Effects of leptin on the differentiation and metabolism of human 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.

Effect of tumor necrosis factor alpha and transforming growth factor beta 1 on plasminogen activator inhibitor-1 secretion from subcutaneous and omental human fat cells in suspension culture.

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are characteristic of the obese state and may contribute to the association between obesity and cardiovascular disease. In this study, we measured the rate of secretion of PAI-1 antigen in isolated subcutaneous and omental abdominal adipocytes from severely obese and non-obese individuals and studied the effect of selected cytokines on PAI-1 release using a suspension culture technique. PAI-1 secretion was approximately 2-fold greater in isolated fat cells from the obese versus non-obese subjects. In addition, PAI-1 mRNA levels were higher in adipose tissue samples from obese versus non-obese individuals (P < .05). PAI-1 release was also approximately 2-fold greater in omental versus subcutaneous adipocytes from both obese and non-obese subjects (each P < .05). A 24-hour exposure to 1 nmol/L tumor necrosis factor alpha (TNF-alpha) slightly increased PAI-1 release from both subcutaneous and omental adipocytes (30% +/- 21% and 17% +/- 18%, respectively, nonsignificant [NS]). Transforming growth factor beta 1 (TGF-beta1) induced a significant dose-dependent increase of PAI-1 release into the medium. Exposure to 400 pmol/L TGF-beta1 of subcutaneous and omental fat cells from both obese and non-obese individuals elevated PAI-1 secretion by 2-fold. These data provide evidence that human fat cells release a substantial amount of PAI-1 in a depot-specific manner and that TGF-beta1 particularly contributes to the regulation of PAI-1 secretion.

Role of cytokines in the regulation of plasminogen activator inhibitor-1 expression and secretion in newly differentiated subcutaneous human adipocytes.

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-beta1 (TGF-beta1) 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-beta1. This effect is probably mediated by TGF-beta1 type 2 and 3 receptors, which were found to be expressed in cultured human adipocytes. Moreover, TNF-alpha and interkeukin-1beta (IL-1beta) also exerted a stimulatory effect on PAI-1 release and increased PAI-1 mRNA levels. As assessed by a semiquantitative reverse transcription-polymerase chain reaction technique, TGF-beta1 mRNA is expressed by differentiation of human preadipocytes and is moderately upregulated by TNF-alpha and IL-1beta. In conclusion, our results clearly indicate that TGF-beta1 is a potent inducer of PAI-1 production in subcutaneous human adipocytes. In addition, data suggest that TNF-alpha and IL-1beta also have stimulatory effects on PAI-1 protein secretion and may contribute to the elevated PAI-1 levels observed in obesity.

Depot-specific release of leptin from subcutaneous and omental adipocytes in suspension culture: effect of tumor necrosis factor-alpha and transforming growth factor-beta1.

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)-alpha and transforming growth factor (TGF)-beta1 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-alpha and TGF-beta1 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 24h respectively. A 2-h exposure of adipocytes to 1nmol/l TNF-alpha and 400pmol/l TGF-beta1 respectively did not significantly affect leptin secretion. Whereas a 24-h incubation with 1nmol/l TNF-alpha also did not influence leptin secretion from fat cells from both depots, exposure of omental fat cells to 400pmol/l TGF-beta1 for 24h 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-alpha led to a significant decrease in leptin mRNA levels to 70 +/- 8% and 49 +/- 13% of controls respectively. Similarly, TGF-beta1 decreased leptin mRNA expression in newly differentiated human adipocytes to 77 +/- 12% after 24h and to 54 +/- 8% after 48h compared with control cultures. These data provide evidence that long-term exposure of human fat cells to TNF-alpha or TGF-beta1 may suppress leptin expression in human adipose tissue. The inhibitory effect of TGF-beta1 appears to be more pronounced in omental as compared with subcutaneous adipocytes.

Expression pattern of tumour necrosis factor receptors in subcutaneous and omental human adipose tissue: role of obesity and non-insulin-dependent diabetes mellitus.

BACKGROUND: Tumour necrosis factor alpha (TNF) mRNA expression has been reported to be up-regulated in adipose tissue from several rodent models of obesity and diabetes and from obese humans. This elevated expression has been assumed to be associated with the development of insulin resistance. However, the biological signal of TNF may be influenced by the expression of the two TNF receptors: the p60 TNF receptor, TNFR60, and the p80 TNF receptor, TNFR80. DESIGN: The aim of this study was to investigate the mRNA expression pattern of the two TNF receptors and their ligand in two adipose tissue depots of glucose-tolerant obese women [n = 18, body mass index (BMI) 48.2 +/- 8.4 kg m-2], obese women with impaired glucose tolerance or overt non-insulin-dependent diabetes mellitus (NIDDM) (n = 10, BMI 49.1 +/- 11.6 kg m-2) and healthy non-obese control subjects (n = 12, BMI 25.8 +/- 2.7 kg m-2). RNA expression was assessed by a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique. RESULTS: The mean mRNA levels of both TNF receptors were two- to threefold higher in adipose tissue samples from the obese than from the non-obese women (P < 0.01 for each). Although TNFR60 mRNA did not vary within each obese group, there was a wide variation in the levels of TNFR80 mRNA and of TNF mRNA. A comparison of the expression levels between the subcutaneous abdominal and the omental adipose tissue depots showed significantly higher expression in the former. The TNFR60 expression level was positively correlated with BMI and fat cell size, whereas TNFR80 and TNF mRNA levels showed positive associations with serum insulin and triglyceride concentrations. No significant differences in the expression levels were observed between obese individuals with and without impaired glucose tolerance/NIDDM. CONCLUSION: 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.