‐Liss, Inc.     

Tissue leptin and plasma insulin are associated with lipoprotein lipase activity in severely obese patients

The development of metabolic complications of obesity has been associated with the existence of depot-specific differences in the biochemical properties of adipocytes. The aim of this study was to investigate, in severely obese men and women, both gender- and depot-related differences in lipoprotein lipase (LPL) expression and activity, as well as the involvement of endocrine and biometric factors and their dependence on gender and/or fat depot. Morbidly obese, nondiabetic, subjects (9 men and 22 women) aged 41.1+/-1.9 years, with a body mass index (BMI) of 54.7+/-1.7 kg/m(2) who had undergone abdominal surgery were studied. Both expression and activity of LPL and leptin expression were determined in adipose samples from subcutaneous and visceral fat depots. In both men and women, visceral fat showed higher LPL mRNA levels as well as lower ob mRNA levels and tissue leptin content than the subcutaneous one. In both subcutaneous and visceral adipose depots, women exhibited higher protein content, decreased fat cell size and lower LPL activity than men. The gender-related differences found in abdominal fat LPL activity could contribute to the increased risk for developing obesity-associated diseases shown by men, even in morbid obesity, in which the massive fat accumulation could mask these differences. Furthermore, the leptin content of fat depots as well as plasma insulin concentrations appear in our population as the main determinants of adipose tissue LPL activity, adjusted by gender, depot and BMI.     

Maternal Obesity in Sheep Increases Fatty Acid Synthesis,Upregulates Nutrient Transporters,and Increases Adiposity in Adult Male Offspring after a Feeding Challenge

Maternal obesity in women is increasing worldwide. The objective of this study was to evaluate differences in adipose tissue metabolism and function in adult male offspring from obese and control fed mothers subjected to an ad libitum feeding challenge. We developed a model in which obese ewes were fed 150% of feed provided for controls from 60 days before mating to term. All ewes were fed to requirements during lactation. After weaning, F1 male offspring were fed only to maintenance requirements until adulthood (control = 7, obese = 6), when they were fed ad libitum for 12 weeks with intake monitored. At the end of the feeding challenge offspring were given an intravenous glucose tolerance test (IVGTT), necropsied, and adipose tissue collected. During the feeding trial F1obese males consumed more (P < 0.01), gained more weight (P < 0.01) and became heavier (P < 0.05) than F1control males. During IVGTT, Obese F1 offspring were hyperglycemic and hypoinsulinemic (P < 0.01) compared to F1 control F1. At necropsy perirenal and omental adipose depots weights were 47% and 58% greater respectively and subcutaneous fat thickness 41% greater in F1obese vs F1control males (P < 0.05). Adipocyte diameters were greater (P ≤ 0.04) in perirenal, omental and subcutaneous adipose depots in F1obese males (11, 8 and 7% increase vs. control, respectively). When adipose tissue was incubated for 2 hrs with C-14 labeled acetate, subcutaneous, perirenal, and omental adipose tissue of F1 obese males exhibited greater incorporation (290, 83, and 90% increase vs. control, respectively P < 0.05) of acetate into lipids. Expression of fatty acid transporting, binding, and syntheses mRNA and protein was increased (P < 0.05) compared to F1 control offspring. Maternal obesity increased appetite and adiposity associated with increased adipocyte diameters and increased fatty acid synthesis in over-nourished adult male offspring.     

Preadipocyte Number in Omental and Subcutaneous Adipose Tissue of Obese Individuals

Objective: To determine the variation in preadipocyte isolation procedure and to assess the number and function of preadipocytes from subcutaneous and omental adipose tissue of obese individuals. Research Methods and Procedures: The preadipocyte number per gram of adipose tissue in the abdominal‐subcutaneous and abdominal‐omental adipose stores of 27 obese subjects with a BMI of 44 ± 10 kg/m² and an age of 40 ± 9 years was determined. Results: The assessment of the preadipocyte number was found to be labor intensive and error prone. Our data indicated that the number of stromal vascular cells (SVCs), isolated from the adipose tissue by collagenase digestion, was dependent on the duration of collagenase treatment and the size and the origin of the biopsy. In addition, the fat accumulation and leptin production by differentiated SVCs were dependent on the number of adherent SVCs (aSVCs) in the culture plate and the presence of proteins derived from serum and peroxisome proliferator‐activated receptor ligands. Discussion: Using our standardized isolation and differentiation protocol, we found that the number of SVCs, aSVCs, leptin production, and fat accumulation still varied considerably among individuals. Interestingly, within individuals, the number of SVCs, aSVCs, and the leptin production by differentiating aSVCs from both the subcutaneous and the omental fat depots were associated, whereas fat accumulation was not. In obese to severely obese subjects, differences in BMI and age could not explain differences in SVCs, aSVCs, leptin production, and fat accumulation.     

Sex- and depot-specific lipolysis regulation in human adipocytes: interplay between adrenergic stimulation and glucocorticoids

The purpose of this investigation was to explore interactions between adrenergic stimulation, glucocorticoids, and insulin on the lipolytic rate in isolated human adipocytes from subcutaneous and omental fat depots, and to address possible sex differences. Fat biopsies were obtained from 48 nondiabetic subjects undergoing elective abdominal surgery. Lipolysis rate was measured as glycerol release from isolated cells and proteins involved in lipolysis regulation were assessed by immunoblots. Fasting blood samples were obtained and metabolic and inflammatory variables were analyzed. In women, the rate of 8-bromo-cAMP- and isoprenaline-stimulated lipolysis was approximately 2- and 1.5-fold higher, respectively, in subcutaneous compared to omental adipocytes, whereas there was no difference between the two depots in men. Dexamethasone treatment increased the ability of 8-bromo-cAMP to stimulate lipolysis in the subcutaneous depot in women, but had no consistent effects in fat cells from men. Protein kinase A, Perilipin A, and hormone sensitive lipase content in adipocytes was not affected by adipose depot, sex, or glucocorticoid treatment. In conclusion, catecholamine and glucocorticoid regulation of lipolysis in isolated human adipocytes differs between adipose tissue depots and also between sexes. These findings may be of relevance for the interaction between endogenous stress hormones and adipose tissue function in visceral adiposity and the metabolic syndrome.     

