Age-related adipose tissue mRNA expression of ADD1/SREBP1, PPARgamma, lipoprotein lipase, and GLUT4 glucose transporter in rhesus monkeys.

Aging has been shown to have an effect on the capacity to differentiate preadipocytes and on the expression of some genes expressed in adipose tissue. The mRNA levels of adipocyte differentiation-related genes were examined in rhesus monkeys (Macaca Mulatta) ranging in age from 7 to 30 years. The effect of aging on the expression of peroxisome proliferator activated receptor gamma (PPARgamma), adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element binding protein 1 (ADD1/SREBP1), CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL), GLUT4 glucose transporter, and adipsin were examined by slot blot analysis. Significant inverse correlations were observed between age and the mRNA levels of PPARgamma, ADD1/SREBP1, LPL, and GLUT4. The coordinate downregulation of these genes may be linked to the declining fat mass of senescent animals. There was no correlation between age and the mRNA levels of adipsin. The mRNA levels of these genes were not correlated to body weight orfasting plasma insulin. These findings indicate that aging may have an effect on the adipocyte differentiation program and this effect appears to be gene specific.     

Effect of troglitazone on tumor necrosis factor alpha and transforming growth factor beta expression and action in human adipocyte precursor cells in primary culture

Troglitazone is a member of the class of thiazolidinediones that are known to act as insulin-sensitizing agents. Administration of these compounds ameliorates insulin resistance in type 2 diabetic patients, but may also promote weight gain. The main site of action is adipose tissue, where troglitazone binds to and activates the nuclear receptor peroxisome proliferator-activated receptor gamma2. The aim of this study was to investigate whether troglitazone is able to affect the adipose expression and function of tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta). Both TNF-alpha and TGF-beta blocked adipose differentiation in vitro and led to a marked reduction in glycerol-3-phosphate dehydrogenase activity, a marker enzyme of adipose differentiation, by 69% +/- 11% and 75% +/- 15%, respectively. Addition of 2 mumol/L troglitazone significantly reduced this inhibitory effect of both cytokines on glycerol-3-phosphate dehydrogenase activity. Peroxisome proliferator-activated receptor gamma messenger RNA (mRNA) was reduced by TNF-alpha in freshly isolated adipocytes. This effect was completely counteracted by troglitazone, whereas TGF-beta had no immediate effect on peroxisome proliferator-activated receptor gamma mRNA. Moreover, troglitazone alone promoted adipose differentiation in a time- and dose-dependent manner. Troglitazone treatment was found to result in a marked reduction of TNF-alpha mRNA expression in human preadipocytes to 54% +/- 13% compared with untreated cultures. Furthermore, troglitazone was observed to partially antagonize the inhibitory effect of TNF-alpha on insulin-stimulated 2-deoxy-glucose uptake in newly differentiated human fat cells. In conclusion, troglitazone exerts a potent adipogenic activity in human preadipocytes, which may be mediated by suppression of the endogenous production of TNF-alpha and by counteracting the antiadipogenic effect of TGF-beta. In addition, troglitazone improved insulin-stimulated glucose uptake in differentiated fat cells.     

Tumour necrosis factor-alpha exerts dual effects on human adipose leptin synthesis and release

The acute and chronic effects of tumour necrosis factor-alpha (TNF-alpha) on leptin production by human preadipocytes, differentiated preadipocytes, and mature adipocytes have been examined by competitive RT-PCR of leptin mRNA and by western blotting. In preadipocytes, secreted leptin was detectable after 5-day incubation in differentiation medium and this increased 4-fold by day 20. TNF-alpha blocked leptin synthesis during differentiation. In differentiated preadipocytes and mature adipocytes, TNF-alpha treatment resulted in time-dependent decreases in mRNA for leptin and glycerol-3-phosphate dehydrogenase (G3PD). In contrast, TNF-alpha (4-8-h treatment) resulted in a 4-fold increase in leptin release. This effect was lost at 24 h and leptin accumulation in culture medium was decreased 24-48 h after TNF-alpha addition. We conclude that TNF-alpha stimulates the release of preformed leptin from human mature adipocytes and existing differentiated preadipocytes, which may contribute to obesity/infection-linked hyperleptinemia, and that TNF-alpha inhibits leptin synthesis via inhibition of preadipocyte differentiation and induction of adipocyte dedifferentiation.     

