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Short-term gemfibrozil treatment reverses lipid profile and peroxidation but does not alter blood glucose and tissue antioxidant enzymes in chronically diabetic ratsMolecular and Cellular Biochemistry 216: 59–63, 2001.
2001 Kluwer Academic Publishers. Printed in the Netherlands.
Short-term gemfibrozil treatment reverses lipid
profile and peroxidation but does not alter blood
glucose and tissue antioxidant enzymes in
chronically diabetic rats
G. Ozansoy, B. Akin, F. Aktan and Ç. Karasu Ankara University, Faculty of Pharmacy, Departments of Pharmacology and Biochemistry, Tando an, Received 3 May 2000; accepted 20 September 2000 Abstract
In this study, we investigated the efficiency of short-term treatment with gemfibrozil in the reversal of diabetes-induced changeson carbohydrate and lipid metabolism, and antioxidant status of aorta. Diabetes was induced by a single injection of streptozotocin(45 mg/kg, i.p.). After 12 weeks of induction of diabetes, the control and diabetic rats were orally gavaged daily with a dosingvehicle alone or with 100 mg/kg of gemfibrozil for 2 weeks. At 14 weeks, there was a significant increase in blood glucose,plasma cholesterol and triglyceride levels of untreated-diabetic animals. Diabetes was associated with a significant increase inthiobarbituric acid reactive substances (TBARS) in both plasma and aortic homogenates, indicating increased lipid peroxidation.
Diabetes caused an increase in vascular antioxidant enzyme activity, catalase, indicating existence of excess hydrogen perox-ide (H O ). However, superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities in aortas did not signifi- cantly change in untreated-diabetic rats. In diabetic plus gemfibrozil group both plasma lipids and lipid peroxides showed a
significant recovery. Gemfibrozil treatment had no effect on blood glucose, plasma insulin and vessel antioxidant enzyme activity
of diabetic animals. Our findings suggest that the beneficial effect of short-term gemfibrozil treatment in reducing lipid
peroxidation in diabetic animals does not depend on a change of glucose metabolism and antioxidant status of aorta, but this
may be attributed to its decreasing effect on circulating lipids. The ability of short-term gemfibrozil treatment to recovery of
metabolism and peroxidation of lipids may be an effective strategy to minimize increased oxidative stress in diabetic plasma
and vasculature. (Mol Cell Biochem 216: 59–63, 2001)
Key words: gemfibrozil, streptozotocin-diabetes, aorta, superoxide dismutase, catalase, glutathione peroxidase, lipid peroxidation,hyperlipidemia Introduction
we have previously observed that increased free radical pro-duction is responsible for abnormalities in both vasomotor Lipid peroxidation has been implicated in the pathogenesis activity and ultrastructural organisation of aorta in experi- of many degenerative disorders including naturally occurring mental diabetes [4–6]. Under normal conditions, antioxidant and chemically induced diabetes [1, 2]. Oxidation of lipids enzymes, catalase, glutathione peroxidase (GSHPx) and in plasma lipoproteins and in cellular membranes is associ- superoxide dismutase (SOD), offer protection to cell and tis- ated with increased oxidative stress leading to the develop- sues against oxidative injury . If the balance between the ment of cardiovascular disease in diabetes [1, 3]. Accordingly, formation of reactive oxygen species and their elimination Address for offprints: Ç. Karasu, Department of Pharmacology, Faculty of Pharmacy, Ankara University, 06100 Tando an, by endogenous antioxidant defense systems is impaired, oxi- oxidation, were measured by the fluorimetric technique of dative damage of the membrane and changes in the structural and functional integrity of subcellular organelles can occur.
Superoxide dismutase (SOD) activity in aortic homogenates Gemfibrozil is most widely used fibric acid, an effective was measured spectrophotometrically at 560 nm using the cholesterol- and triglyceride-lowering agent, which has been method of Spitz . The reaction mixture in 50 mM phos- shown to be beneficial in the treatment of atherosclerosis .
phate buffer (pH 7.8) consisted of SOD-induced inhibition It has been reported that gemfibrozil renders low-density li- of the reduction of nitro blue tetrazolium, using a free radi- poprotein (LDL) less susceptible to oxidative modification cal-generating system of 0.1 nM xanthine and an amount of , decreases the rate of LDL oxidation in vivo and in vitro xanthine oxidase to produce a rate of absorbance change of [10, 11], potentiates fibrinolysis , and increases the lev- els of high-density lipoprotein cholesterol (HDL-C) .
