Mechanisms contributing to angiotensin II regulation of body weight.

Previous studies in our laboratory have implicated adipose tissue as a potential site for local angiotensin II (ANG II) synthesis. However, functions of ANG II in adipose tissue and the impact of ANG II on body weight regulation are not well defined. To study the effect of ANG II on body weight, a chronic ANG II infusion model was used. In study 1, a low dose of ANG II (175 ng.kg-1.min-1) was infused into rats for 14 days. Plasma ANG II levels were not elevated after 14 days of infusion. ANG II-infused rats did not gain weight over the 14-day protocol and exhibited a lower body weight than controls on day 8. Food intake was not altered, but water intake was increased in ANG II-infused rats. Blood pressure gradually increased to significantly elevated levels by day 14. Thermal infrared imaging demonstrated an increase in abdominal surface temperature. Measurement of organ mass demonstrated site-specific reductions in white adipose tissue mass after ANG II infusion. In study 2, the dose-response relationship for ANG II infusion (200, 350, and 500 ng.kg-1.min-1) was determined. Body weight (decrease), blood pressure (increase), white adipose mass (decrease), plasma ANG II levels (increase), and plasma leptin levels (decrease) were altered in a dose-related manner after ANG II infusion. In study 3, the effect of ANG II infusion (350 ng.kg-1.min-1) was examined in rats treated with the vasodilator hydralazine. Hydralazine treatment normalized blood pressure in ANG II-infused rats. The effect of ANG II to decrease body weight was augmented in hydralazine-treated rats. These results demonstrate that low levels of ANG II infusion regulate body weight through mechanisms related to increased peripheral metabolism and independent of elevations in blood pressure.

Role of cytochrome P450 2C9 and an allelic variant in the 4'-hydroxylation of (R)- and (S)-flurbiprofen.

Flurbiprofen is a chiral non-steroidal anti-inflammatory drug used in the treatment of pain or inflammation. The primary routes of biotransformation for (R)- and (S)-flurbiprofen are oxidation (presumably cytochrome P450) and conjugation. To date, the specific cytochrome P450 (P450) involved in the oxidative metabolism of this compound (specifically 4'-hydroxylation) has not been elucidated. Experiments were conducted to characterize the kinetic parameters (Km and Vmax) for the 4'-hydroxylation of (R)- and (S)-flurbiprofen in human liver microsomes, to determine if enantiomeric interactions occur when both enantiomers are present, and to identify the specific P450 form(s) involved in this reaction. In human liver microsomes, the Km and Vmax (mean +/- SD) for (R)-4'-hydroxy-flurbiprofen formation were 3.1 +/- 0.8 microM and 305 +/- 168 pmol.min-1.mg protein)-1, respectively. In comparison, the Km and Vmax (mean +/- SD) for (S)-4'-hydroxy-flurbiprofen formation were 1.9 +/- 0.4 microM and 343 +/- 196 pmol.min-1.mg protein-1, respectively. Enantiomeric interaction studies revealed a decrease in Km and Vmax for both enantiomers and an apparent loss of stereoselectivity. Racemic-warfarin, tolbutamide, alpha-naphthoflavone and erythromycin were studied as potential inhibitors of this process. The estimated Ki values for the inhibition of (R)- and (S)-4'-hydroxy-flurbiprofen formation by racemic-warfarin were 2.2 and 4.7 microM. This reaction was also inhibited by tolbutamide. In contrast, erythromycin and alpha-naphthoflavone had no appreciable effect on 4'-hydroxy-flurbiprofen formation. cDNA-expression of individual forms was used to determine which P450 was involved in 4'-hydroxy-flurbiprofen formation. P450 2C9 and an allelic variant (R144C) readily catalyzed the formation of 4'-hydroxy-flurbiprofen. P450 1A2 was also active albeit with a turnover rate 1/140th that of P450 2C9R144C (P450s 2C8, 2E1 and 3A4 were not active toward either enantiomer). The results of these studies indicate that the enantiomers of flurbiprofen may exhibit stereoselectivity with respect to enzyme affinity but have roughly equal maximum formation velocities. Additionally, these two enantiomers may compete for the enzyme resulting in lower maximum velocities for both enantiomers. Finally, of those P450 forms examined, only P450 2C9 and an allelic variant catalyzed the 4'-hydroxylation of both (R)- and (S)-flurbiprofen.