Reduced serum dipeptidyl peptidase-IV after metformin and pioglitazone treatments.

Dipeptidyl peptidase-IV (DPP-IV) regulates metabolism by degrading incretins involved in nutritional regulation. Metformin and pioglitazone improve insulin sensitivity whereas glyburide promotes insulin secretion. Zucker diabetic rats were treated with these antidiabetic agents for 2 weeks and DPP-IV activity and expression were determined. Serum DPP-IV activity increased whereas tissue activity decreased as the rats aged. Treatment of rats with metformin, pioglitazone, and glyburide did not alter DPP-IV mRNA expression in liver or kidney. Metformin and pioglitazone significantly (P<0.05) reduced serum DPP-IV activity and glycosylated hemoglobin. Glyburide did not lower DPP-IV activity or glycosylated hemoglobin. Regression analysis showed serum DPP-IV activity correlated with glycosylated hemoglobin (r=0.92) and glucagon-like peptide-1 levels (r=-0.49). Metformin, pioglitazone, and glyburide had no effect on serum DPP-IV activity in vitro, indicating these are not competitive DPP-IV inhibitors. We propose the in vivo inhibitory effects observed with metformin and pioglitazone on serum DPP-IV activity results from reduced DPP-IV secretion.

Discrimination against purine-pyrimidine mispairs in the polymerase active site of DNA polymerase I: a structural explanation.

We previously identified five derivatives of Klenow fragment DNA polymerase that have lower fidelity because of amino acid substitutions in the polymerase active site. One of these has alanine substituted for the invariant Glu-710, whose side chain interacts with the deoxyribose of the incoming dNTP. Here we show that the E710A enzyme has reduced fidelity for five of the 12 possible mismatches. All but one of these involve misinsertion of pyrimidines, including two transition mismatches A-dCTP and G-dTTP. In contrast, E710A polymerase error rates for the reciprocal C-dATP and T-dGTP transition mismatches were similar to those of the wild-type enzyme. The kinetics of formation of correct base pairs and transition mismatches by the wild-type and E710A polymerases, combined with information on the structure of the DNA polymerase active site and the asymmetry of wobble base pairs, provides a plausible explanation for the differential effects of the E710A mutation on fidelity. The data suggest that the Glu-710 side chain plays a pivotal role in excluding wobble base pairs between template pyrimidines and purine triphosphates by steric clash. Moreover, this same side chain enhances the stability of incoming correct dNTPs, such that loss of this interaction on removal of the side chain leads to lower selectivity against mismatches involving incoming pyrimidines.

The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.

GPR40 is a member of a subfamily of homologous G protein-coupled receptors that include GPR41 and GPR43 and that have no current function or ligand ascribed. Ligand fishing experiments in HEK293 cells expressing human GPR40 revealed that a range of saturated and unsaturated carboxylic acids with carbon chain lengths greater than six were able to induce an elevation of [Ca(2+)](i), measured using a fluorometric imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent fatty acid tested, with a pEC(50) of 5.7. G protein coupling of GPR40 was examined in Chinese hamster ovary cells expressing the G alpha(q/i)-responsive Gal4-Elk1 reporter system. Expression of human GPR40 led to a constitutive induction of luciferase activity, which was further increased by exposure of the cells to eicosatriynoic acid. Neither the constitutive nor ligand-mediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G alpha(q/11.) Expression analysis by quantitative reverse transcription-PCR showed that GPR40 was specifically expressed in brain and pancreas, with expression in rodent pancreas being localized to insulin-producing beta-cells. These data suggest that some of the physiological effects of fatty acids in pancreatic islets and brain may be mediated through a cell-surface receptor.