Catalase was measured spectrophotometrically by the These mechanisms have been suggested to mediate its ben- method of Aebi . Cleaned and minced aortas were ho- eficial effects in reducing or preventing of atherogenic risk mogenized in 3 volumes of 50 mM phosphate buffer (pH 7) and cardiovascular disease [9, 11, 13]. The aim of the present with Tirton X-100. Twenty µM of homogenate was added to study was to investigate whether the changes in lipid metabo- a cuvette containing the same phosphate buffer to make fi- lism and aorta antioxidant enzyme profile in chronically dia- nal volume of 2.0 ml. The reaction was started by the addi- betic rats could be reversed by acute gemfibrozil treatment.
tion of 1.0 mL of 30 mM H O . The rate of decomposition of H O was read at 20oC against a blank containing prepared enzyme solution but no substrate in absorbancy at 240 nm.
Materials and methods
The activity of tissues is expressed as k/sec/mg protein, wherek is the first order rate constant.
Animals, induction of diabetes and gemfibrozil treatment The method of Lawrence was used to measure glutathione peroxidase (GSH-Px) activity . In this method, GSHPx The ‘Animal Care Ethics Committee’ of Ankara University has activity is coupled to NADPH utilization, and the production approved the study. Male Wistar rats weighing 200–250 g were of NADP+ was measured spectrophotometrically at 340 nm.
divided into the following groups: (1) Untreated-control rats The assay mixture consisted of 76 mM phosphate buffer with (n = 8); (2) gemfibrozil treated-control rats (n = 9); untreated- EDTA and NaN (pH 7.0), 0.150 mg 10,000 × g supernatant diabetic rats (n = 9); gemfibrozil treated-diabetic rats (n = 10).
protein of tissue, 0.1 mM NADPH, 4.0 mM GSH and 1.5 U Diabetes was induced by streptozotocin (45 mg/kg, i.p.) in- glutathion reductase in a final volume of 500 µL. The reac- jection. Control animals were injected with the vehicle alone.
tion was started by addition of 3.0 mM H O . GSHPx activ- Two days after streptozotocin injection, development of dia- ity was expressed as µmol of NADPH oxidized to NADP+/ betes was confirmed from tail vein blood glucose, and rats with blood glucose levels of 250 mg/dl or above were con-sidered to be diabetic. Twelve weeks after diabetes induc-tion, gemfibrozil was given for 2 weeks (100 mg/kg/day in carboxymethylcellulose 10%, per oral). The treatment doseof gemfibrozil was chosen according to a previous study .
All chemicals were purchased from Sigma Chemical (St.
Rats were maintained under standard housing conditions for Louis, MO, USA). Results are expressed as the mean ± S.E.M.
14 weeks before experiments were conducted.
Statistical analysis was carried out using one-way analysisof variance followed by Neuman-Keul’s test. Results wereconsidered significantly different if p < 0.05.
Blood glucose concentrations were measured by an Ames glucometer (Glucometer III, Bayer Diagnostics, France).
Plasma immunoreactive insulin concentrations were deter- Body weights and blood glucose levels mined by standard radioimmunoassay technique using Coat-A-Count (DPC) insulin kit available from Diagnostic Products Initial and final body weights and blood glucose levels of ani- Corporation, USA. Plasma triglyceride and cholesterol con- mals are presented in Table 1. The baseline weight at the be- centrations were measured using a commercially available ginning of the study was similar in all groups. At the end of the treatment period, untreated-diabetic rats exhibited loss of Thiobarbituric acid reactive substances (TBARS), as an body weight. There was no significant difference between ini- index of malondialdehyde production and hence lipid per- tial and final body weight levels in gemfibrozil treated group.
Table 1. Body weights and blood glucose levels of animals Data are means ± S.E.M. **p < 0.01; ***p < 0.001 vs. untreated-control.
