Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice.

AIMS/HYPOTHESIS: We previously observed hyperglycaemia, hyperinsulinaemia, insulin resistance and obesity in Gpx1-overexpressing mice (OE). Here we determined whether these phenotypes were eliminated by diet restriction, subsequently testing whether hyperinsulinaemia was a primary effect of Gpx1 overexpression and caused by dysregulation of pancreatic duodenal homeobox 1 (PDX1) and uncoupling protein-2 (UCP2) in islets. METHODS: First, 24 male OE and wild-type (WT) mice (2 months old) were given 3 g (diet-restricted) or 5 g (full-fed) feed per day for 4 months to compare their glucose metabolism. Thereafter, several mechanistic experiments were conducted with pancreas and islets of the two genotypes (2 or 6 months old) to assay for beta cell mass, reactive oxygen species (ROS) levels, mitochondrial membrane potential (Deltapsi(m)) and expression profiles of regulatory proteins. A functional assay of islets was also performed. RESULTS: Diet restriction eliminated obesity but not hyperinsulinaemia in OE mice. These mice had greater pancreatic beta cell mass (more than twofold) and pancreatic insulin content (40%) than the WT, along with an enhanced Deltapsi(m) and glucose-stimulated insulin secretion in islets. With diminished ROS production, the OE islets displayed hyperacetylation of H3 and H4 histone in the Pdx1 promoter, elevated PDX1 and decreased UCP2. CONCLUSIONS/INTERPRETATION: Overproduction of the major antioxidant enzyme, glutathione peroxidase 1, caused seemingly beneficial changes in pancreatic PDX1 and UCP2, but eventually led to chronic hyperinsulinaemia by dysregulating islet insulin production and secretion.

Synthesis and evaluation of fluorine-18 labeled glyburide analogs as beta-cell imaging agents.

Glyburide is a prescribed hypoglycemic drug for the treatment of type 2 diabetic patients. We have synthesized two of its analogs, namely N-[4-[beta-(2-(2'-fluoroethoxy)-5-chlorobenzenecarboxamido)ethyl]benzenesulfonyl]-N'-cyclohexylurea (2-fluoroethoxyglyburide, 8b) and N-[4-[beta-(2-(2'-fluoroethoxy)-5-iodobenzenecarboxamido)ethyl]benzenesulfonyl]-N'-cyclohexylurea (2-fluoroethoxy-5-deschloro-5-iodoglyburide, 8a), and their fluorine-18 labeled analogs as beta-cell imaging agents. Both F-18 labeled compound 8a and compound 8b were synthesized by alkylation of the corresponding multistep synthesized hydroxy precursor 4a and 4b with 2-[(18)F]fluoroethyl tosylate in DMSO at 120 degrees C for 20 minutes followed by HPLC purification in an overall radiochemical yield of 5-10% with a synthesis time of 100 minutes from EOB. The octanol/water partition coefficients of compounds 8a and 8b were 141.21 +/- 27.77 (n = 8) and 124.33 +/- 21.61 (n = 8), respectively. Insulin secretion experiments of compounds 8a and 8b on rat islets showed that both compounds have a similar stimulating effect on insulin secretion as that of glyburide. In vitro binding studies showed that approximately 2% of compounds 8a and 8b bound to beta TC3 and Min6 cells and that the binding was saturable. Preliminary biodistribution studies in mice showed that the uptake of both compounds 8a and 8b in liver and small intestine were high, whereas the uptake in other organs studied including pancreas were low. Additionally, the uptake of compound 8b in vivo was nonsaturable. These results tend to suggest that compounds 8a and 8b may not be the ideal beta-cell imaging agents.

Insulin gene transfer enhances the function of human islet grafts.

