Eberhard Heinze

Insulin and Cortisol Promote Leptin Production in Cultured Human Fat Cells

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.

Contribution of androgens to the gender difference in leptin production in obese children and adolescents.

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+/-14.1 vs. 17.2+/-12.6 ng/ml, P < 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.

Human fetal and adult chondrocytes. Effect of insulinlike growth factors I and II, insulin, and growth hormone on clonal growth.

Abstract Clonal proliferation of freshly isolated human fetal chondrocytes and adult chondrocytes in response to human insulinlike growth factors I and II (IGF I, IGF II), human biosynthetic insulin, and human growth hormone (GH) was assessed. IGF I (25 ng/ml) stimulated clonal growth of fetal chondrocytes (54 +/- 12 colonies/1,000 inserted cells, mean +/- 1 SD), but IGF II (25 ng/ml) was significantly more effective (106 +/- 12 colonies/1,000 inserted cells, P less than 0.05, unstimulated control: 14 +/- 4 colonies/1,000 inserted cells). In contrast, IGF I (25 ng/ml) was more effective in adult chondrocytes (42 +/- 6 colonies/1,000 inserted cells) than IGF II (25 ng/ml) (21 +/- 6 colonies/1,000 inserted cells; P less than 0.05, unstimulated control: 6 +/- 3 colonies/1,000 inserted cells). GH and human biosynthetic insulin did not affect clonal growth of fetal or adult chondrocytes. The clonal growth pattern of IGF-stimulated fetal and adult chondrocytes was not significantly changed when chondrocytes were first grown in monolayer culture, harvested, and then inserted in the clonal culture system. However, the adult chondrocytes showed a time-dependent decrease of stimulation of clonal growth by IGF I and II. This was not true for fetal chondrocytes. The results are compatible with the concept that IGF II is a more potent stimulant of clonal growth of chondrocytes during fetal life, whereas IGF I is more effective in stimulating clonal growth of chondrocytes during postnatal life.

Thyroid Autoimmunity in Children and Adolescents with Type 1 Diabetes mellitus

Type 1 diabetes is often associated with additional autoimmune phenomena. However, data reported on the frequency of thyroid autoimmunity differ vastly. Therefore, the prevalence of thyroid autoantibodies was evaluated at a large pediatric diabetes center in Southern Germany. 2,305 determinations (TPO and TG, ELISA) were performed in 495 patients with type 1 diabetes (234 boys, 261 girls; age at last measurement: 15.4 ± 0.3 years, duration of diabetes 7.5 ± 0.2 years). The prevalence of elevated thyroid antibodies increased dramatically with age: from 3.7% in patients less than 5 years of age up to 25.3% in the age group 15–20 years (p < 0.0001). For children older than 10 years, girls were significantly more affected than boys (p < 0.0001). Thyroid autoimmunity tended to be more prevalent in the subgroup of patients with the HLA type DR3/DR4 compared to patients with other HLA types (p = 0.08). In children older than 10 years, basal TSH concentrations were significantly elevated in antibody-positive patients (p < 0.05). In conclusion, thyroid autoimmunity is prevalent in children and adolescents with type 1 diabetes. Adolescent girls and young women are especially affected. Yearly routine determinations of thyroid antibodies are therefore recommended.

Turner syndrome: Final height, glucose tolerance, bone density and psychosocial status in 25 adult patients

The information available on the medical and psychosocial status of patients with Turner syndrome beyond the paediatric age group is scarce. We therefore studied 25 unselected women with cytogenetically proven Turner syndrome (age 20–50 years), who never received any growth-promoting therapy, and ten control women (25–48 years). In addition to anthropometric measurements, an oral glucose tolerance test was performed, auto-antibodies to endocrine tissues were studied, bone mineral density of the forearm was measured by single photon densitometry, and information about the psychosocial distress of the patients was obtained. Adult height averaged 148.7±1.1 cm (mean±SE), which was 16 cm below the mean of adult women from a similar background. In Turner patients, final height correlated significantly with mid-parental height (final height=0.67×MPH+32.1;r=0.69). Body mass index was increased in Turner patients (25.6±1.3 kg/m2) compared to controls (21.4±0.6;P<0.006). Six patients (25%) had impaired glucose tolerance or overt diabetes mellitus (one patient). Insulin release was augmented but delayed in the Turner group, and the area under the insulin stimulation curve was correlated to body mass index (r=+0.54,P<0.01). Thyroid antibodies were detected in nine patients (37.5%). On average, bone density of the forearm was only marginally reduced compared to the agedependent normal range. All women were employed, while only one of the Turner women was married. As a group, the subjects expressed greater distress due to infertility compared to short stature. These data demonstrate a high degree of impaired glucose tolerance and hyperinsulinism in adult Turner women, which—together with increased body fat—potentially increase the cardiovascular risk for these patients. In contrast, early osteoporosis as well as subjective dissatisfaction with attained height seem to be of secondary importance.

