Determinants of Hydroperoxide Detoxification in Diabetic Rat Intestine: Effect of Insulin and Fasting on the Glutathione Redox Cycle Ryuichi Iwakiri, Carol Ann Rhoads, and Tak Yee Aw The capacity for hydroperoxide detoxification in diabetic (DM) intestine was studied in streptozocin-indUced DM rats by quantification of the intestinal glutathione (GSH} redox cycle, a key cellular pathway for peroxide elimination. A role for luminal glucose in regulation of redox cycle activity was examined in insulin-treated or 24.hour-fasted DM animals. Intestinal activities Of the redox enzymes, GSH peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase (G6PD), were significantly decreased by 17 hours' insulin treatment, whereas only G6PD was decreased by fasting. Mucosal GSH levels were also markedly decreased under these conditions. These results are consistent with an overall Suppression of intestinal GSH redox cycle function by short-term administration of insulin. Insulin treatment for 7 consecutive days increased hepatic G6PD activity by fourfold but was without effect on intestinal G6PD, suggesting tissue specificity in insulin regulation of G6PD. The rate of metabolism of tert-butyl hydroperoxide (tBH) in isolated enterocytes was low in the absence of substrates (0.51 4 0.07 nmol / 10s cells / min) but was increased fivefold by exogenous glucose (2,70 +- 0.11 nm01/ 10s cells / min), indicating that glucose availability is an important contributor to intestinal detoxification of toxic hydroperoxides. Collectively, the current results show that GSH redox cycle enzymes in DM intestine are under coordinate insulin control, and that this control appears to be downregulated by short-term insulin treatment. Copyright © 1995 by W,B. Saunders Company p REVIOUS STUDIES have shown that an elevated level of serum oxidized !ipoprotein is associated with human and experimental diabetes mellitus (DM). 1-3 This enhanced circulating lipid peroxide level is implicated in DM complications) Although the mechanism for increased plasma accumulation of peroxides is unclear, recent studies suggest that an increase in absorption of dietary oxidized lipids may be primarily responsible for the elevated plasma levels of lipid peroxides observed in the streptozocin- induced DM rat. 1 A contributing factor to an enhanced peroxide uptake and subsequent peroxide transport into the lymph could be a decreased intracellular hydroperoxide metabolism 4-6 in DM intestine. We have recently shown that the giutathione (GSH) redox cycle7,,8 (Fig 1) is an important cellular pathway for detoxification of lipid hydroperoxides by rat small intes- tine. 4-6 The rapid and efficient reduction of hydroperoxides is accomplished by the coordinate function of GSH peroxi- dase, GSSG reductase, and the supply of GSH, a major cellular reductant (Fig 1). Thus, regulation of intestinal redox enzyme activities and of GSH supply could ultimately control the output of hydroperoxides into the systemic circulation. In addition, a key step in the supply of GSH is the continuous regeneration of GSH from GSSG at the expense of NADPH (Fig 1). A major contributor to the total cell NADPH pool is the pentose phosphate pathway, 9 and glucose utilization by this pathway is regulated by glucose-6-phosphate dehydrogenase (G6PD), the rate- From the Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport, LA. Submitted October 28, 1994; accepted February 15, 1995. Supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant No. DK-44510. T.Y~A. is a recipient of an American Heart AssociatiOn Established Investigatorship Award. Address reprint requests to Tak Yee Aw, PhD, Department of Physiology and Biophysics, Louisiana State UniversityMedical Center, Shreveport, LA 71130-3932. Copyright © 1995 by W.B. Saunders Company 0026-0495/95/4411-0016503.00/0 limiting Step. 1° Hence, elimination Of hydroperoxides by DM Small intestine could depend on the function of G6PD and the availability of glucose. Because tissUe utilization of glucose is altered by insulin insufficiency in DM, this change in tissue glucose status could have a significant impact on the overall regulation of hydroperoxide detoxification in the DM intestine. The objective of the current study is therefore to quantify the determinants of hydroperoxide detoxification in the DM intestine. To address this objective, we used the streptozocin-induced diabetic rat as the animal model and defined intestinal detoxification capacity in terms of tissue enzyme activities and reductant levels of each component of the GSH redox cycle. In addition, we have directly quantified the rate of hydroperoxide metabolism without or with glucose supplements in isolated enterocytes. The results provide important insights into the function of the GSH redox cycle in the DM intestine, and underscore the importance of maintaining glucose status to support redox cycle activity in intestinal hydroperoxide detoxification in DM animals. MATERIALS AND METHODS Materials Streptozocin, tert-butyi hydroperoxide (tBH), and the glucose assay kit were purchased from Sigma Chemical (St Louis, MO). Ultralente insulin and lente insulin were from Novo Nordisk Pharmaceuticals (Bagsvaerd, Denmark). The protein dye reagent was obtained from Bio Rad (Hercules, CA). All other chemicals were of reagent grade and were purchased from local sources. Induction of DM in Rats Male Sprague-Dawley rats (170 to 200 g) were maintained ad libitum on standard rat chow for 5 days before streptozocin treatments. Streptozocin was dissolved in 0.01 mol/L citrate buffer, pH 4.5, and each rat was injected intraperitoneally (70 mg/kg) within 5 minutes after drug preparation. On day 7 after streptozo- cin administration, attainment of DM was confirmed by measure- ments of plasma glucose level. Animals with plasma glucose levels of 12 mmol/L or greater were considered DM, and typically70% to 80% of the rats developed DM under these conditions. Rats that 1462 Metabolism, Voi 44, No 11 (November), 1995: pp 1462-i468