Acute and selective regulation of glyceroneogenesis and cytosolic phosphoenolpyruvate carboxykinase in adipose tissue by thiazolidinediones in type 2 diabetes.

AIMS/HYPOTHESIS: Regulation of glyceroneogenesis and its key enzyme cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) plays a major role in the control of fatty acid release from adipose tissue. Here we investigate the effect of rosiglitazone on the expression of genes involved in fatty acid metabolism and the resulting metabolic consequences. MATERIALS AND METHODS: Rosiglitazone was administered to Zucker fa/fa rats for 4 days and to 24 diabetic patients for 12 weeks, then mRNA expression for the genes encoding PEPCK-C, mitochondrial PEPCK, adipocyte lipid-binding protein, glycerol kinase, lipoprotein lipase and glycerol-3-phosphate dehydrogenase was examined in s.c. adipose tissue by real-time RT-PCR. Glyceroneogenesis was determined using [1-(14)C]pyruvate incorporation into lipids. Cultured adipose tissue explants from overweight women undergoing plastic surgery were incubated with rosiglitazone for various times before mRNA determination and analysis of PEPCK-C protein, activity and glyceroneogenesis. RESULTS: Rosiglitazone administration to rats induced the expression of the gene encoding PEPCK-C mRNA (PCK1) and PEPCK-C activity in adipose tissue with a resulting 2.5-fold increase in glyceroneogenesis. This was accompanied by an improvement in dyslipidaemia as demonstrated by the decrease in plasma NEFAs and triacylglycerol. In rosiglitazone-treated diabetic patients, PCK1 mRNA was raised 2.5-fold in s.c. adipose tissue. Rosiglitazone treatment of adipose tissue explants from overweight women caused a selective augmentation in PCK1 mRNA which reached a maximum of 9-fold at 14 h, while mRNA for other genes remained unaffected. Experiments with inhibitors showed a direct and transcription-only effect, which was followed by an increase in PEPCK-C protein, enzyme activity and glyceroneogenesis. CONCLUSIONS/INTERPRETATION: These results favour adipocyte glyceroneogenesis as the initial thiazolidinedione-responsive pathway leading to improvement in dyslipidaemia.

Treatment of streptozotocin-induced diabetic rats with vanadate and phlorizin prevents the over-expression of the liver insulin receptor gene.

Administration of vanadate, an insulinomimetic agent, has been shown to normalize the increased number of insulin receptors in the liver of streptozotocin-induced diabetic rats. In the present study, the effects of vanadate on various steps of expression of the liver insulin receptor gene in diabetic rats have been analyzed and compared with those of phlorizin, a glucopenic drug devoid of insulinomimetic properties. Livers of rats killed 23 days after streptozotocin injection showed a 30-40% increase in the number of cell surface and intracellular insulin receptors, a 50-90% increase in the levels of 9.5 and 7.5 kb insulin receptor mRNA species, and a 20% decrease in the relative abundance of the A (exon 11-) insulin receptor mRNA isotype. Daily administration of vanadate or phlorizin from day 5 to day 23 prevented the increase in insulin receptor number and mRNA level, and vanadate treatment also normalized receptor mRNA isotype expression. Unlike observations in vivo, vanadate and phlorizin differentially affected the expression of the insulin receptor gene in Fao hepatoma cells. Vanadate treatment (0.5 mmol/l for 4 h) decreased the levels of the 9.5 and 7.5 kb insulin receptor transcripts by at least twofold, without affecting the relative abundance of the A insulin receptor mRNA isotype. In contrast, phlorizin treatment (5 mmol/l for 4 h) slightly increased or did not affect the levels of the 9.5 and 7.5 kb insulin receptor transcripts respectively, and increased by twofold the relative expression of the A insulin receptor mRNA isotype. It is suggested that, although mediated in part by a reversal of hyperglycemia, normalization of liver insulin receptor gene expression by vanadate treatment in diabetic rats may also involve a direct inhibitory effect of this drug on gene expression.

