OBJECTIVE:To study the relation between body mass index (BMI) and age on the one hand and total number of human (pre-) adipocytes and preadipocyte differentiation capacity on the other hand. SUBJECTS: In total, 189 women undergoing surgical mammary reduction, age range 16-73 y, BMI range 19.7-39.7 kg/m 2 . MEASUREMENTS: Differentiation of preadipocytes in primary culture was assessed by morphological criteria, and determination of glycerol-3-phosphate dehydrogenase after stimulation of the cells by standardized adipogenic conditions containing isobutyl-methylxanthine, troglitazone or both compounds. The total number of stromal cells (ie preadipocytes) and fat cells per gram of adipose tissue and per body as well as mature fat cell volume were calculated from isolated stromal cells and adipocytes, respectively, and anthropometric measures. RESULTS: BMI correlated positively to age, mature fat cell size and total number of adipocytes and stromal cells per body (r varying from 0.22 to 0.54, each Po0.05). In contrast, BMI correlated negatively to the number of adipocytes and stromal cells per gram of adipose tissue and the capacity of preadipocytes to differentiate (r varying from À0.20 to À0.37, each Po0.05). No significant correlation was observed between BMI and the ratio of stromal cells to adipocytes. The sample was also divided into three groups: BMI o25 kg/m 2 (lean), BMI 25-29.9 kg/m 2 (overweight) and BMI Z30 kg/m 2 (obese). The overweight group showed a larger fat cell size but no increase in total fat cell or stromal cell number when compared to the lean subjects. The obese subjects showed larger stromal and fat cell numbers when compared to the lean subjects. Age did not independently correlate to the number of stromal cells or adipocytes per gram of adipose tissue or total body, nor with the capacity of preadipocytes to undergo differentiation and the ratio of stromal cells to adipocytes. CONCLUSION: There seems to be a constant ratio between the number of adipose tissue stromal cells and adipocytes independently of BMI and age in humans. During adipose tissue expansion, there seems to be both a continuous increase in fat cell size, and in stromal cell and adipocyte number, but the increase in fat cell size apparently precedes the increase in fat cell number. The differentiation capacity of the stromal cells appears to decrease with increasing BMI.

Summary Tumour necrosis factor alpha (TNFa) has been found to cause a delipidation of fat cells and a decrease of the adipose tissue mass. In the present study, we tried to elucidate some of the mechanisms responsible for this phenomenon by investigating the action of TNFa on specific pathways which are involved in lipid storage. Cultured stromal cells from human adipose tissue were induced to differentiate into adipose cells by exposure to adipogenic factors and subsequently used for studying the effects of TNFa on fat cell metabolism. Presence of 5 nmol/l TNFa for 24 h resulted in a complete loss of the stimulatory effect of insulin on 2-deoxy-glucose transport. This inhibitory action was paralleled by a decrease of GLUT4 protein and mRNA levels. The amount of cellular GLUT4 protein was reduced by 49 + 3 % after a 24-h exposure and by 82 + 18 % after a 72-h exposure to 5 nmol/1 TNFt~. GLUT4 mRNA was almost undetectable after a 24-h incubation with 5 nmol/1 TNFct In a similar time-dependent manner, TNFa dramatically reduced the lipoprotein lipase mRNA content of the cells. Furthermore, incubation of cultured human fat ceils with TNFa resulted in a marked dose-dependent stimulation of lipolysis, assessed by glycerol release, by up to 400 % above controis, which became apparent after a 6-h exposure at the earliest. These data suggest that TNFtx induces a catabolic state in human adipose tissue which includes a loss of the stimulatory effect of insulin on glucose transport. These multiple actions of TNFa may contribute to the loss of adipose tissue observed during cachexia in man. [Diabetologia (1995) 38: 764-771]

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Abstract.We investigated the effects of epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) on the differentiation of human adipocyte precursor cells and some metabolic aspects of newly formed fat cells kept in primary culture. Exposure of stromal cells from human adipose tissue to EGF (O.O1-lOOngmL-l) resulted in a dose-and time-dependent decrease in the number of developing fat cells and the activity of glycerol-3-phosphate dehydrogenase (GPDH), a marker of adipose differentiation. Continuous presence of EGF completely blocked lipid accumulation with a EDso in the range of 0.2ngmL-I. This inhibitory action of EGF was associated with a potent stimulation of cell proliferation, up to 8-fold compared with cultures in the absence of EGF. PDGF (0.1-50ngmL-I) and FGF (O.l-lOOngmL-') provoked a less marked suppression of GPDH activities which was significant at concentrations of 10 ngmL-' and higher. A 12 day exposure to EGF of differentiated cells was followed by a suppression of GPDH and, again, a significant increase in cell number. Concomitantly, a distinct loss of cellular lipids was observed in the newly formed adipocytes. This effect could be partly explained by a stimulation of lipolysis, since EGF caused an increase of glycerol in the culture medium. Addition of PDGF or FGF to newly developed fat cells had no effect on lipolysis but, at higher concentrations, also decreased GPDH activity. These findings suggest that EGF is a potent inhibitor of adipose cell differentiation at physiological concentrations, and may be involved in the control of human adipose tissue development and function.

