Identification of depot-specific human fat cell progenitors through distinct expression profiles and developmental gene patterns

Anatomically separate fat depots differ in size, function, and contribution to pathological states, such as the metabolic syndrome. We isolated preadipocytes from different human fat depots to determine whether the basis for this variation is partly attributable to differences in inherent properties of fat cell progenitors. We found that genome-wide expression profiles of primary preadipocytes cultured in parallel from abdominal subcutaneous, mesenteric, and omental fat depots were distinct. Interestingly, visceral fat was not homogeneous. Preadipocytes from one of the two main visceral depots, mesenteric fat, had an expression profile closer to that of subcutaneous than omental preadipocytes, the other main visceral depot. Expression of genes that regulate early development, including homeotic genes, differed extensively among undifferentiated preadipocytes isolated from different fat depots. These profiles were confirmed by real-time PCR analysis of preadipocytes from additional lean and obese male and female subjects. We made preadipocyte strains from single abdominal subcutaneous and omental preadipocytes by expressing telomerase. Depot-specific developmental gene expression profiles persisted for 40 population doublings in these strains. Thus, human fat cell progenitors from different regions are effectively distinct, consistent with different fat depots being separate mini-organs.     

Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes

Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.     

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.     

Sex‐ and Depot‐Dependent Differences in Adipogenesis in Normal‐Weight Humans

To elucidate cellular mechanisms of sex‐related differences in fat distribution, we determined body fat distribution (dual‐energy X‐ray absorptiometry and single‐slice abdominal computed tomography (CT)), adipocyte size, adipocyte number, and proportion of early‐differentiated adipocytes (aP2⁺CD68^?) in the stromovascular fraction (SVF) in the upper and lower body of normal‐weight healthy men (n = 12) and premenopausal women (n = 20) (age: 18–49 years, BMI: 18–26 kg/m²). Women had more subcutaneous and less visceral fat than men. The proportion of early differentiated adipocytes in the subcutaneous adipose tissue SVF of women was greater than in men (P = 0.01), especially in the femoral depot, although in vitro adipogenesis, as assessed by peroxisome proliferator activated receptor‐γ (PPARγ) expression, was not increased in femoral preadipocytes cultured from women compared with men. In women, differentiation of femoral preadipocytes was less than that of abdominal subcutaneous preadipocytes (P = 0.04), and femoral subcutaneous preadipocytes tended to be more resistant to tumor necrosis factor‐α (TNFα)–induced apoptosis (P = 0.06). Thus, turnover and utilization of the preadipocyte pool may be reduced in lower vs. the upper‐body fat in women. Collectively, these data indicate that the microenvironment, rather than differences in inherent properties of preadipocytes between genders, may explain the gynoid obesity phenotype and higher percent body fat in women compared to men.     

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.     

Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells

Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression in?vivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots.     

Interleukin-6 release from human abdominal adipose cells is regulated by thyroid-stimulating hormone: effect of adipocyte differentiation and anatomic depot

Adipose cells are extrathyroidal targets of thyroid-stimulating hormone (TSH). TSH stimulates interleukin-6 (IL-6) release from adipocytes. We examined TSH responsiveness as a function of stage of differentiation or adipose tissue depot in cultured adipose cells and determined the effect of TSH on extrathyroidal IL-6 production in vivo. Stromal preadipocytes, isolated from human abdominal subcutaneous or omental adipose tissue, and their differentiated counterparts were studied. IL-6 protein concentration in the medium was measured after TSH stimulation. Basal IL-6 release was greater for preadipocytes than differentiated adipocytes, whether derived from subcutaneous or omental fat depots. A depot-dependent effect (omental > subcutaneous) on basal IL-6 release was observed for preadipocytes (1.6-fold, P < 0.05); a similar trend for differentiated adipocytes was not significant (6.2-fold, P > 0.05). IL-6 responsiveness to TSH was observed upon differentiation, but only for subcutaneous adipocytes (1.9-fold over basal, P < 0.001). To determine if TSH could stimulate IL-6 release from extrathyroidal tissues in vivo, we measured serum IL-6 levels from five thyroidectomized patients who received recombinant human (rh) TSH and found that levels increased by threefold on days 3 and 4 (P < 0.05) after its administration. Our data demonstrate that stage of differentiation and fat depot origin affect basal and TSH-stimulated IL-6 release from adipose cells in culture. Furthermore, rhTSH elevates serum IL-6 response in thyroidectomized patients, indicating an extrathyroidal site of TSH action.     

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.     

