These results indicate that severe obesity in women is characterized by increased amounts of the two TNF receptor mRNAs. The role of this up-regulation for the development of obesity-associated insulin resistance remains to be elucidated.

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.

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Objective: Leptin, the product of the ob gene, is overexpressed in human obesity and increased serum leptin levels are closely correlated with adipose tissue mass, but the regulation of leptin production is not completely understood. The aim of this study was to characterize the role of tumor necrosis factor (TNF)-a and transforming growth factor (TGF)-b1 in depot-specific secretion of leptin from cultured human adipocytes. Design and methods: We measured the leptin concentrations in the culture medium of omental and subcutaneous abdominal adipocytes taken from severely obese individuals and kept in suspension culture, and studied the effect of TNF-a and TGF-b1 on leptin release. Leptin protein was measured by radioimmunoassay, leptin mRNA was assessed by reverse transcriptase (RT)-PCR relative to a housekeeping gene. Results and conclusion: Leptin secretion from subcutaneous fat cells was 2-to 3-fold higher than that from omental fat cells after incubation for 2 and 24 h respectively. A 2-h exposure of adipocytes to 1 nmol/l TNF-a and 400 pmol/l TGF-b1 respectively did not significantly affect leptin secretion. Whereas a 24-h incubation with 1 nmol/l TNF-a also did not influence leptin secretion from fat cells from both depots, exposure of omental fat cells to 400 pmol/l TGF-b1 for 24 h resulted in a significant inhibitory effect (by 33%) on leptin secretion (P<0.05). A 24-and 48-h exposure of in vitro differentiated human adipocytes to TNF-a led to a significant decrease in leptin mRNA levels to 70 Ϯ 8% and 49 Ϯ 13% of controls respectively. Similarly, TGF-b1 decreased leptin mRNA expression in newly differentiated human adipocytes to 77 Ϯ 12% after 24 h and to 54 Ϯ 8% after 48 h compared with control cultures. These data provide evidence that long-term exposure of human fat cells to TNF-a or TGF-b1 may suppress leptin expression in human adipose tissue. The inhibitory effect of TGF-b1 appears to be more pronounced in omental as compared with subcutaneous adipocytes.

BACKGROUND:Leptin is an adipose protein regulating food intake in the hypothalamus. Animal studies have suggested that leptin also acts in an auto-=paracrine fashion on adipose cell function. OBJECTIVE: The aim of this study was to investigate the effects of leptin on the differentiation and metabolism of cultured human adipocytes. MATERIAL: Adipose tissue from young healthy, lean women (body mass index (BMI) < 27 kg=m 2 ) undergoing elective mammary reduction surgery and young obese individuals (BMI > 40 kg=m 2 ) undergoing laparoscopic gastric banding. METHODS: Human preadipocytes in primary culture were induced to undergo differentiation by defined adipogenic factors. Mature adipocytes were isolated by collagenase digestion and kept in culture suspension. Glycero-3-phosphate dehydrogenase (GPDH) activity was used as a marker of adipose differentiation; glucose uptake, lipolysis and PAI-1 secretion were measured as parameters of fat cell function. RESULTS: Human preadipocytes and adipocytes from lean and obese subjects expressed the long leptin receptor isoform and two of the three short forms as assessed by polymerase chain reaction (PCR). Leptin at a supraphysiological concentration induced a transient increase of GPDH activity, but had no effect on glucose uptake and PAI-1 secretion from human adipocytes. In addition, basal and isoproterenol-stimulated lipolysis as well as the antilipolytic action of insulin in human adipocytes was not significantly affected by leptin exposure. CONCLUSION: In contrast to animal data, the results of our experiments do not demonstrate significant effects of leptin on main metabolic functions of human adipocytes arguing against a local auto-=paracrine action of leptin in human adipose tissue.