OBJECTIVE:To develop and to characterize a human preadipocyte cell strain with high capacity for adipose differentiation serving as a model for studying human adipocyte development and metabolism in vitro. METHODS: Cells were derived from the stromal cells fraction of subcutaneous adipose tissue of an infant with Simpson ± Golabi ± Behmel syndrome (SGBS). Adipose differentiation was induced under serum-free culture conditions by exposure to 10 nM insulin, 200 pM triiodothyronine, 1 mM cortisol and 2 mM BRL 49653, a PPARg agonist. RESULTS: During the differentiation process SGBS cells developed a gene expression pattern similar to that found in differentiating human preadipocytes with a characteristic increase in fat cell-speci®c mRNAs encoding lipoprotein lipase (LPL), glycero-3-phosphate dehydrogenase (GPDH), GLUT4, leptin and others. Differentiated SGBS cells exhibited an increase in glucose uptake upon insulin stimulation and in glycerol release upon catecholamine exposure. SGBS adipocytes were morphologically, biochemically and functionally identical to in vitro differentiated adipocytes from healthy subjects. However, while preadipocytes from healthy control infants rapidly lost their capacity to differentiate after a few cell divisions in culture, SGBS cells maintained their differentiation capacity over many generations: upon appropriate stimulation 95% of SGBS cells of generation 30 developed into adipocytes. A mutation in the glypican 3 gene was not detected in the patient. Thus, it remains unclear whether the molecular alteration in SGBS cells is also responsible for the high differentiation capacity and further investigations are required. CONCLUSION: The human cell strain described here provides an almost unlimited source of human preadipocytes with high capacity for adipose differentiation and may, therefore, represent a unique tool for studying human fat cell development and metabolism. International Journal of Obesity (2001) 25, 8 ± 15
Recent studies demonstrated significantly higher serum leptin concentrations in females as compared with males, even after correction for differences in body fat mass. The aim of our study was to measure serum leptin concentrations in a large group of obese children and adolescents to determine the possible role of sex steroid hormones on both leptin serum concentrations and production in human adipocytes. Obese girls were found to have significantly higher leptin concentrations than boys at the same degree of adiposity (25.2 Ϯ 14.1 vs. 17.2 Ϯ 12.6 ng/ml, P Ͻ 0.001). In a multiple regression analysis with age and body mass index (percent body fat) as fixed variables, it turned out that testosterone had a potent negative effect on serum leptin in boys, but not in girls. In vitro experiments using newly developed human adipocytes in primary culture showed that both testosterone and its biologically active metabolite dihydrotestosterone are able to reduce leptin secretion into the culture medium by up to 62%. Using a semiquantitative reverse transcriptase-PCR method, testosterone was found to suppress leptin mRNA to a similar extent. These results suggest that, apart from differences in body fat mass, the higher androgen concentrations in obese boys are responsible for the lower leptin serum concentrations compared with obese girls. ( J. Clin. Invest. 1997. 100:808-813.) Key words: androgens • gender difference • human adipose tissue • leptin
The aim of this study was to investigate the regulation of leptin expression and production in cultured human adipocytes using the model of in vitro differentiated human adipocytes. Freshly isolated human preadipocytes did not exhibit significant leptin mRNA and protein levels as assessed by reverse transcriptase (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). However, during differentiation induced by a defined adipogenic serum-free medium, cellular leptin mRNA and leptin protein released into the medium increased considerably in accordance with the cellular lipid accumulation. In fully differentiated human fat cells, insulin provoked a dose-dependent rise in leptin protein. Cortisol at a near physiological concentration of 10(-8) mol/l was found to potentiate this insulin effect by almost threefold. Removal of insulin and cortisol, respectively, was followed by a rapid decrease in leptin expression, which was reversible after readdition of the hormones. These results clearly indicate that both insulin and cortisol are potent and possibly physiological regulators of leptin expression in human adipose tissue.
