Acarbose reduces elevated testosterone serum concentrations in hyperinsulinaemic premenopausal women: a pilot study.

In seven hyperinsulinaemic, hypertestosteronaemic premenopausal patients, we tested the effect of an attenuation of insulin serum concentrations by long-term treatment with the enteral disaccharidase inhibitor acarbose on serum concentrations of total and free testosterone, dehydroepiandrosterone sulphate (DHEAS) and sex hormone-binding globulin (SHBG). The subjects showed typical features of hyperinsulinaemia-hypertestosteronaemia syndrome, including elevated concentrations of insulin and testosterone, normal concentrations of DHEAS and suppressed SHBG concentrations. The patients were orally treated with an initial dosage of 50 mg acarbose/ day, which was gradually increased to a maximum of 300 mg/day. Blood was sampled at week 6 (under a dosage of 150 mg acarbose/day) and at week 20 of treatment. A significant reduction in the increase of glucose and insulin concentrations, determined after administration of a standard oral 100 g glucose load, was found at week 6 (P < 0.02, P < 0.00007) and at week 20 (P < 0.04, P < 0.003) of therapy and was associated with a significant decrease of total and free testosterone at week 20 (P < 0.04, P < 0.01). Concentrations of both DHEAS and SHBG remained nearly unchanged. In conclusion, it was shown that a decline of ovarian hypertestosteronaemia was achieved in association with a flattening of the postprandial glucose and insulin increase by long-term treatment with acarbose. Side effects were limited to abdominal distension and flatulence and were absent using a low dosage of 50 mg acarbose/ meal (3 x 50 mg/day).

Obesity and hypertestosteronaemia are independently and synergistically associated with elevated insulin concentrations and dyslipidaemia in pre-menopausal women.

The association of obesity and hypertestosteronaemia with elevated insulin concentration and dyslipidaemia was studied in 15 non-obese and 15 obese, hypertestosteronaemia patients; 14 non-obese and 10 obese, normotestosteronaemic subjects served as controls. Data were subjected to multivariate analysis. Enhanced body mass index (BMI kg/m2) resulted in a significant elevation of basal insulin (b-Ins), glucose-stimulated (delta) insulin (del-Ins), triglycerides (TG), very low density lipoprotein (VLDL), low density lipoprotein (LDL), and LDL/high density lipoprotein (HDL) ratio, and in a significant reduction of HDL. Furthermore, it was shown that BMI was positively correlated with TG, VLDL, LDL and LDH/HLD ratio, and negatively correlated with HDL in the normotestosteronaemic groups. Hypertestosteronaemia was associated with a significant increase of del-Ins, VLDL and LDL/HDL ratio, and with a significant decrease of HDL concentration. Testosterone was directly associated with del-Ins and LDL/HDL ratio, and inversely related to HDL in the non-obese groups. Summation effects of obesity and hypertestosteronaemia were found for del-Ins and VLDL. The data suggest that obesity and hypertestosteronaemia are independently and jointly associated with insulin resistance and dyslipidaemia, indicating an increased risk for coronary heart disease. The highest risk rate was found in obese hypertestosteronaemic patients. Serum testosterone may be a useful marker in detecting metabolic disorders connected with cardiovascular risk.

Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.

Production of recombinant rat interleukin-6 in Escherichia coli using a novel highly efficient expression vector pGEX-3T.

Interleukin-6 (IL-6) is one of the most important mediators of the acute phase reaction in liver. For the production of recombinant rat IL-6 in Escherichia coli, a previously isolated cDNA coding for the rat IL-6 was cloned into the modified novel expression vector pGEX-3T. The IL-6 cDNA was highly expressed as a fusion protein with the glutathione S-transferase (GST) at its C-terminus and rat IL-6 at its N-terminus. The GST-IL-6 fusion protein was controlled by a tac-promoter and could be induced very efficiently by isopropyl-beta-D-thiogalactopyranoside. The synthesized GST-IL-6 fusion protein was insoluble and precipitated intracellularly in E. coli. Using an advanced technique, the insoluble protein was solubilized and purified to homogeneity by affinity chromatography using immobilized glutathione in a one-step procedure.

Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography.

A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.

Cloning and expression of antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen in Escherichia coli.

Autoantibodies directed against the 68-kDa (U1) ribonucleoprotein antigen are mainly found in sera of patients with mixed connective tissue disease. The corresponding cDNA was fragmented into four regions coding for the major antigenic epitopes A', B', C' and D'. All the epitopes were subcloned and expressed as fusion proteins with the glutathione S-transferase in Escherichia coli using the novel expression system pGEX that allows very high yields of recombinant proteins after a single-step purification. The sera of patients with the autoimmune disease were analyzed for the expressed recombinant proteins by an immunoblotting technique. All positive sera showed a patient-specific behavior and could be divided into four groups regarding recognition of the four antigenic epitopes of the 68-kDa (U1) ribonucleoprotein antigen. The epitope B' was reactive to all patient sera positively tested and classified as the marker antigenic epitope for the mixed connective tissue disease.

Impaired insulin-induced RNA synthesis secondary to a genetically defective insulin receptor.

The stimulation of glucose uptake and RNA synthesis by insulin was studied in cultured fibroblasts from patients with an inherited affinity defect of the insulin receptor. Additional cell cultures were set up of two patients with Alstrøm syndrome, a genetic disease with insulin resistance but normal insulin receptor, and of eight healthy individuals. In receptor-defective patients we found a corresponding defect of the insulin-mediated stimulation of RNA synthesis but a normal stimulation of glucose uptake. We conclude that both postreceptor effects are controlled by different insulin-directed mechanisms.

Three different classes of oral antidiabetic drugs do not increase insulin binding and insulin-induced RNA synthesis in human fibroblast cultures.

We describe insulin binding and insulin-mediated RNA synthesis on seven human fibroblasts strains in culture initiated from skin biopsies in the presence of three oral antidiabetic agents, Metformin, Gliquidone, and a non-sulfonylurea antidiabetic drug (B X DF 591 ZW), which belong to different chemical classes. At least at a nontoxic pharmacologic concentration of 1 microgram/ml these drugs did not influence the number and affinity of insulin receptors, nor did they increase the insulin-mediated RNA synthesis. We conclude that cultured fibroblasts do not represent the proper target for an evaluation of an extrapancreatic action of oral antidiabetic drugs.

Heterozygous carriers for Bloom syndrome exhibit a spontaneously increased micronucleus formation in cultured fibroblasts.

Cultured fibroblasts of homozygotes and heterozygotes for Bloom syndrome exhibit an enhanced formation of micronuclei. The number of spontaneously occurring micronuclei permit clear separation of heterozygotes from either normal controls or homozygous patients without overlap between these groups. The observed differences could not be enhanced further by the addition of various mutagens. We conclude from the increased chromosomal damage that heterozygotes for Bloom syndrome may have a higher risk for malignant diseases.