The role of ghrelin and growth hormone secretagogues receptor on rat adipogenesis

Recent research progress indicates a close link between ghrelin, a natural ligand of GH secretagogues receptor (GHS-R), and both the metabolic balance and body composition. To clarify the involvement of ghrelin and GHS-R in the process of adipogenesis, we measured the expression of GHS-R and peroxisome proliferator-activated receptor gamma 2 (PPAR-gamma 2) mRNA in rat adipocytes using semiquantitative RT-PCR methods. The levels of GHS-R mRNA increased by up to 4-fold in adipose tissue from epididymal and parametrial regions as the rat aged from 4-20 wk and were significantly elevated during the differentiation of preadipocytes in vitro. Ghrelin (10(-8) M for 10 d) stimulated the activity of glycerol-3-phosphate dehydrogenase and the differentiation of rat preadipocytes in vitro. Ghrelin treatment also significantly increased the levels of PPAR-gamma 2 mRNA in primary cultured rat differentiated adipocytes. In addition, isoproterenol (10(-8) M, 40 min)-stimulated lipolysis was significantly reduced by simultaneous ghrelin treatment in a dose-dependent manner in vitro. In conclusion, the expression of GHS-R in rat adipocytes increases with the age and during adipogenesis. Ghrelin in vitro stimulates the differentiation of preadipocytes and antagonizes lipolysis. Ghrelin may therefore play an important role in the process of adipogenesis in rats.     

A novel selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor prevents human adipogenesis

Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating cortisol concentrations are normal in most patients with metabolic syndrome (MS). At a pre-receptor level, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisol from cortisone locally within adipose tissue, and inhibition of 11beta-HSD1 in liver and adipose tissue has been proposed as a novel therapy to treat MS by reducing hepatic glucose output and adiposity. Using a transformed human subcutaneous preadipocyte cell line (Chub-S7) and human primary preadipocytes, we have defined the role of glucocorticoids and 11beta-HSD1 in regulating adipose tissue differentiation. Human cells were differentiated with 1.0 microM cortisol (F), or cortisone (E) with or without 100 nM of a highly selective 11beta-HSD1 inhibitor PF-877423. 11beta-HSD1 mRNA expression increased across adipocyte differentiation (P<0.001, n=4), which was paralleled by an increase in 11beta-HSD1 oxo-reductase activity (from nil on day 0 to 5.9+/-1.9 pmol/mg per h on day 16, P<0.01, n=7). Cortisone enhanced adipocyte differentiation; fatty acid-binding protein 4 expression increased 312-fold (P<0.001) and glycerol-3-phosphate dehydrogenase 47-fold (P<0.001) versus controls. This was abolished by co-incubation with PF-877423. In addition, cellular lipid content decreased significantly. These findings were confirmed in the primary cultures of human subcutaneous preadipocytes. The increase in 11beta-HSD1 mRNA expression and activity is essential for the induction of human adipogenesis. Blocking adipogenesis with a novel and specific 11beta-HSD1 inhibitor may represent a novel approach to treat obesity in patients with MS.     

Regulation of differentiation of sheep subcutaneous and abdominal preadipocytes in culture.

Factors regulating the differentiation of sheep subcutaneous and abdominal (omental or perirenal) preadipocytes from foetal lambs, suckling lambs and fattening sheep have been investigated using a serum-free cell culture system. Differentiation was assessed by changes in the activity of the enzyme glycerol 3-phosphate dehydrogenase. Insulin or IGF-I was essential for differentiation. Dexamethasone, a lipid supplement (Excyte) and the thiazolidinedione, rosiglitazone (BRL 49653) (a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist) all enhanced preadipocyte differentiation, whereas fibroblast growth factor and GH were inhibitory. The most effective combination was insulin, triiodothyronine, dexamethasone and rosiglitazone. Under suboptimal conditions, preadipocytes from fattening sheep differentiated less well than cells from suckling and foetal lambs. Also, under suboptimal conditions, abdominal preadipocytes did not differentiate as well as subcutaneous preadipocytes. However, age and depot differences were minimised when cells were cultured with insulin, triiodothyronine, rosiglitazone and either dexamethasone or lipid. The results suggest that variation in the ability to produce the natural ligand for PPAR-gamma contributes to depot- and age-specific differences in the ability of preadipocytes to differentiate.     

