Oxidative damage and altered antioxidant enzyme activities in the small intestine of streptozotocin-induced diabetic rats

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Abstract

The small intestine exhibits numerous morphological and functional alterations during diabetes. Oxidative stress, a factor implicated in the pathogenesis of diabetic complications may contribute towards some of these alterations. We therefore investigated the occurrence of oxidative stress in the small intestine during diabetes by measuring the extent of oxidative damage as well as the status of the antioxidant defense system. Significant increases in lipid peroxidation (four-fold) as measured by TBARS and protein oxidation (38%) as measured by protein carbonyl content were observed after 6 weeks of diabetes. A distinct elevation in the activities of catalase (123.9%) and superoxide dismutase (71.9%) and a decline in the activity of glutathione peroxidase (67.7%) were also observed. The steady state mRNA levels of these enzymes measured by RT-PCR were, however, unchanged suggesting the absence of transcriptional control. In contrast, no changes in the levels of protein and non-protein thiols as well as the activities of glutathione reductase and glutathione-S-transferase were detected. Interestingly, decreases in the activities of xanthine oxidase (XO; 25.7%) and xanthine dehydrogenase (XDH; 42.6%) indicate that they do not contribute significantly to oxidative damage. The results thus reveal the occurrence of oxidative stress in the small intestine during diabetes and suggest its possible involvement in some of the accompanying functional alterations.

Introduction

Diabetes mellitus results in severe metabolic imbalances and pathological changes in many tissues. Oxidative stress is believed to play a role in the development of complications in these tissues (Baynes & Thorpe, 1996). Recently, it has been proposed that carbonyl stress, i.e. the increase in reactive carbonyl compounds derived from oxidative and non-oxidative reactions, leads to increased chemical modification of proteins and at a later stage, to oxidative stress and tissue damage. A deficit in the detoxification of carbonyl compounds by the enzymes of the glyoxalase pathway and aldose reductase is believed to be partly responsible for carbonyl stress and consequent oxidative stress (Baynes & Thorpe, 1999).

The cellular antioxidant status determines the susceptibility to oxidative damage and is usually altered in response to oxidative stress (Halliwell & Gutteridge, 1999). Alterations in the antioxidant enzyme activities and increased oxidative damage have been demonstrated in different tissues of diabetic animals (Kakkar, Kalra, Mantha, & Prasad, 1995). In addition to alterations in activities, changes in the mRNA expression of the antioxidant enzymes have also been reported (Cederberg et al., 2000, Kamata and Kobayashi, 1996, Reddi and Bollineni, 1997).

Diabetes is accompanied by several morphological and functional changes in the small intestinal mucosa (Brasitus & Dudeja, 1985; Fedorak, 1990, Feingold et al., 1982; Feingold et al., 1990, Sharma and Sivakami, 1998, Zoubi et al., 1995). These include hyperplasia and hypertrophy of the epithelial cells (Zoubi et al., 1995), elevated levels of digestive enzymes (Sharma & Sivakami, 1998), increased absorption of sugars, amino acids (Fedorak, 1990), enhanced endogenous synthesis of cholesterol (Feingold et al., 1982) and triglycerides (Feingold et al., 1990) and decreased fluidity of the brush border membrane (Brasitus & Dudeja, 1985). The intestinal mucosa is also vulnerable to oxidative stress on account of the constant exposure to reactive oxygen species (ROS) generated by the luminal contents such as oxidized food debris, transition metals like iron and copper, bacterial metabolites, bile acids and salivary oxidants (Halliwell, Zhao, & Whiteman, 2000). Though ROS-mediated injury to the small intestine has been demonstrated in several conditions such as ischemia/reperfusion (Halliwell & Gutteridge, 1999), inflammatory bowel disease (Halliwell & Gutteridge, 1999), surgical stress (Prabhu, Anup, & Balasubramanian, 2000), radiation enteritis (Mutlu-Turkoglu et al., 2000), iron supplementation (Srigiridhar & Nair, 1998) and zinc deficiency (Virgili et al., 1999), there is no information regarding its occurrence during diabetes. In addition there is inadequate information about the small intestinal antioxidant status during diabetes.

The present study was therefore undertaken to determine whether the small intestine is subjected to oxidative damage during diabetes as well as to examine the accompanying changes in antioxidant status in order to understand its role in the pathogenesis of the disease. The nature of regulation of the primary antioxidant enzymes catalase, SOD and GPx was explored by measuring their mRNA levels using RT-PCR. Additionally, the status of the reactive carbonyl compounds detoxifying enzymes, glyoxalases I and II as well as aldose reductase was examined to study the contribution of carbonyl stress, if any, to oxidative damage during diabetes.

Section snippets

Materials

Bovine serum albumin (BSA), Tris(hydroxymethyl)aminomethane (Tris), steptozotocin (STZ), pyrogallol, thiobarbituric acid (TBA), 2,4-dinitrophenylhydrazine (DNPH), guanidine hydrochloride, reduced glutathione (GSH), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), oxidized glutathione (GSSG), nicotinamide adenine dinucleotide reduced phosphate (NADPH), methyl glyoxal, S-d-lactoylglutathione, d-glyceraldehyde, nicotinamide adenine dinucleotide (NAD), xanthine,

Induction of diabetes

Diabetes was induced in male Wistar rats (200–250 g) by a single intraperitonial injection of streptozotocin at a dose of 75 mg/kg body weight in 0.1 M citrate buffer, pH 4.5. Rats showing blood glucose values above 300 mg/dl and persistent glucosuria were selected for the study. Control and diabetic rats were provided food and water ad libitum and maintained for a period of 6 weeks at the end of which, they were weighed, sacrificed and immediately opened surgically and the small intestines were

Results

At the end of 6 weeks, the intestines of the diabetic rats were significantly longer and the mucosal yield higher compared to controls. Significant increases in both lipid peroxidation and the protein carbonyl content confirm the occurrence of oxidative damage in diabetic rats. The increase in lipid peroxidation (four-fold) was greater than the increase (38%) in protein carbonyl content (Table 2). A reduction in the activities of XO and XDH by 25.7 and 42.6% (Table 3) indicate that these

Discussion

The results of our study demonstrate the occurrence of oxidative damage in the small intestine during experimental diabetes. It is likely to be both the cause and consequence of some of the associated changes in intestinal function. The observed increases in lipid peroxidation levels in the small intestine are in agreement with similar findings in other tissues (Kakkar et al., 1995). Lipid peroxidation may bring about protein damage and inactivation of membrane bound enzymes either through

Acknowledgements

The award of Junior Research Fellowship by the Lady Tata Trust, Mumbai and the Senior Research Fellowship by the Council for Scientific and Industrial Research, Government of India, to V.M.B, is gratefully acknowledged. The authors thank Mr. Chiradeep Sarkar for the help with the RT-PCR.

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