Attenuated metabolism is a hallmark of obesity as revealed by comparative proteomic analysis of human omental adipose tissue

Obesity is recognized as an epidemic health problem worldwide. In humans, the accumulation of omental rather than subcutaneous fat appears to be tightly linked to insulin resistance, type 2 diabetes and cardiovascular disease. Differences in gene expression profiles in the adipose tissue comparing non-obese and obese subjects have been well documented. However, to date, no comparative proteomic studies based on omental fat have investigated the influence of obesity in protein expression. In this work, we searched for proteins differentially expressed in the omental fat of non-obese and obese subjects using 2D-DIGE and MS. Forty-four proteins, several of which were further studied by immunoblotting and immunostaining analyses, showed significant differences in the expression levels in the two groups of subjects. Our findings reveal a clearly distinctive proteomic profile between obese and non-obese subjects which emphasizes: i) reduced metabolic activity in the obese fat, since most down-regulated proteins were engaged in metabolic pathways; and ii) morphological and structural cell changes in the obese fat, as revealed by the functions exerted by most up-regulated proteins. Interestingly, transketolase and aminoacylase-1 represent newly described molecules involved in the pathophysiology of obesity, thus opening up new possibilities in the study of obesity.     

Glucocorticoid-induced androgen inactivation by aldo-keto reductase 1C2 promotes adipogenesis in human preadipocytes

Adipogenesis and lipid storage in human adipose tissue are inhibited by androgens such as DHT. Inactivation of DHT to 3α-diol is stimulated by glucocorticoids in human preadipocytes. We sought to characterize glucocorticoid-induced androgen inactivation in human preadipocytes and to establish its role in the antiadipogenic action of DHT. Subcutaneous and omental primary preadipocyte cultures were established from fat samples obtained in subjects undergoing abdominal surgeries. Inactivation of DHT to 3α/β-diol for 24 h was measured in dexamethasone- or vehicle-treated cells. Specific downregulation of aldo-keto reductase 1C (AKR1C) enzymes in human preadipocytes was achieved using RNA interference. In whole adipose tissue sample, cortisol production was positively correlated with androgen inactivation in both subcutaneous and omental adipose tissue (P < 0.05). Maximal dexamethasone (1 μM) stimulation of DHT inactivation was higher in omental compared with subcutaneous fat from men as well as subcutaneous and omental fat from women (P < 0.05). A significant positive correlation was observed between BMI and maximal dexamethasone-induced DHT inactivation rates in subcutaneous and omental adipose tissue of men and women (r = 0.24, n = 26, P < 0.01). siRNA-induced downregulation of AKR1C2, but not AKR1C1 or AKR1C3, significantly reduced basal and glucocorticoid-induced androgen inactivation rates (P < 0.05). The inhibitory action of DHT on preadipocyte differentiation was potentiated following AKR1C2 but not AKR1C1 or AKR1C3 downregulation. Specifically, lipid accumulation, G3PDH activity, and FABP4 mRNA expression in differentiated preadipocytes exposed to DHT were reduced further upon AKR1C2 siRNA transfection. We conclude that glucocorticoid-induced androgen inactivation is mediated by AKR1C2 and is particularly effective in omental preadipocytes of obese men. The interplay between glucocorticoids and AKR1C2-dependent androgen inactivation may locally modulate adipogenesis and lipid accumulation in a depot-specific manner.     

Perilipin Expression in Human Adipose Tissues: Effects of Severe Obesity,Gender, and Depot

Objective: Perilipins are phosphoproteins that are localized to the surface of triacylglycerol droplets within adipocytes where they regulate the rate of lipolysis. We sought to determine the effects of severe obesity and depot [omental (Om) vs. subcutaneous (Sc)] on perilipin expression in the adipose tissue of individuals. Research Methods and Procedures: Samples of Om and Sc adipose tissues obtained at surgery from severely obese subjects and fat aspirations from nonobese subjects were analyzed for perilipin protein and mRNA levels by Northern and Western analysis. Results: Perilipin A (periA) was the major perilipin expressed in adipose tissues. periA mRNA relative abundance was significantly lower in Sc adipose tissue from severely obese compared to that from nonobese subjects. Western blotting of adipose tissue extracts showed that periA protein levels expressed relative to tissue protein or fat cell surface area were significantly lower (～ ?40%) in abdominal Sc adipose tissue from severely obese compared to that from nonobese subjects. However, the calculated mass of perilipin per fat cell did not differ between the two groups. Perilipin mRNA levels were higher in Sc compared to Om adipose tissue from obese individuals (p < 0.025; n = 26; 17 women, 9 men); however, periA protein levels did not differ. In addition, perilipin protein, but not mRNA, levels were higher in Sc adipose tissue from obese men than from women (p < 0.025). Discussion: Variations in perilipin expression may contribute to the higher basal lipolytic rates observed in obese compared to nonobese individuals and in obese women compared to obese men.     

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.