Differentiation dependent biphasic regulation of adipsin gene expression by insulin and insulin-like growth factor-1 in 3T3-F442A adipocytes

Adipsin is a serine protease with complement factor D activity that is synthesized by adipocytes and secreted into the blood stream. Expression of adipsin is deficient in models of genetic (ob/ob, db/db) and acquired (monosodium glutamate-lesioned) obesity, but the cellular mechanisms responsible for this deficiency are unknown. Because hyperinsulinemia is frequently associated with obesity, we evaluated the effects of this hormone and insulin-like growth factor 1 (IGF-1) on adipsin secretion and adipsin messenger RNA (mRNA) levels in 3T3-F442A adipocytes. In the present study, we report that in fully differentiated adipocytes (after 11 days post confluence), insulin exposure progressively decreases adipsin secretion by 40%, 67%, and 78% after 2, 4, and 6 days of treatment. The inhibition of adipsin secretion by insulin is the result of a corresponding decrease in adipsin mRNA and is specific since two other differentiation-dependent fat cell mRNAs encoding aP2 (a fatty acid binding protein) and glycerophosphate dehydrogenase (GPD), are unaffected. Insulin suppresses adipsin gene expression via high affinity insulin receptors, because physiological levels of insulin produce this effect, and dose-response curves for insulin stimulation of 2-deoxyglucose uptake and glucose utilization are similar to insulin's effect on adipsin. In contrast, insulin when present during days 1-8 post confluence (during differentiation) markedly increases adipsin secretion and adipsin mRNA levels. This stimulation is due to the ability of insulin to accelerate differentiation as evidenced by corresponding increases in aP2 and GPD mRNAs as well. Insulin and IGF-1 are equipotent in this effect, suggesting that both insulin and IGF-1 receptors can mediate this response. In summary, during the differentiation of 3T3-F442A adipocytes, insulin stimulates adipsin gene expression by accelerating differentiation. As the cells become mature adipocytes, they acquire some differentiation-dependent factor, which couples insulin receptor stimulation to inhibition of adipsin gene expression. This model should aid our search for the molecular links between insulin receptor stimulation and altered gene expression.     

Preadipocyte 11beta-hydroxysteroid dehydrogenase type 1 is a keto-reductase and contributes to diet-induced visceral obesity in vivo

Glucocorticoid excess promotes visceral obesity and cardiovascular disease. Similar features are found in the highly prevalent metabolic syndrome in the absence of high levels of systemic cortisol. Although elevated activity of the glucocorticoid-amplifying enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) within adipocytes might explain this paradox, the potential role of 11beta-HSD1 in preadipocytes is less clear; human omental adipose stromal vascular (ASV) cells exhibit 11beta-dehydrogenase activity (inactivation of glucocorticoids) probably due to the absence of cofactor provision by hexose-6-phosphate dehydrogenase. To clarify the depot-specific impact of 11beta-HSD1, we assessed whether preadipocytes in ASV from mesenteric (as a representative of visceral adipose tissue) and sc tissue displayed 11beta-HSD1 activity in mice. 11beta-HSD1 was highly expressed in freshly isolated ASV cells, predominantly in preadipocytes. 11beta-HSD1 mRNA and protein levels were comparable between ASV and adipocyte fractions in both depots. 11beta-HSD1 was an 11beta-reductase, thus reactivating glucocorticoids in ASV cells, consistent with hexose-6-phosphate dehydrogenase mRNA expression. Unexpectedly, glucocorticoid reactivation was higher in intact mesenteric ASV cells despite a lower expression of 11beta-HSD1 mRNA and protein (homogenate activity) levels than sc ASV cells. This suggests a novel depot-specific control over 11beta-HSD1 enzyme activity. In vivo, high-fat diet-induced obesity was accompanied by increased visceral fat preadipocyte differentiation in wild-type but not 11beta-HSD1(-/-) mice. The results suggest that 11beta-HSD1 reductase activity is augmented in mouse mesenteric preadipocytes where it promotes preadipocyte differentiation and contributes to visceral fat accumulation in obesity.     