The streptozotocin-induced diabetic animals showed con- enzyme activities in diabetic aorta. Control rats received two sistent fasting hyperglycaemia throughout the study. Gem- weeks treatment with gemfibrozil showed similar values in fibrozil treatment did not induce a significant fall in blood oxidative stress markers when compared with untreated-con- glucose levels of diabetic animals and had no effect on body weight and blood glucose levels of control rats.
Plasma insulin levels, lipid metabolism and oxidative Our experiments have demonstrated that TBARS levels, as an index of lipid peroxidation, are increased in aorta andplasma of untreated-diabetic rats. This is concomitant with Plasma insulin levels of untreated- or gemfibrozil treated- augmented plasma cholesterol and triglycerides, and is con- diabetic rats were found to be significantly decreased when sistent with the earlier reports [4, 5, 19]. The increased lipid compared with control rats (Table 2).
peroxidation, firstly, implies the increased levels of reactive Plasma cholesterol and triglyceride concentrations mark- oxygen species (i.e. superoxide, hydrogen peroxide and hy- edly increased in untreated-diabetic rats, as expected. As droxyl radical) that could be due to their increased produc- shown in Table 2, short-term gemfibrozil treatment com- tion or decreased destruction. Hyperglycemia may lead to an pletely reversed plasma triglyceride and cholesterol levels of increase in the production of reactive oxygen species mainly diabetic animals. Gemfibrozil treatment of control rats did through glucose autooxidation, non-enzymatic protein gly- not induce significant changes in plasma insulin, cholesterol cation, increased sorbitol pathway and depletion of some non- and triglyceride concentrations (Table 2).
enzymatic or enzymatic scavengers [1, 3, 20]. Furthermore, Lipid peroxidation levels, measured as TBARS, was higher hyperlipidemia alone may be unique reason for the enhanced in the aorta and plasma of untreated-diabetic rats than con- levels of TBARS in diabetics since previous research has trol rats. Gemfibrozil treatment normalized plasma and aorta directly linked hyperlipidemia with increased serum and tis- lipid peroxidation levels in diabetic animals (Table 2).
sue concentrations of lipid peroxidation products [21, 22].
SOD and GSHPx activities were not significantly different In order to evaluate antioxidant status, superoxide dismutase in aorta of untreated- or gemfibrozil treated-diabetic versus (SOD), catalase and glutathione peroxidase (GSHPx) activi- control animals. In contrast, diabetes resulted in a significant ties in aortic homogenates were measured, and a significant increase in aorta catalase activity (Table 2). Short-term gem- increase in vascular catalase activity was observed without fibrozil treatment did not significantly change antioxidant significant changes in either SOD or GSHPx in diabetic rats.
Table 2. Plasma insulin, cholesterol, triglyceride levels and oxidative stress markers of animals Gemfibrozil treated-control Gemfibrozil treated-diabetic Aorta Superoxide dismutase (U/mg protein) Aorta glutathione peroxidase (nmol/dk/mg protein) Data are means ± S.E.M. **p < 0.01; ***p < 0.001 vs. untreated-control.
The selective increase in catalase but not SOD or GSHPx of diabetic hypertriglyceridemia [26, 30]. We conclude that has previously been reported in diabetic blood vessels  in addition to the management of diabetic dyslipidemia, and in other diabetic tissue . Increased catalase activ- gemfibrozil may provide a useful therapeutic option in the ity could be a compensatory mechanism in response to in- reversal of oxidative stress in diabetes mellitus.
creased oxidative stress, more specifically, peroxidativestress, due to elevated production of hydrogen peroxide(H O ) in vessels. It has been reported that the preferential References
pathway for detoxification of H O in blood vessels is de- pendent on the concentration of H O . Catalase has been implicated in the detoxification of high H O concentrations 1. Hunt JV, Smith CCT, Wolff SP: Autoxidative glycosylation and possi- ble involvement of peroxides and free radicals in LDL modification whereas GSHPx is effective at lower concentrations of H O by glucose. Diabetes 39: 1420–1424, 1990 . Thus, in the present of a high H O concentration, the 2. Kakkar R, Kalra J, Mantha SV, Prasad K: Lipid peroxidation and ac- induction of catalase in diabetic vessels is an expected result.