Familial hyperproinsulinaemia due to a mutation substituting histidine for arginine at position 65 in proinsulin: identification of the mutation by restriction enzyme mapping.

UNLABELLED: Familial hyperproinsulinaemia is a rare genetic disorder characterized by point mutations in the insulin gene which impair the conversion of proinsulin to insulin. We report here three members of a two-generation Caucasian family in whom this syndrome was identified by unexplained hyperinsulinism associated with normal glucose tolerance and normal insulin sensitivity. Plasma insulin immunoreactivity showed a reduced affinity for the insulin receptor and eluted mainly, on Biogel chromatography, at the position of proinsulin. Analysis of the PCR-amplified insulin gene by restriction enzyme mapping revealed a new recognition site for the enzyme Nla III, indicating a Arg65 to His mutation. Sequence analysis of exon 3 confirmed this mutation in one allele of the gene. CONCLUSION: This study reports a two-generation European-Caucasian family with hyperproinsulinaemia due to a substitution of His for Arg at position 65 in proinsulin, the seventh now identified worldwide and the second from Europe. The mutation generated a new restriction site on the insulin gene suggesting the usefulness of restriction enzyme mapping as a screening procedure.

Vanadate, but not insulin, inhibits insulin receptor gene expression in rat hepatoma cells.

Insulin and vanadate treatments have recently been shown to reverse the overexpression of the hepatic insulin receptor (IR) gene in streptozotocin-induced diabetic rats. To better understand the mechanisms underlying these effects, the abilities of insulin and vanadate to affect IR gene expression have been comparatively examined in Fao hepatoma cells, an insulin-responsive cell line. Exposure of Fao cells to insulin (1 microM) or vanadate (500 microM) for 24 h led to a 2-fold decrease in IR number in total cellular membranes. Insulin treatment did not affect IR messenger RNA (mRNA) level regardless of time of exposure and concentration. In contrast, vanadate treatment caused a time- and dose-dependent decrease in IR mRNA level, which was maximal (4-fold change) after a 24-h exposure to 500 microM vanadate and was fully reversible. Insulin treatment increased from 28 to 39% the relative expression of isotype A IR mRNA, but vanadate treatment did not significantly affect this parameter. Vanadate treatment did not modify mRNA half-life (3.5 h) in 5, 6 dichlorobenzimidazole riboside-treated cells but decreased by 4-fold the transcriptional activity of the IR gene. These data show for the first time that, although both insulin and vanadate decrease total cellular IR number in Fao cells, only vanadate decreases IR mRNA level. It does so by inhibiting transcription of the IR gene, suggesting an action on the gene promoter which could be mediated by changes in the level of expression and/or of phosphorylation of trans-acting factors.

[Regulation of insulin receptor expression and its gene]. / Régulation de l'expression du récepteur de l'insuline et de son gène.

The insulin receptor is a membrane macromolecule whose expression on the cell surface is essential for cell sensitivity to insulin. Current knowledge on the regulation of expression of the insulin receptor and its gene in human and animal cells is presented. Although ubiquitously distributed, the insulin receptor and its messenger RNA (mRNA) are mainly expressed in metabolically active cells such as hepatocytes and adipocytes. Two receptor isoforms, generated by alternative splicing of exon 11, have been identified. Isoform B (exon 11+) predominates in liver and adipocytes, and isoform A (exon 11-) in brain, spleen and leukocytes. In vivo and in several cell models, the expression of the insulin receptor and/or its mRNA is under positive regulation by glucocorticoid hormones and negative regulation by insulin. Glucocorticoid hormones stimulate receptor gene transcription and receptor protein synthesis. Insulin stimulates receptor protein degradation and, in certain cell types, decreases receptor mRNA level. Vanadate (an insulinomimetic agent) corrects, in vivo, the hyperexpression of the liver receptor observed in experimental insulinopenic diabetes, but its effects on receptor expression in vitro are complex and vary with the cell type. In vivo the insulin receptor and/or its mRNA are expressed early in fetal development with a high level, in liver, of isoform A. Maximal expression is reached at the end of gestation and then decreases after birth. In several cell models, receptor protein and/or mRNA expression is affected by cell growth and/or differentiation. Several cis- and trans-acting factors regulating the expression of the human insulin receptor gene and its response to glucocorticoid hormones have been identified.