Preadipocyte conversion to macrophage. Evidence of plasticity

Preadipocytes are present throughout adult life in adipose tissues and can proliferate and differentiate into mature adipocytes according to the energy balance. An increasing number of reports demonstrate that cells from adipose lineages (preadipocytes and adipocytes) and macrophages share numerous functional or antigenic properties. No large scale comparison reflecting the phenotype complexity has been performed between these different cell types until now. We used profiling analysis to define the common features shared by preadipocyte, adipocyte, and macrophage populations. Our analysis showed that the preadipocyte profile is surprisingly closer to the macrophage than to the adipocyte profile. From these data, we hypothesized that in a macrophage environment preadipocytes could effectively be converted into macrophages. We injected labeled stroma-vascular cells isolated from mouse white adipose tissue or 3T3-L1 preadipocyte cell line into the peritoneal cavity of nude mice and investigated changes in their phenotype. Preadipocytes rapidly and massively acquired high phagocytic activity and index. 60-70% of preadipocytes also expressed five macrophage-specific antigens: F4/80, Mac-1, CD80, CD86, and CD45. These values were similar to those observed for peritoneal macrophages. In vitro experiments showed that cell-to-cell contact between preadipocytes and peritoneal macrophages partially induced this preadipocyte phenotype conversion. Overall, these results suggest that preadipocyte and macrophage phenotypes are very similar and that preadipocytes have the potential to be very efficiently and rapidly converted into macrophages. This work emphasizes the great cellular plasticity of adipose precursors and reinforces the link between adipose tissue and innate immunity processes.     

Regional uptake of meal fatty acids in humans

Two protocols were performed to study meal fatty acid metabolism. In protocol 1, 14 patients scheduled for elective intra-abdominal surgery (11 undergoing bariatric surgery for severe obesity) consumed a meal containing [3H]triolein in the evening before surgery. This allowed us to measure adipose tissue lipid specific activity (SA) in mesenteric and omental, deep and superficial abdominal subcutaneous adipose tissue. Intra-abdominal adipose tissue lipid SA was greater than subcutaneous lipid SA. There were no significant differences between mesenteric and omental or between deep and superficial abdominal subcutaneous adipose tissue. In protocol 2, meal fatty acid oxidation and uptake into subcutaneous and omental adipose tissue ([3H]triolein) were measured in six normal, healthy volunteers. Meal fatty acid oxidation (3H2O generation) plus that remaining in plasma ( approximately 1%) plus uptake into upper body subcutaneous, lower body subcutaneous, and visceral fat allowed us to account for 98 +/- 6% of meal fatty acids 24 h after meal ingestion. We conclude that omental fat is a good surrogate for visceral fat and that abdominal subcutaneous fat depots are comparable with regard to meal fatty acid metabolic studies. Using [3H]triolein, we were able to account for virtually 100% of meal fatty acids 24 h after meal ingestion. These results support the meal fatty acid tracer model as a way to study the metabolic fate of dietary fat.     

Plasma cells and Fc receptors in human adipose tissue--lipogenic and anti-inflammatory effects of immunoglobulins on adipocytes

We have previously reported high immunoglobulin expression in human omental adipose tissue. The aim of this work was to investigate plasma cell density and Fc receptor (FcR) expression in human adipose tissue depots and in vitro effects of immunoglobulins on adipocyte function. Plasma cell density was higher in the visceral compared to the subcutaneous depot (10.0+/-1.56% and 5.2+/-0.98%, respectively, n=20, p<0.05). Microarray analysis revealed expression of four FcR genes in adipose tissue; FCGR2A, FCGR2B, FCER1G, and FCGRT. FCGR2A was highly expressed in adipocytes in both depots and this was verified by immunohistochemistry. Expression of IL-1beta and IL-6 was markedly reduced in adipocytes after incubation with the Fc moiety of immunoglobulin G (Fc) (p<0.01). Furthermore, Fc stimulated adipocyte lipogenesis as potently as insulin (p<0.05), but did not influence lipolysis. In conclusion, immunoglobulins produced by plasma cells in human adipose tissue could influence adipocyte metabolism and cytokine production.     

CD36 is important for adipocyte recruitment and affects lipolysis

Objective: The scavenger receptor CD36 facilitates the cellular uptake of long‐chain fatty acids. As CD36‐deficiency attenuates the development of high fat diet (HFD)‐induced obesity, the role of CD36‐deficiency in preadipocyte recruitment and adipocyte function was set out to characterize. Design and Methods: Fat cell size and number were determined in gonadal, visceral, and subcutaneous adipose tissue of CD36^?/? and WT mice after 6 weeks on HFD. Basal lipolysis and insulin‐inhibited lipolysis were investigated in gonadal adipose tissue. Results: CD36^?/? mice showed a reduction in adipocyte size in all fat pads. Gonadal adipose tissue also showed a lower total number of adipocytes because of a lower number of very small adipocytes (diameter <50 μm). This was accompanied by an increased pool of preadipocytes, which suggests that CD36‐deficiency reduces the capacity of preadipocytes to become adipocytes. Regarding lipolysis, in adipose tissue from CD36^?/? mice, cAMP levels were increased and both basal and 8‐bromo‐cAMP stimulated lipolysis were higher. However, insulin‐mediated inhibition of lipolysis was more potent in CD36^?/? mice. Conclusions: These results indicate that during fat depot expansion, CD36‐deficiency negatively affects preadipocyte recruitment and that in mature adipocytes, CD36‐deficiency is associated with increased basal lipolysis and insulin responsiveness.     