RAS阻断剂对人前脂肪细胞分化和胰岛素敏感性的影响

目的 探讨肾素-血管紧张素系统(RAS)阻断剂对体外原代培养的人内脏和外周来源的前脂肪细胞分化和胰岛素敏感性的影响. 方法 自16例行电切开腹部手术的健康成年女性腹部皮下和网膜分离前脂肪细胞.分为4组,即无干预的正常对照(NC)组、噻唑烷二酮类约物吡格列酮(Pioglitazone)组、血管紧张素转换酶抑制剂(ACEI)类药物贝那普利(Benazapril)组和血管紧张素Ⅱ受体拮抗剂(ARB)类药物替米沙坦(Telmisartan)组,诱导分化共14 d.观察细胞活力、细胞内脂质含量和前脂肪细胞分化标志物--甘油-3-磷酸脱氧酶的活性.细胞的胰岛素敏感性通过葡萄糖消耗试验测定. 结果 对照组皮下来源的前脂肪细胞活力高于网膜,脂质含量反而低于网膜来源细胞,二者胰岛素敏感性无差别.与对照组相比,贝那普利、替米沙坦和吡格列酮均能明显提高网膜和皮下来源前脂肪细胞的细胞活力和脂质含量,并提高细胞的胰岛素敏感性;其中,替米沙坦组网膜前脂肪细胞的上述各指标均高于吡格列酮组.在网膜,替米沙坦和吡格列酬组的葡萄糖消耗量分别为(5.567±1.612)mmol/L和(4.418±1.572)mmol/L,P=0.020;皮下则相反,两组葡萄糖消耗量分别为(5.335±1.461)mmol/L和(7.506±1.615)mmol/L,P<0.01. 结论 RAS阻断剂(替米沙坦和贝那普利)可促进人前脂肪细胞分化并改善细胞的胰岛素敏感性,且较之吡格列酮在内脏发挥优势性作用.     

Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptor gamma2

To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17beta-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (x2) of 17beta-estradiol (0.01 microM) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 microM) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17beta-estradiol (0.01 microM), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariectomized female rats, 17beta-estradiol significantly increased (x1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor gamma2 was up-regulated by 17beta-estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 beta-estradiol (0.01 microM) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 microM) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor gamma2 expression (estrogens).     

Häufigkeit terminal differenzierter Fibroblasten in der Synovialmembran von Rheumapatienten

The metabolic activation of synovial fibroblasts (SF) and their expression of matrix degrading enzymes and inflammatory cytokines contributes to the pathology of rheumatoid arthritis (RA). It is remarkable that SF of RA patients do not proliferate at higher rates when compared to SF of other patients, but they are resistant to apotposis inducing signals. The chronic inflammation in RA causes fibrosis of the synovial tissue and fibrosis has been associated with terminal differentiation. Therefore we investigated if there are increased numbers of terminally differentiated fibroblasts in the RA synovium and if there is a correlation between terminal differentiation of SF and increased levels of expression of interleukins and matrix metalloproteinases. We analyzed specimen of four RA patients, two patients with osteoarthritis (OA) and two healthy donors suffering from joint injuries. By use of RT-PCR techniques we examined mRNA expression of two genes in SF which are associated with terminal differentiation, p16INK4a and p21-cip. In addition, we labelled differentiated fibroblasts using the SA-beta-galaktosidase assay and investigated differences in protein expression patterns of factor PIVa and the tropomyosin 1 and 2 molecules. We report that the number of terminally differentiated fibrolasts are not increased in the synovial membrane of RA patients. On the contrary we show that the synovia of the much younger patients has higher levels of terminally differentiated fibroblasts. Consequently, the fibrosis of synovial tissues in RA patients at later stages of disorder is not associated with proliferation and differentiation of the fibroblasts but rather a consequence of chronic inflammation.     