Development of height and weight in children with diabetes mellitus: report on two prospective multicentre studies, one cross-sectional, one longitudinal.

Optimal regimen for insulin therapy should lead to normal longitudinal growth and weight gain in children with diabetes mellitus. However, reports published so far indicate that this goal of paediatric diabetology is currently not achieved in a considerable number of patients. In a cross-sectional sample of 89 children with insulin dependent diabetes mellitus (IDDM) for more than 3 years, we found the relation of height to weight to be significantly different compared to 102 healthy school children of similar age. Using bivariate analysis, body shape in these children with diabetes was shifted towards small and obese (P<0.05) compared to control children. We subsequently initiated a longitudinal study and followed children from the onset of diabetes for the following 3 years, recording height, weight and bone age as well as glycosylated haemoglobin and daily insulin requirement. At diagnosis, height SDS was identical in children with IDDM (+0.04±0.10) compared to control children (−0.07±0.10; M±SE), while weight SDS was −0.26±0.10 in children with diabetes (controls: +0.01±0.1). Bone age was identically retarded in newly diagnosed IDDM children (−0.73±0.12 SDS) and in our control group of children from the same regional background (−0.50±0.12; n.s.). In this group of children with diabetes mellitus followed prospectively, height to weight relationship differed from controls after 2 and after 3 years of the disease (P<0.05). At 2 years, body size in children with diabetes was shifted towards taller and heavier compared to controls, while at 3 years, the relation of height to weight was even more abnormal with increased obesity but a reduction of standardized height. This is the same relation encountered in the cross-sectional sample of children with a duration of diabetes beyond 3 years. These data demonstrate that even modern insulin therapy does not guarantee normal development of height and weight in children with IDDM.

Mitogenic and antiadipogenic properties of human growth hormone in differentiating human adipocyte precursor cells in primary culture

Children with GH deficiency have enlarged fat cells but a reduced number of fat cells compared with healthy children. After treatment with human GH (hGH) both fat cell volume and number are shifted toward normal. To clarify the role of hGH in fat cell formation in human adipose tissue, we investigated the effect of hGH on the proliferation and the differentiation of cultured human adipocyte precursor cells obtained from five children and 10 adults. In a chemically defined serum-free medium treatment of adipocyte precursor cells with hGH led to an increase in IGF-I production and a stimulation of cell proliferation, which could be blocked by a MAb raised against human IGF-I. hGH dose-dependently reduced the number of differentiating cells and suppressed the expression of glycerol-3-phosphate dehydrogenase (GPDH), a marker of adipose differentiation. No significant differences in the hGH effects on proliferation and differentiation capacities were seen between cultures obtained from children and adults. In newly differentiated adipocytes, hGH inhibited glucose uptake and lipogenesis, and stimulated lipolysis. Scatchard analysis of hGH competition experiments using 125I-labeled hGH yielded a linear plot with an apparent Kd of 1.08 nM and an estimated number of 7000 hGH receptors per cell. These data suggest that hGH is able to enlarge the human adipocyte precursor pool via induction of IGF-I synthesis but exhibits a direct antiadipogenic activity. hGH is also able to reduce fat cell volume by reducing lipogenesis and increasing lipolysis.

Reduced pancreatic insulin release and reduced peripheral insulin sensitivity contribute to hyperglycaemia in cystic fibrosis.