Tumor necrosis factor (TNF)-alpha is postulated to play a major role in the pathogenesis of obesity-linked insulin resistance, probably resulting from an interaction with insulin signaling pathways. This cross talk has now been investigated in human adipocytes at the level of phosphatidylinositol (PI) 3-kinase, and the TNF receptors (TNFRs) mediating these processes have been identified. Equilibrium binding studies using human adipocytes from mammary tissue indicated the presence of two populations of TNFR with apparent affinity constants of 13 pmol/l and 1.6 nmol/l, respectively. Interaction of TNF-alpha with insulin signaling was determined by quantification of insulin receptor substrate (IRS)-1-associated PI 3-kinase activity. Under control conditions, PI 3-kinase was activated about 10-fold in response to insulin (10[-7] mol/l, 5 min). Preincubation of adipocytes with 5 nmol/l TNF-alpha for 15 min resulted in a 60-70% reduction of insulin action, reaching a stable inhibition (40%) after longer incubation with the cytokine. The inhibitory action of TNF-alpha was dose-dependent, already detectable at 10 pmol/l, and was correlated to inhibition of tyrosine phosphorylation of IRS-1 with an unaltered autophosphorylation of the insulin receptor beta-subunit. The modulation of insulin signaling by TNF-alpha was found to be paralleled by a comparable inhibition of insulin-stimulated glucose transport. An agonistic TNFR1 antibody completely mimicked the inhibitory action of TNF-alpha on insulin signaling, whereas at 100 pmol/l TNF-alpha, a nonagonistic p80 TNFR antibody, was shown to ameliorate the inhibitory action of the cytokine. These findings indicate that in human adipocytes, low concentrations of TNF-alpha induce a rapid inhibition of insulin signaling at the level of PI 3-kinase. We suggest that under these conditions, the p80 TNFR is essential for initiating the intracellular cross talk that involves signaling by the p60 TNFR.

Objective: Interleukin (IL)-18, an important mediator of innate immunity and strong risk factor for the development of cardiovascular disease, was shown recently to be elevated in obesity. The aim of our study was to investigate whether human adipocytes produce IL-18. Methods: Human adipose tissue was obtained from lean women undergoing elective plastic surgery and from obese individuals undergoing laparoscopic surgery (gastric banding). Preadipocytes from mammary adipose tissue were isolated and differentiated under defined adipogenic conditions. IL-18 expression was analyzed by real-time reverse transcriptase PCR, ELISA and immunocytochemistry. Results: Human preadipocytes of all differentiation stages spontaneously secreted IL-18. In parallel significant amounts of IL-18 mRNA were detected. Freshly isolated mature adipocytes from subcutaneous and omental depots also released IL-18. IL-18 release from adipocytes from obese donors was about 3-fold higher compared to adipocytes from non-obese donors. Conclusions: We conclude that human adipose tissue produces IL-18 and thereby contributes to systemic IL-18 concentrations. This finding supports the concepts that adipocytes behave as primitive immune cells and that IL-18 may mediate some of the detrimental complications of obesity such as cardiovascular disease and type 2 diabetes.

Abstract-Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are characteristic for obesity and are associated with increased risk of thromboembolic complications. PAI-1 recently was reported to be expressed and secreted by human adipocytes, but little is known about regulation of PAI-1 in human adipose tissue. Therefore, we examined the effects of selected cytokines present in adipose tissue on expression and secretion of PAI-1 in in vitro, differentiated subcutaneous human adipocytes in primary culture. Transforming growth factor-␤1 (TGF-␤1) increased PAI-1 secretion in a dose-and time-dependent manner. PAI-1 protein increased by 3.2-fold and PAI-1 mRNA by 1.9-fold after a 6-hour exposure to 400 pmol/L TGF-␤1. This effect is probably mediated by TGF-␤1 type 2 and 3 receptors, which were found to be expressed in cultured human adipocytes. Moreover, TNF-␣ and interkeukin-1␤ (IL-1␤) also exerted a stimulatory effect on PAI-1 release and increased PAI-1 mRNA levels. As assessed by a semiquantitative reverse transcriptionpolymerase chain reaction technique, TGF-␤1 mRNA is expressed by differentiation of human preadipocytes and is moderately upregulated by TNF-␣ and IL-1␤. In conclusion, our results clearly indicate that TGF-␤1 is a potent inducer of PAI-1 production in subcutaneous human adipocytes. In addition, data suggest that TNF-␣ and IL-1␤ also have stimulatory effects on PAI-1 protein secretion and may contribute to the elevated PAI-1 levels observed in obesity.