Differentiation of human adipocyte precursors in a chemically defined serum-free medium

Stromal-vascular cells from the inguinal fat tissue of human (age range 1.5 month-27 years), were able to undergo adipose conversion when cultured in a medium containing insulin, transferrin and triiodothyronine. Between 10 and 20 per cent of the cells changed their morphology and accumulated lipid droplets within 10 to 15 days. In most cultures, differentiated cells were present in clusters. These clustered cells were shown by indirect immunofluorescence to contain lipoprotein lipase (located in the Golgi region) and by histochemistry to contain glycerol-3-phosphate dehydrogenase. The occurrence of both enzymes was assessed directly by determining enzyme activities and the synthesis of triacylglycerol was demonstrated by incorporation of [U-14C]glucose into lipids. Foreskin fibroblasts did not display any of these phenotypes. The development of a serum-free, chemically defined medium for the differentiation of diploid adipocyte precursors from human should be of interest for the characterization of factors involved in the stimulation or inhibition of the differentiation process.     

Comparison of messenger RNA distribution for 60 proteins in fat cells vs the nonfat cells of human omental adipose tissue

The messenger RNA (mRNA) distribution of 60 proteins was examined in the 3 fractions obtained by collagenase digestion (fat cells and the nonfat cells comprising the tissue remaining after collagenase digestion [matrix] and the stromovascular cells) of omental adipose tissue obtained from morbidly obese women undergoing bariatric surgery. Fat cells were enriched by at least 3-fold as compared with nonfat cells in the mRNAs for retinol binding protein 4, angiotensinogen, adipsin, glutathione peroxidase 3, uncoupling protein 2, peroxisome proliferator-activated receptor gamma, cell death-inducing DFFA-like effector A, fat-specific protein 27, 11beta-hydroxysteroid dehydrogenase 1, glycerol channel aquaporin 7, NADPH:quinone oxidoreductase 1, cyclic adenosine monophosphate phosphodiesterase 3B, glyceraldehyde-3-phosphate dehydrogenase, insulin receptor, and amyloid A1. Fat cells were also enriched by at least 26-fold in the mRNAs for proteins involved in lipolysis such as hormone-sensitive lipase, lipoprotein lipase, adipose tissue triglyceride lipase, and FAT/CD36. The relative distribution of mRNAs in cultured preadipocytes was also compared with that of in vitro differentiated adipocytes derived from human omental adipose tissue. Cultured preadipocytes had far lower levels of the mRNAs for inflammatory proteins than the nonfat cells of omental adipose tissue. The nonfat cells were enriched by at least 5-fold in the mRNAs for proteins involved in the inflammatory response such as tumor necrosis factor alpha, interleukin lbeta, cyclooxygenase 2, interleukin 24, interleukin 6, and monocyte chemoattractant protein 1 plus the mRNAs for osteopontin, vaspin, endothelin, angiotensin II receptor 1, butyrylcholinesterase, lipocalin 2, and plasminogen activator inhibitor 1. The cells in the adipose tissue matrix were enriched at least 3-fold as compared with the isolated stromovascular cells in the mRNAs for proteins related to the inflammatory response, as well as osteopontin and endothelial nitric oxide synthase. We conclude that the mRNAs for inflammatory proteins are primarily present in the nonfat cells of human omental adipose tissue.     