AIMS/HYPOTHESIS: Recent success in islet transplantation renews the hope for the complete cure of patients afflicted with Type 1 diabetes. However, in the Edmonton series, two to four pancreas donors were required to obtain a sufficient islet mass to reverse the diabetes of each patient. In view of the donor shortage, this represents a major obstacle preventing greater application of islet transplantation to diabetic patients. We hypothesised that increasing the expression of the insulin gene in transplanted islets would augment their capacity for insulin production, thereby allowing reversal of diabetes with a reduced islet mass. METHODS: We used a replication defective adenovirus to deliver the human proinsulin gene (Ad-Ins) to isolated human islets. The function of Ad-Ins-transduced human islets was compared to islets transduced with a control vector (Ad-lacz). RESULTS: Ad-Ins-transduced islets produced two to three times more insulin than normal islets or those infected with Ad-lacz, as assessed by in vitro perifusion tests of glucose stimulated insulin release. When transplanted, Ad-Ins-transduced islets normalised the blood glucose of diabetic immunodeficient NOD-Scid mice, and less than half as many Ad-Ins islets were required for reversal of diabetes than when normal islets were transplanted. CONCLUSION/INTERPRETATION: Our results suggest a simple and effective approach that could enhance the efficiency of islet transplantation for treatment of diabetes in humans.

Acetylcholine affects rat liver metabolism via type 3 muscarinic receptors in hepatocytes.

Although the role of acetylcholine (Ach) in hepatic glucose metabolism is well elucidated, it is still unclear if it influences gluconeogenesis, glycogenolysis and high-energy phosphate metabolism, and if it does what the mechanisms of this influence are. Therefore, using isolated perfused rat liver as a model, we have studied the effect of Ach on oxygen consumption, synthesis of glucose from lactate and pyruvate, glycogen formation, mitochondrial oxidative phosphorylation and ATP-synthesis. We have established that effects of Ach on oxygen consumption depend on its concentration. When used at a concentration of 10(-7) M, Ach exerts maximum stimulatory effect, while its infusion at 10(-6) M causes a decrease of oxygen consumption by the liver. Moreover, when used at a concentration of 10(-6) M or 10(-7) M, Ach increases rates of glucose production from the gluconeogenic substrates lactate and pyruvate, leading to enhanced glycogen content in perfused liver. It was also shown that Ach possesses a stimulating effect on alanine and aspartate aminotransferases. As detected by 31P NMR spectroscopy, continuous liver perfusion with pyruvate and lactate in the presence of Ach leads to a significant decrease of ATP level, implying enhanced energy requirements for gluconeogenesis under these conditions. Elimination of the described effects of Ach by atropine, the antagonist of muscarinic receptors, and identification of the type 3 muscarinic receptors (m3) in isolated hepatocytes as well as in whole liver, imply that Ach may exert its effect on liver metabolism through m3 receptors.

Tissue-specific deletion of Foxa2 in pancreatic beta cells results in hyperinsulinemic hypoglycemia.

We have used conditional gene ablation to uncover a dramatic and unpredicted role for the winged-helix transcription factor Foxa2 (formerly HNF-3 beta) in pancreatic beta-cell differentiation and metabolism. Mice that lack Foxa2 specifically in beta cells (Foxa2(loxP/loxP); Ins.Cre mice) are severely hypoglycemic and show dysregulated insulin secretion in response to both glucose and amino acids. This inappropriate hypersecretion of insulin in the face of profound hypoglycemia mimics pathophysiological and molecular aspects of familial hyperinsulinism. We have identified the two subunits of the beta-cell ATP-sensitive K(+) channel (K(ATP)), the most frequently mutated genes linked to familial hyperinsulinism, as novel Foxa2 targets in islets. The Foxa2(loxP/loxP); Ins.Cre mice will serve as a unique model to investigate the regulation of insulin secretion by the beta cell and suggest the human FOXA2 as a candidate gene for familial hyperinsulinism.

[The results of sentinel epidemiological surveillance on the level of HIV prevalence among injection narcotic abusers in the city of Poltava]. / Rezul'taty "dozornogo" épidnadzora za urovnem rasprostranennosti VICh sredi in''ektsionnykh potrebitele narkotikov v g. Poltava.

The sentinel epidemiological surveillance on the level of spread of HIV infection among injecting drug users in Poltava, taking part in the program of harm reduction, was carried out. The results of the surveillance revealed that the level of HIV infection among drug users was 37.8%, the level of HIV infection among females being higher than that among males. These data indicate that the level of HIV infection rises with the increase of age and the time of drug consumption. Still in the age group of 30 years and older and among drug addicts using drugs for 7 years and longer the lowest level of HIV infection was noted.