BACKGROUND: Recent studies reported an increased prevalence of type II diabetes mellitus in obese children and adolescents, especially in specific ethnic subgroups. The aim of this study was to determine the prevalence of type II diabetes mellitus and impaired glucose regulation in a large group of Caucasian children and adolescents with obesity living in Germany. PATIENTS AND METHODS: A total of 520 subjects (237 boys, 283 girls) (mean age: 14.072.0 y (range 8.9-20.4 y)) with a BMI497th percentile, BMI-SDS: 2.770.5 (range 1.9-4.6), who were consecutively admitted to an in-patient obesity unit participated in the study. A 2-h oral glucose tolerance test (1.75 mg of glucose per kilogram of body weight) was performed before entering a weight-loss program and capillary blood glucose concentrations were measured. Patients were categorized into normal glucose regulation, impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and diabetes. In addition, fasting venous blood was taken to determine the circulating insulin, C-peptide and lipids. Insulin resistance was estimated by homeostatic model assessment. RESULTS: Type II diabetes was present in 1.5% (n ¼ 8) of the patients, two patients were admitted with already diagnosed type II diabetes and six patients were identified with yet unknown diabetes. IFG was detected in 3.7% (n ¼ 19) and IGT in 2.1% (n ¼ 11) of the patients. All together, in 6.7% (n ¼ 35) (95% confidence interval: 4.7-9.2%) of the patients, impaired glucose regulation (IFG, IGT) or diabetes was identified. These patients had a higher BMI-SDS, higher levels of fasting insulin and Cpeptide and a higher insulin resistance index than the patients with normal glucose regulation. Risk factors for the occurrence of impaired glucose regulation were a BMI-SDS42.5 as well as a positive parents' history for diabetes. CONCLUSIONS: This is the first report on the prevalence of type II diabetes in a large cohort of Caucasian children and adolescents with obesity living in Europe. Impaired glucose regulation and type II diabetes were present in a substantial proportion of the patients studied. Screening for diabetes in severely obese children and adolescents (BMI-SDS42.5) is therefore recommended. Patients identified with impaired glucose regulation need specific treatment programs in order to prevent progression to diabetes.
On the basis of the positive outcome of animal experiments, several large placebo-controlled trials are underway and aiming for the first time at the prevention of an immune-mediated disease, type 1 diabetes. The first of these trials, The Deutsche Nicotinamide Intervention Study (DENIS), evaluated the clinical efficacy of high doses of nicotinamide in children at high risk for IDDM. Nicotinamide has been shown to protect beta-cells from inflammatory insults and to improve residual beta-cell function in patients after onset of IDDM. Individuals at high risk for developing IDDM within 3 years were identified by screening the siblings (age 3-12 years) of patients with IDDM for the presence of high titer (> or =20 Juvenile Diabetes Foundation [JDF] U) islet cell antibodies. Probands (n = 55) were randomized into placebo and nicotinamide (slow release, 1.2 g x m(-2) x day(-1)) receiving groups and followed prospectively in a controlled clinical trial using a sequential design. Rates of diabetes onset were similar in both groups throughout the observation period (maximum 3.8 years, median 2.1 years). This sequential design provides a 10% probability of a type II error against a reduction of the cumulative diabetes incidence at 3 years from 30 to 6% by nicotinamide. The trial was terminated when the second sequential interim analysis after the eleventh case of diabetes showed that the trial had failed to detect a reduction of the cumulative diabetes incidence at 3 years from 30 to 6% (P = 0.97). The group receiving nicotinamide exhibited decreased first-phase insulin secretion in response to intravenous glucose (P = 0.03). No other side effects were observed. We conclude that in this subgroup of diabetes-prone individuals at very high risk and with an assumed rapid disease progression, nicotinamide treatment did not cause a major decrease or delay of diabetes development. However, the data do not exclude the possibility of a less strong, but potentially meaningful, risk reduction in this cohort, or a major clinical effect of nicotinamide in individuals with less risk of progression to IDDM than studied here.
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