甘露糖-6-磷酸对脂肪细胞分化的影响及其分子机制初探

目的 探讨甘露糖-6-磷酸(M6P)对脂肪细胞分化的影响及其分子机制.方法 油红O染色、测定TG及甘油含量观察M6P对3T3-L1前脂肪细胞株脂肪细胞分化进程的影响.激光共聚焦显微镜下观察组织蛋白酶K(CTSK)酶活性变化,四甲基偶氮唑盐比色法(MTT)检测前脂肪细胞增殖情况,同时RT-PCR检测M6P受体(M6PR)转录表达.结果 经不同浓度M6P干预后,油红O染色逐渐变浅,当M6P 10 mmol/L时细胞完全不被染色.同时TG合成及甘油含量明显下降,有剂量依赖关系,浓度分别为5 mmol/L和8 mmol/L时即与对照组差异有统计学意义(P＜0.05).脂肪细胞分化进程中的不同时间点均可检测到M6PR mRNA表达.加入M6P后CTSK酶活性呈剂量依赖性被抑制,10 mmol/L时,CTSK酶活性被完全抑制.MTT显示M6P加入后细胞增殖,10 mmol/L的M6PA值达到0.057±0.091,与对照组相比增加了62.9%(P＜0.05).结论 在3T3-L1前脂肪细胞中同样有M6PR表达,M6P可能通过竞争结合M6PR抑制CTSK酶活性,从而影响脂肪细胞分化进程.     

Expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue is not increased in human obesity

Central obesity is associated with increased morbidity and mortality. Preadipocyte proliferation and differentiation contribute to increases in adipose tissue mass, yet the mechanisms that underlie these processes remain unclear. Patients with glucocorticoid excess develop a reversible form of central obesity, but circulating cortisol levels in idiopathic obesity are invariably normal. We have hypothesized that the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), by converting inactive cortisone to active cortisol in adipose tissue, might be an important autocrine regulator of fat mass. Paired omental and sc fat biopsies were obtained from 32 women (median age, 43 yr; range, 28-65; median body mass index, 27.5 kg/m(2); range, 19.7-39.2) undergoing elective abdominal surgery. 11beta-HSD1 activity and mRNA levels were assessed in whole tissue and in isolated preadipocytes and adipocytes using specific enzyme assays and real-time PCR. Preadipocyte proliferation was measured using tritiated thymidine incorporation. Whole adipose tissue 11beta-HSD1 mRNA levels did not differ between omental and sc samples (P = 0.73). In addition, mRNA levels did not correlate with body mass index (omental: r = 0.1; P = 0.6; sc: r = 0.15; P = 0.4). In keeping with earlier studies, 11beta-HSD1 mRNA levels were higher in omental compared with sc preadipocytes. However, in cultured omental preadipocytes, 11beta-HSD1 activity inversely correlated with body mass index (r = -0.47; P = 0.03). In omental preadipocytes, both cortisol and cortisone decreased proliferation (P < 0.05). Inhibition of 11beta-HSD1 with glycyrrhetinic acid partially reversed the cortisone-induced decrease in preadipocyte proliferation (P < 0.05). Enhanced preadipocyte proliferation within omental adipose tissue as a consequence of decreased 11beta-HSD1 mRNA levels and activity may contribute to increases in visceral adipose tissue mass in obese patients.     

Comparison of human abdominal subcutaneous versus omental preadipocyte differentiation in primary culture

Excess intra-abdominal fat is associated with a higher risk for type 2 diabetes mellitus and cardiovascular disease, yet little is known about what influences regional adipose tissue accumulation. Adipocytes arise from specialized fibroblast-like preadipocytes within the adipose tissue stromal-vascular compartment. The aim of our study was to determine if there are variations in preadipocyte differentiation between abdominal subcutaneous (SC) and omental (OM) preadipocytes. Abdominal SC and OM preadipocytes were isolated from adipose tissue obtained from 18 subjects (7 men, 11 women), undergoing elective abdominal surgery, by collagenase treatment and filtration/centrifugation. Preadipocytes were placed in culture and then differentiated for 3 weeks in a serum-free medium containing insulin, dexamethasone, isobutylmethylxanthine, and carbaprostacyclin. The cells were then harvested for measurement of cytosolic glycerol phosphate dehydrogenase (GPDH), a marker of terminal differentiation. Data are expressed as a differentiation index (DI), which was the log of the SC/OM ratio of GPDH values for each patient (calculated as 0 for an equivalent SC v OM responses). The mean DI for the group (n = 18) was 0.04, with a 95% confidence interval (CI) of -0.11 to 0.20. The mean DI for men was 0.07 (95% CI, -0.06 to 0.19), and that for women was 0.03 (95% CI, -0.21 to 0.27). This indicates that SC versus OM preadipocyte differentiation responses were not significantly different from each other, either for the group as a whole or when divided by gender. Overall, 8 subjects had a DI favoring SC preadipocyte differentiation, compared to 11 subjects with a DI reflecting greater OM preadipocyte differentiation. There was no correlation of the DI with body mass index or age. Our results indicate that preadipocytes from the abdominal SC adipose tissue depot do not uniformly differentiate more than those from the OM depot.     