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.     

Antagonistic effects of thiazolidinediones and cytokines in lipotoxicity

Ectopic lipid accumulation is promoted by obesity and an impaired ability to accumulate triglycerides in the subcutaneous depots. The adipose tissue is dysregulated in hypertrophic obesity, i.e., when the adipose cells have become enlarged. In some individuals, however, obesity is a consequence of a recruitment of new adipocytes, i.e., a hyperplastic obesity. This form of obesity is usually not associated with the metabolic complications and is termed “obese but metabolically normal”. We here review recent findings showing that hypertrophic obesity is associated with an impaired differentiation of committed preadipocytes. This may be a primary (genetic?) event, thus leading to hypertrophic fat cells and the associated inflammation. However, it is also possible that the inflammation is a primary event allowing, in particular, TNFα to inhibit preadipocyte differentiation. TNFα, instead, promotes a partial transdifferentiation of the preadipocytes to assume a macrophage-like phenotype. PPARγ activation promotes adipogenesis but can apparently not overcome the impaired preadipocyte differentiation seen in hypertrophic obesity.     

Using gene expression to predict differences in the secretome of human omental vs. subcutaneous adipose tissue

The objective of this study was to characterize differences in the secretome of human omental compared with subcutaneous adipose tissue using global gene expression profiling. Gene expression was measured using Affymetrix microarrays (Affymetrix, Santa Clara, CA) in subcutaneous and omental adipose tissue in two independent experiments (n = 5 and n = 3 independent subjects; n = 16 arrays in total, 2 for each subject). Predictive bioinformatic algorithms were employed to identify secreted proteins. Microarray analysis identified 22 gene probe sets whose expression was significantly different with a fold change (FC) greater than 5 in expression in both experiments between omental and subcutaneous adipose tissue. Using bioinformatic predictive programs 11 of these 22 probe sets potentially coded for secreted proteins. Pathway network analysis of the secreted proteins showed that three of the proteins are part of a common pathway network. These proteins gremlin 1 (GREM1), pleiotrophin (PTN), and secretory leukocyte peptidase inhibitor (SLPI) are expressed respectively 43×, 23×, and 5× in omental adipose tissue relative to subcutaneous adipose tissue as determined by real-time PCR. The presence of GREM1, PTN, and SLPI protein in human adipose tissue was confirmed by western blotting. All three proteins are expressed in the human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell line. The expression of GREM1, PTN, and SLPI changed with the differentiation of the preadipocytes into mature adipocytes. Gene expression coupled with predictive bioinformatic algorithms have identified several genes coding for secreted proteins which are expressed differently in omental adipose tissue compared to subcutaneous adipose tissue proving a valid alternative approach to help further define the adipocyte secretome.     

Depot specific differences during adipogenesis of porcine stromal-vascular cells

Recently a role of adipose tissue as an endocrine organ secreting factors involved in the regulation of whole-body energy homeostasis has emerged. Preadipocytes in different fat depots have distinct adipogenic potential and the metabolic activity differs between mature adipocytes of different depot origins. Here we describe the proliferation and differentiation of stromal-vascular cells derived from subcutaneous and visceral fat depots of adult pigs. We demonstrate that subcutaneous porcine preadipocytes proliferate more actively and that individual subcutaneous adipocytes have a more rapid accumulation of triacylglycerols than visceral cells. During differentiation, subcutaneous and visceral preadipocytes showed similar gene expression patterns with increased expression of adiponectin (APM1), adipocyte-specific fatty acid binding protein (FABP4), catalase (CAT), and peroxisome proliferator-activated receptor gamma 2 (PPARG2). Furthermore, initial data showing depot-originated effects on the expression of CAT, carnitine palmitoyl transferase 1B (CPT1B) and FABP4 suggest possible depot specific differences in the function and metabolism of mature porcine adipocytes.     

Human adipose tissue endothelial cells promote preadipocyte proliferation

Adipogenesis is preceded by development of a microvascular network, and optimal functioning of adipose tissue as an energy store and endocrine organ is dependent on extensive vascularization. We have examined the role of endothelial cell-derived factors that influence the proliferation of human preadipocytes. Microvascular endothelial cells and preadipocytes were isolated from human omental and subcutaneous adipose tissue biopsies by use of a developed procedure of collagenase digest, immunoselection, and differential trypsinization. Conditioned medium from microvascular endothelial cell cultures promoted the proliferation of preadipocytes (P = <0.001) and (to a lesser extent) other cell types. No depot-specific differences in mitogenic capacity of microvascular endothelial cell medium or of preadipocyte response were observed. These results indicate that adipose tissue endothelial cells secrete soluble adipogenic factor(s).