tivity of antioxidant enzymes in diabetic rats. Mol Cell Biochem 151: In addition to this, the reason for an increase in catalase but not GSHPx may also be related to location of each enzyme, 3. Chisolm GM, Irwin KC, Pen MS: Lipoprotein oxidation and lipopro- tein-induced cell injury in diabetes. Diabetes 41(suppl 2): 61–66, 1992 as reported previously . The catalase is located mainly 4. Karasu Ç, Ozansoy G, Bozkurt O, Erdo gan D, Ömero glu S: Antioxi- in the peroxisomal compartment as same as fatty acyl CoA dant and triglyceride lowering effects of vitamin E associated with the oxidase, is an H O -generating enzyme in the presence of prevention of abnormalities in the reactivity and morphology of aorta fatty acid and keton body metabolism increasing states such from streptozotocin-diabetic rats. Metabolism 46: 872–879, 1997 as diabetes. The observation of increased catalase without a 5. Karasu Ç, Ozansoy G, Bozkurt O, Erdo an D, Ömero in isoprenaline-induced endothelium-dependent and -independent re- significant change in SOD activity has been reported to be laxation of aorta in long-term STZ-diabetic rats: Reversal effect of possible , since the rate constant for the degradation of dietary vitamin E. Gen Pharmacol 29: 561–567, 1997 H O by catalase is lower than the rate constant for the dis- 6. Karasu Ç: Increased activity of H O in aorta isolated from chronically mutation of superoxide anion radicals to form H O by SOD.
streptozotocin-diabetic rats: Effects of antioxidant enzymes and en- Increased H O is known to inactivate SOD, thus an increase zyme inhibitors. Free Rad Res Biol 27: 16–27, 1999 7. Volkovova K, Charvathova V, Jurcovicova M, Koszeghyova L, Bobek in catalase activity due to oxyradicals may protect SOD in- P: Antioxidative state of myocardium and kidneys in acute diabetic In this study, we have shown for the first time that short- 8. Yuan J, Tsai MY, Hunninghake DB: Changes in composition and distri- term gemfibrozil treatment initiated 12 weeks after induction bution of LDL subspecies in hypertriglyceridemic and hypercholester- of diabetes is able to reverse hyperlipidemia and the excess olemic patients during gemfibrozil therapy. Atherosclerosis 110: 1–11,1994 lipid peroxidation in plasma and vascular tissue without sig- 9. Yoshida H, Ishikawa T, Ayaori M, Shige H, Ito S, Suzukawa M, nificant changes in glucose metabolism and vessel antioxidant Nakamura H: Beneficial effect of gemfibrozil on the chemical com- enzymes in diabetic rats. It has been shown that gemfibrozil position and oxidative susceptibility of low density lipoprotein: A increases lipoprotein lipase activity, increases the clearance randomized, double-blind, placebo-controlled study. Atherosclerosis of very low-density lipoprotein (VLDL) triglycerides and 10. Kawamura M, Hase K, Miyazaki S: In vitro inhibition of low density some times enhances LDL clearance [8, 10, 11]. In addition, lipoprotein oxidation by Gemfibrozil metabolite. Clin Chem 42: 644– lipid-lowering effect of gemfibrozil was reported to be due to a decrease in the plasma triglyceride concentration by in- 11. Hoogerbrugge N, Kerkhofs LG, Jensen H: Gemfibrozil decreases auto- hibiting the VLDL synthesis . It seems that, the benefi- antibodies against oxidized low-density lipoprotein in men with com- cial effect of gemfibrozil on the oxidation of lipids is mainly bined hyperlipidemia. J Intern Med 243: 355–359, 1998 12. Fuji S, Sobel BE: Direct effects of gemfibrozil on the fibrinolytic sys- linked with its hypolipidemic effect. In accordance with our tem. Diminution of synthesis of plasminogen activator inhibitor type.