Evidence for protein and mRNA TSHr expression in fibroblasts from patients with thyroid-associated ophthalmopathy (TAO) after adipocytic differentiation

OBJECTIVE: Thyroid-associated ophthalmopathy (TAO) is a chronic autoimmune disorder characterized by an increased volume of adipose/connective tissue in the human orbit. DESIGN: The aim of this study was to investigate the thyrotropin receptor (TSHr) expression in orbital fibroblasts from TAO patients undergoing adipocytic differentiation. METHODS: Retro-ocular tissue and skin were obtained from five patients undergoing orbital decompression surgery for TAO and placed in culture. Proliferating fibroblasts were subjected to adipocytic differentiation for 10 days. Total RNA was isolated from fibroblasts and was reverse transcribed. TSHr mRNA levels were determined by real-time PCR. cAMP was determined by radioimmunoassay (RIA) after fibroblast incubation with the substances to test. RESULTS: Orbital differentiated fibroblasts became rounded and acquired lipid droplets. The amount of TSHr mRNA in these fibroblasts was higher than fibroblasts not subjected to adipocytic differentiation. Immunocytochemical analysis showed TSHr protein in differentiated orbital fibroblasts. Differentiated orbital fibroblasts stimulated with bovine (b) TSH showed a cAMP production greater than that in paired undifferentiated cultures. A specific thyroid-inhibiting antibody (TBAb) inhibited cAMP production after bTSH challenge, and a thyroid-stimulating antibody (TSAb) stimulated cAMP production in differentiated fibroblasts. CONCLUSIONS: We suggest that orbital fibroblasts subjected to adipocytic differentiation increase TSHr expression that responds specifically to bTSH and TSAb stimulation, and to TBAb inhibition.     

Comparison of proliferation and differentiation capacity of human adipocyte precursor cells from the omental and subcutaneous adipose tissue depot of obese subjects

Upper body obesity is characterized by an expansion of the visceral adipose tissue and is associated with an increased susceptibility for type 2 diabetes and cardiovascular disease. In order to get a better understanding of the regulation of body fat distribution, the aim of the present study was to compare adipocyte development between the omental and subcutaneous adipose tissue region in obese subjects. Therefore, the proliferation and differentiation capacity in primary cultures of adipose tissue-derived stromal cells were compared between the 2 depots in a group of 29 obese individuals, of which 21 were women. Proliferation of the cells was stimulated using fetal calf serum (FCS) and assessed by counting the cell number in the culture dishes. Differentiation of preadipocytes was assessed in parallel by morphological criteria and determination of glycerol-3-phosphate dehydrogenase (GPDH) after stimulation by standardized adipogenic conditions. Stromal cells from the subcutaneous adipose tissue region proliferated faster (doubling time, 4 +/- 1 days) than those from the omental region (doubling time, 5 +/- 1 days), whereas there was no regional difference in adipose differentiation with any of the adipogenic media. The same findings were observed when men were excluded from the analysis. Interestingly, there were more endothelial cells in the cultures from the omental tissue as compared to those from the subcutaneous tissue, but there was no correlation between endothelial cell contamination and proliferation capacity, suggesting that the regional difference in proliferation capacity was not due to regional differences in the amount of endothelial cells. In addition, we found a negative correlation between donor age and proliferation of subcutaneous cells but not of omental cells, possibly explaining the greater capacity for adipose tissue growth in the omental as compared to the subcutaneous depot with aging. In conclusion, there may exist regional differences in adipose tissue growth with regard to proliferation capacity, whereas there are apparently no significant differences in in vitro differentiation capacity between subcutaneous and omental preadipocytes.