Objective: Circulating leptin, the product of the ob gene, is known to be closely correlated with adipose tissue mass, but it is also subject to short-term regulation by a variety of hormones including catecholamines. The aim of this study was to investigate the contribution of the three b-adrenergic receptors to leptin secretion from cultured human adipocytes. Design and methods: The model of in vitro differentiated human subcutaneous adipocytes was used in this study. The presence of the b-adrenoceptor subtypes was studied by RT-PCR. The functional role of the receptor subtypes was determined by stimulation of lipolysis by selective b-adrenergic agonists and by measuring glycerol release. Leptin secretion into the medium of cultured human adipocytes from young normal-weight females was measured by radioimmunoassay. Results and conclusion: In a ®rst set of experiments, the expression of the three b-adrenergic receptor subtypes in cultured human adipocytes was demonstrated. To test their functional activity, the effect of the b-adrenoceptor agonists isoproterenol (non-selective agonist), dobutamine (b 1 -selective), fenoterol (b 2 -selective) and the b 3 -selective agonists BRL 37344 and CGP 12177 was studied. All agonists exhibited a dose-and time-dependent stimulation of glycerol release into the medium in a rather uniform manner. Isoproterenol rapidly reduced leptin secretion from cultured subcutaneous adipocytes in a dose-dependent fashion. Incubation with 10 À6 mol/l isoproterenol for 24 h resulted in a reduction of the leptin concentration by 48% (P < 0.01). A similar, but less pronounced suppressing effect was seen for dobutamine and fenoterol, whereas both BRL 37344 and CGP 12177 were not effective. These data provide evidence that catecholamines are able to suppress leptin release from differentiated human adipocytes, supporting the concept that leptin secretion is acutely regulated by surrounding hormones. This inhibition is obviously mediated via b 1 -and b 2 -adrenergic receptors.

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.