interpretation is a previous observation which showed that antioxidant effect of gemfibrozil was related to two independ- 13. Kanters SD, Algra A, de Bruit TW, Erkelens DW, Banga JD: Intensive ent phenomena: the reduction of the amount of substrate for lipid-lowering strategy in patients with diabetes mellitus. Diabetes Med the oxidation process due to its hypolipidemic activity, and 14. Haubenwallner S, Essenburg AD, Barnett BC, Pape ME, DeMattos the alteration in the type of fatty acids transported by the RB, Krause BR, Minton LL, Auerbach BJ, Newton RS, Leff T et al.: lipoproteins towards an enrichment in species resistant to the Hypolipidemic activity of select fibrates correlates to changes in he- oxidation process . Furthermore, gemfibrozil can affect patic apolipoprotein C-III expression: A potential physiologic basis for lipoprotein oxidizability by inhibiting their susceptibility to their mode of action. J Lipid Res 36: 2541–2551, 1995 oxidation, and in vivo metabolites of gemfibrozil has been 15. Yagi K: Lipid peroxides and human disease. Chem Phys Lipids 45: reported to inhibit VLDL, LDL and HDL oxidation by free 16. Spitz DR, Oberley LW: An assay for superoxide dismutase activity in radical scavenging activity of themselves . Gemfibrozil mammalian tissue homogenates. Anal Biochem 179: 8–18, 1989 has recently been proposed to be suitable for the treatment 17. Aebi H. Catalase in vitro. Meth Enzymol 105: 121–126, 1983 18. Lawrence RA, Burk RF: Glutathione peroxidase activity in selenium- 26. Tikkanen MJ, Laakso M, Ilmonen M, Helve E, Kaarasalo E, Kilkki E, deficient rat liver. Biochem Biophys Res Commun 71: 952–958, 1976 Saltavo J: Treatment of hypercholesterolemia and combined hyper- 19. Kakkar R, Mantha SV, Kalra J, Prasad K: Time course study of oxida- lipidemia with simvastatin and gemfibrozil in patients with NIDDM.
tive stress in aorta and heart of diabetic rat. Clin Sci 91: 441–448, 1996 20. West IC: Radicals and oxidative stress in diabetes. Diabet Med 17:171– 27. Yang CY, Gu ZW, Xie YH, Valentinova NV, Yang M, Yeshurun D, Quion JA, Gotto AM Jr: Effects of gemfibrozil on very-low-density 21. Kubow S, Goyette N, Kermasha S, Stewart-Phillip J, Koshi KG: Vi- lipoprotein composition and low-density lipoprotein size in patients tamin E inhibits fish-oil induced hyperlipidemia and tissue lipid with hypertriglyceridemia or combined hyperlipidemia. Atherosclero- peroxidation in hamsters. Lipid 31: 839–847, 1996 22. De La Cruz JP, Quintero L, Villalobolos MA, Sanches dela Caesta F: 28. Vasques M, Merlos M, Adzet T, Laguna JC: Decreased susceptibility Lipidperoxidation and glutathione system in hyperlipidemic rabbits: to copper-induced oxidation of rat-lipoproteins after fibrate treatment: Influence of olive oil administration. Biochem Biophys Acta 1485: 36– Influence of fatty acid composition. Br J Pharmacol 117: 1155–1162, 23. Pieper GM, Jordan M, Dodlinger LA, Adams MB, Rosa AM: Per- 29. Aviram M, Rosenblat M, Bisgaier CL, Newton RS: Atorvastatin and oxidative stress in diabetic blood vessels. Reversal by pancreatic islet gemfibrozil metabolites, but not the parent drugs, are potent antioxi- transplantation. Diabetes 44: 884–889, 1995 dants against lipoprotein oxidation. Atherosclerosis 138: 271–280, 24. Wohaieb SA, Godin DV: Alterations in free radical tissue-defence mechanisms in streptozotocin-induced diabetes in rat. Effect of insu- 30. Avogaro A, Piliego T, Catapano A, Miola M, Tiengo A: The effect of lin treatment. Diabetes 36: 1014–1018, 1987 gemfibrozil on lipid profile and glucose metabolism in hypertrigly- 25. Doroshow JC, Locker GY, Myers CE: Enzymatic defenses of the mouse ceridaemic well-controlled non-insulin-dependent diabetic patients.
heart against reactive oxygen metabolites. J Clin Invest 65: 128–135, 1980
The Upper Airway Resistance Syndrome and the Single Girl Madge began her workday differently from the hundreds of others that had gone before. Avoiding the coffee room (the ‘gathering place’), she took a circuitous path past the PFT Lab and the copy machine, hoping to avoid the inevitable. Prior to leaving work the night before, she had gingerly placed the ‘Note-from-Hell’ in Alliso