W. M. Teller

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.

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.

17α-Hydroxyprogesterone, 4-Androstenedione, and Testosterone Profiled by Routine Stable Isotope Dilution/Gas Chromatography-Mass Spectrometry in Plasma of Children

ABSTRACT: Using stable isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS), a physicochemical method, we have profiled the plasma steroids 17α-hydroxyprogesterone, 4-androstenedione, and testosterone in normal children of various age groups. Comparison of our values with those obtained by direct immunologic assays and those using an extraction or purification step showed that immunoassays in general overestimate steroid concentrations. This was especially true for plasma samples in the neonatal period and was most expressed for the concentrations of 17α-hydroxyprogesterone. Our study demonstrated the applicability of ID/GC-MS to routine clinical steroid analysis. The application of ID/GC-MS is recommended whenever problems from matrix effects or cross-reactivity are likely to arise or suspicious results by immunoassays need to be rechecked.

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.

Androgen metabolism assessment by routine gas chromatography/mass spectrometry profiling of plasma steroids: part 1, unconjugated steroids

Using gas chromatography/mass spectrometry we have developed a method for the simultaneous determination of six plasma steroids: testosterone, 4-androstenedione, 17 alpha-hydroxyprogesterone, 5 alpha-androstane-3 alpha,17 beta-diol, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone. For each analyte, a deuterium-labeled internal standard was used for quantification. Due to the high isotopic purity of our standards, no complex corrections for isotope contributions were necessary. The procedure provides a sensitive and specific technique with good accuracy and precision.

Serum concentrations of procollagen I C-terminal propeptide, osteocalcin and insulin-like growth factor-I in patients with non-lethal osteogenesis irnperfecta

Serum concentrations of procollagen I C‐terminal propeptide (PICP) were studied in 74 patients with various forms of non‐lethal osteogenesis imperfecta and 27 unaffected family members. Using the standard deviation (SD) score, PICP concentrations were found to be — 1 SD in 16%, between — 1 and —2 SD in 26% and — 2 SD in 58% of the patients with osteogenesis imperfecta compared to healthy controls. PICP values were lowest in osteogenesis imperfecta type I (‐ 2.4 ± 0.4 SD, n= 37) followed by type III (‐1.9 ± 0.5 SD, n = 13) and type IV (‐ 1.3 ± 0.7 SD, n= 20). Four patients with osteogenesis imperfecta with an atypical clinical course had normal or even elevated levels which may indicate heterogeneity in the underlying primary defects. In osteogenesis imperfecta type I, PICP concentrations proved to be a helpful serum marker for pedigree screening. Osteocalcin was high in 25 of 28 patients with osteogenesis imperfecta in the first decade but only in 1 of 18 older patients. Insulin‐like growth factor‐I was within the normal range in 53 cases of osteogenesis imperfecta, decreased in 2 and elevated in 3 patients. We conclude that PICP concentration is a useful parameter in the clinical management of osteogenesis imperfecta, including the assessment of future therapeutic interventions.

Defective collagen fibril formation and mineralization in osteogenesis imperfecta with congenital joint contractures (Bruck syndrome)

We describe a male patient with osteogenesis imperfecta (OI) who was born with contractures of the knee, elbow and ankle joints. During the first 4 years he suffered from recurrent fractures. He has white sclerae, mild dentinogenesis imperfecta, multiple wormian bones, severe scoliosis and short stature. Morphological analysis of cortical bone revealed typical characteristics of OI including varying width of the osteoid, swollen mitochondria and a dilated endoplasmic reticulum of the osteoblasts. Collagen fibrils of the osteoid had a varying diameter, a feature not found in typical OI patients. Analysis of compact bone showed that the size of apatite crystals and the extractability of collagen with pepsin were markedly elevated compared to controls and other OI type III and IV patients. Lysyl hydroxylation of collagen from the organic bone matrix and the electrophoretic mobility of collagen α1(I)- and α2(I)-chains were normal. Our results provide evidence that this patient belongs to a subtype of OI. The biochemical studies indicate that the underlying defect involves defective fibril-formation of collagen type I leading to an altered mineralization of bone.