High-performance liquid chromatography of monoamines on phenyl-bound sorbents using organic free mobile phases.

Tryptophan and its metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindolacetic acid, as well as dopamine, homovanilic acid and 2,3-dihydroxyphenylacetic acid, were separated on phenyl bound silica gel using isocratic elution with phosphate buffer. The method was successfully transferred to several other phenyl HPLC columns from different manufacturers simply by adjusting the pH of the buffer. The method has been validated by the determination of the level of monoamines in rat hypothalamus.

[Role of calcium ions in the mechanism of action of acetylcholine on energy metabolism in rat liver mitochondria]. / Rol' ioniv Ca mekhanizmi diï atsetylkholinu na enerhetychnyi obmin v mitokhondriiakh pechinky shchura.

It is shown that administration of acetylcholine to animals (50 micrograms per 100 g of body weight) leads to the activation of respiration and oxidative phosphorylation in the rat liver mitochondria under oxidation of alpha-ketoglutarate; this effect depends on the concentration of calcium ions in the incubation medium of mitochondria. The rate of ADP-stimulated respiration of mitochondria of experimental animals reaches its maximum level under lower concentrations of Ca2+ than in the control animals. The results of investigation of dependence of acetyl choline effect on respiration of mitochondria on the concentration of alpha-ketoglutarate in calcium and calcium-free incubation medium have shown that the half-maximum effect of acetylcholine is observed in calcium medium at lower concentration of the substrate than in calcium-free medium. The latter indicates to the increase of affinity of alpha-ketoglutarate dehydrogenase to alpha-ketoglutarate under these conditions. It is found out that acetylcholine (1.10(-8) M) increases the rate of ADP- and Ca(2+)-stimulated respiration of mitochondria of isolated perfused rat liver, while mutual effect of verapamyl and niphedipin removes this effect.

[The epidemiological characteristics of HIV infection in Ukraine]. / Osobennosti épidemiologii VICh-infektsii v Ukraine.

The paper presents results of screening tests involving different groups of the population in the Ukraine, for antibodies to HIV, findings from epidemiologic investigation into cases with HIV-infection, with a view to outlining epidemiologic situation and assessing effectiveness of antiepidemic measures. HIV-transmission through sexual contacts is the commonest way of HIV-transportation in the Ukraine (66.1% of all cases of infection). An attempt is made of giving a short-term prognosis of epidemic situation in the Ukraine over a period of up to 1995. A conclusion is reached on the necessity of strengthening preventive measures with the view of influencing the unsafe forms of sexual behaviour.

[Mechanism of reciprocal effects of acetylcholine on oxidation of alpha-ketoglutarate and succinate in heart and liver mitochondria. Factors influencing detection of the acetylcholine effect]. / Mekhanizm retsiproknoï diï atsetylkholinu na okysneniia alpha-ketohlutaratu i suktsynatu v mitokhondriiakh sertsiia i pechinky. Faktory, shcho vplyvaiut' na vyiavlennia efektu atsetylkholinu.

Effect of acetylcholine administration (50 mg/100 g) on phosphorylating oxidation in the liver and heart mitochondria of rats and guinea pigs has been studied. The reciprocal effect of acetylcholine on alpha-ketoglutarate and succinate oxidation (acceleration and inhibition, respectively) was observed. In both cases, the ADP/O coefficient increased. The effect was specific for two substrates and was absent when pyruvate, isocitrate, malate and glutamate were oxidized. When a mixture of glutamate and malate was oxidized producing alpha-ketoglutarate by transamination, acetylcholine stimulated respiration and that effect was abolished by aminooxyacetate. The extent of acetylcholine effects depended on the substrate concentration, duration of storage of isolated mitochondria, the type of the tissue and species of an animal. The described increase in oxidative phosphorylation efficiency by acetylcholine administration corresponded to physiological action of the blood and tissue acetylcholine in the organism which promoted saving of oxygen consumption.