Isolation and characterisation of porcine sorbin.

Sorbin has been isolated from extracts of porcine upper intestine, and the biological activity in absorbing water and electrolytes utilized to monitor the purification procedure. Pure sorbin was obtained in a yield of about 1 mg/Mg boiled intestine. The protein chain has 153 amino acid residues and the primary structure was determined by analyses of CNBr-cleaved fragments and four enzymatic digests. The protein has a free N-terminal Met and an amidated C-terminal Ala. No structural similarity was observed with other known proteins in data bases, but several segments have special properties and the C-terminal half is rich in Pro and Arg.

Characterization of arsenopyrite oxidizing Thiobacillus. Tolerance to arsenite, arsenate, ferrous and ferric iron.

Two strains of Thiobacillus, T. ferrooxidans and T. thiooxidans, have been isolated from a bacterial inoculum cultivated during a one-year period in a 1001 continuous laboratory pilot for treatment of an arsenopyrite/pyrite concentrate. The optimum pH for the growth of both strains has been found to be between 1.7 and 2.5. Because of the high metal toxicity in bioleach pulps, the tolerance of T. ferrooxidans and T. thiooxidans with respect to iron and arsenic has been studied. The growth of both strains is inhibited with 10 g/l of ferric ion, 5 g/l of arsenite and 40 g/l of arsenate. 20 g/l of ferrous iron is toxic to T. ferrooxidans but 30 g/l is necessary to impede the growth of T. thiooxidans.

Dual effect of bombesin and gastrin releasing peptide on gastric emptying in conscious cats.

The effect of bombesin (BBS) and gastrin releasing peptide (GRP) on gastric emptying was studied in conscious cats. This effect was measured simultaneously with antral motility. Acid and pepsin secretions as well as blood hormonal peptide release were additionally measured. A dual effect was observed. First, BBS and GRP slowed gastric emptying of liquids, while antral motility was decreased, then after 60 minutes of continuous intravenous infusion, antral motility returned to basal values and gastric emptying effect reversed. The mechanism of this peculiar action is independent of gastrin, pancreatic polypeptide, somatostatin and motilin release and most probably connected with a cholinergic stimulation induced by the peptides, the late predominance of which counterbalances the inhibitory effect of bombesin-like peptides on antral motility.

Effect of porcine gastrin releasing peptide on gastric secretion and motility and the release of hormonal peptides in conscious cats.

The effect of porcine gastrin releasing peptide (GRP) was compared to those of bombesin (BBS) and pentagastrin (PG) in conscious cats. GRP and BBS augmented acid and pepsin secretions, as well as antral motility with an early effect comparable to that produced by pentagastrin with an elevation of low amplitude contractions and a diminution of high amplitude contractions. BBS and GRP increased plasma gastrin and pancreatic polypeptide (PP) levels and decreased motilin levels measured by a C terminus-directed antiserum. In all cases, BBS and GRP displayed parallel dose-response curves. PG showed slight differences in the slopes of the dose-response curves slopes of the dose-response curves except for acid secretion stimulation where no difference was noted (PG was the most effective) and for pepsin stimulation where the difference was large (PG was much less effective). According to the different targets studied, BBS was 4 to 9 times more potent than GRP, 6 to 200 times more than PG. Gastrin release, elicited by the lowest ED50 of both BBS and GRP, should be considered as their primary effect in the cat.

Computer-assisted method for identification of Bacillus species isolated from liquid antacids.

One hundred nineteen species of Bacillus were isolated from five heavily contaminated liquid antacids and their constituent chemicals. The 66 different reaction profiles obtained were expressed in probability figures and stored in a computer. A total of 13 Bacillus species were identified, with B. coagulans, B. licheniformis, B. subtilis, and B. polymyxa present at particularly high frequencies. The potential advantage of using a computer in the identification of aerobic sporeformers is demonstrated.