Traditional opinion holds that patients with cystic fibrosis (CF) develop impaired glucose tolerance or diabetes due to insulinopenia caused by fibrosis of the pancreas. However, studies on the dynamics of insulin secretion and peripheral insulin action have yielded confliciting results. We studied 18 patients with CF (9 ♂, 9 ♀, age 15–29 years) and 17 healthy control subjects (8 ♂, 9 ♀, 20–32 years). Oral glucose tolerance tests and combined i.v.-glucose-tolbutamide-tests were performed on separate days in fasting subjects. Bergmans “Minimal Model” was used to quantitate both peripheral insulin sensitivity (SI) and insulin-independent glucose disposal (glucose effectiveness; SG). Based on National Diabetes Data Group criteria, 4 patients were classified as diabetic 922%; CF-DM), 3 patients (17%) had impaired glucose tolerance (CF-IGT) while glucose metabolism was normal in 11 patients (61%; CF-NGT). Irrespective of the degree of glucose tolerance, the insulin response to oral glucose was not reduced but delayed, up to 60 min in the CF-IGT/DM group. First-phase insulin release (0–10 min) after i.v.-glucose was significantly lower in CF patients (29% of healthy controls;P<0.0001), with no difference between the CF-NGT and CF-IGT/DM groups. Insulin release following tolbutamide injection was only marginally reduced in CF patients (64% of controls). In contrast, SI was significantly reduced in the subgroup of CF patients with abnormal glucose metabolism (CF-IGT/DM: 0.97±0.16·10−4 l/min/pmol; control group: 1.95±0.25;P<0.05).ConclusionThe early insulin release is reduced in response to i.v.-glucose, while in the oral glucose tolerance test, insulin secretion is quantitatively preserved, but delayed. Reduced peripheral insulin sensitivity is a major factor for impaired glucose tolerance and diabetes mellitus in CF patients.

Objective assessment of smoking habits by urinary cotinine measurement in adolescents and young adults with type 1 diabetes. Reliability of reported cigarette consumption and relationship to urinary albumin excretion.

OBJECTIVE To examine the relationship of objective smoking status to age, sex, longterm metabolic control, and urinary albumin excretion. Patients with type 1 diabetes who smoke are at increased risk to develop diabetic microvascular and macrovascular complications. While this has repeatedly been demonstrated in adults, smoking habits have rarely been investigated in adolescents. RESEARCH DESIGN AND METHODS Urinary cotinine excretion has been determined by radioimmunoassay in 238 adolescents and young adults with type 1 diabetes. This biochemical parameter of nicotine use was related to age, to the number of cigarettes allegedly consumed per day, and to urinary albumin excretion. RESULTS A total of 46 patients (19.3%) with urinary cotinine values >500 ng/ml were classified as smokers. In 26 patients (10.9%), cotinine values between 100 and 500 ng/ml were found (infrequent smokers or environmental nicotine exposure), while the remaining 166 patients excreted <100 ng/ml of cotinine in the urine (nonsmokers). Smokers were significantly older (20.2 ± 0.6 years [mean ± SE]) compared with the intermediate group (18.3 ± 0.7 years) or with nonsmokers (15.9 ± 0.4 years; P < 0.0001, Wilcoxons signed-rank test). Of 46 smokers, 12 denied smoking cigarettes entirely, and among biochemically defined smokers, no correlation was present between urinary cotinine excretion and the reported number of cigarettes consumed per day. Urinary albumin excretion was significantly higher in smokers compared with nonsmokers (P < 0.003). CONCLUSIONS These data demonstrate that cigarette smoking is common among German adolescents and young adults with type 1 diabetes in this study. Many patients deny nicotine use or refuse to disclose their smoking habits. Increased urinary albumin excretion is consistent with an increased risk of nephropathy in subjects with diabetes who smoke. Pediatricians in charge of adolescents with type 1 diabetes should actively discuss the risk of nicotine consumption with their patients.

Patterns of growth in human septal cartilage: a review of new approaches

Recent results of the growth activities of the septal cartilage in children, adolescents, and adults are reviewed. Cartilaginous biopsies were obtained during septoplasty . Matrix synthesis was measured by in vitro incorporation of labeled sulphate; DNA synthesis was measured by in vitro incorporation of labeled thymidine. The cell density of the septal cartilage was determined by cell counting, and chondrocytes were grown in culture in an assay to determine the number of chondrocytes capable of proliferation. With data from these 4 types of experiments 5 different areas could be distinguished in human septal cartilage: (1) anterior free end, (2) suprapremaxillary area, (3) central area, (4) posterior area, and (5) caudal prolongation. Metabolic activities, degree of cell replication, and proliferative capacity are highest in childhood in all areas; they decline with age, but remain surprisingly high in the central area and in the anterior free end, even in adults. These results help to explain some clinical observations and may help the rhinosurgeon in his decision to resect the septal cartilage, especially in children.