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Indian Journal of Pharmacology, Vol. 43, No. 3, May-June, 2011, pp. 330-335 Research Article Effect of alcohol on blood glucose and antioxidant enzymes in the liver and kidney of diabetic rats KR Shanmugam1, K Mallikarjuna2, K Sathyavelu Reddy1 1 Division of Molecular Biology, Department of Zoology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India Correspondence Address: K Sathyavelu Reddy, Division of Molecular Biology, Department of Zoology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India, ksreddy2008@hotmail.com Date of Submission: 23-Sep-2009 Code Number: ph11085 DOI: 10.4103/0253-7613.81504 Abstract Objective: Diabetes mellitus affects every organ in the man including eyes, kidney, heart, and nervous system. Alcohol consumption is a widespread practice. As the effects of chronic alcohol consumption on diabetic state have been little studied, this study was conducted with the objective of evaluating the effect of alcohol in diabetic rats.Materials and Methods: For this study, the rats were divided into five groups (n = 6 in each group): normal control (NC), alcohol treatment (At), diabetic control (DC), diabetic plus alcohol treatment (D + At), diabetic plus glibenclamide treatment (D + Gli). Alcohol treatment was given to the diabetic rats for 30 days. During the period the blood glucose levels, and body weight changes were observed at regular intervals. The antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels were assayed in the liver and kidney tissues. Results: The blood glucose levels were significantly (P < 0.001) elevated and body weight significantly (P < 0.001) decreased in alcohol-treated diabetic rats. SOD and CAT activities were decreased and the MDA level increased significantly (P < 0.001) in alcohol-treated diabetic rats. Histopathological studies showed that alcohol damages the liver and kidney tissues in diabetic rats. Conclusion: These finddings concluded that the consumption of alcohol in diabetic rats worsens the condition. So the consumption of alcohol by diabetic subjects may be potentially harmful. Keywords: Diabetes, alcohol, antioxidant enzymes, blood glucose levels, rats Introduction The prevalence of diabetes mellitus has increased in recent years, particularly type 2 diabetes, which is related to obesity and sedentary lifestyle. [1] The total number of people globally affected by diabetes was reported to be 171 million in 2000 and is projected to be 366 million by 2030, and the vast majority of these cases will be of type 2 diabetes. [2] Free radical production has been reported to be increased in patients with diabetes mellitus and hyperglycemia appears to be the contributing factor for the generation of reactive oxygen species (ROS) which lower the concentrations of antioxidant enzymes. [3] Oxidative stress induced by a high glucose concentration plays a central role in complications of diabetes. [4],[5] Oxidative stress induces the production of highly reactive oxygen radicals that are toxic to cells and has been attributed to protein glycation and/or glucose auto-oxidation owing to a hyperglycemic environment. [6] Oxygen radicals also interact with the lipid bilayer and produce lipid peroxides particularly in cell membranes. [5] Lipid peroxidation of cellular structures is thought to play an important role in complications of diabetes. [6] Alcoholic beverages are used universally and alcohol is the world′s most widely used psychoactive drug, but chronic, excessive alcohol consumption leads to permanent organ damage or death. Alcohol is rapidly oxidized in the liver tissue to acetaldehyde and acetate by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), respectively. [7] Further alcohol consumption and diabetes disturb the balance between the pro- and antioxidant systems of the organism, thereby leading to the oxidative stress by generating free radicals or ROS, [8] which results in liver and kidney injury. There are limited reports on the influence of alcohol on a diabetic state. A J-shaped relationship between alcohol consumption and diabetes has been reported by Hoffmeister et al. [9] Chronic excessive consumption of alcohol may lead to deleterious effects upon many organs and metabolism. [10] Diabetogenic effects of alcohol include its contribution to obesity, induction of pancreatitis, and disturbance of carbohydrate and glucose metabolism. [11] However, the harmful effects of alcohol abuse on the functions of liver, kidney, heart, immune system, and central and peripheral neural system are not yet known. [12] There are no reports on the effect of alcohol on antioxidant enzymes and blood glucose in diabetic rats. The purpose of this study was to investigate the effect of alcohol in streptozotocin (STZ)-induced diabetic rats by measuring blood glucose levels and assaying the antioxidant enzymes activities in liver and kidney tissues. Materials and Methods Animals Male Wistar albino rats aged 6 months and weighing 180 ± 20 g were obtained from Indian Institute of Science, Bangalore, Karnataka, India. The rats were housed in clean polypropylene cages having six rats per cage and maintained in a temperature-controlled room (27 ± 2°C) with a photoperiod of a 12-h light and 12-h dark cycle. The rats were given standard pellet diet (Lipton Rat Feed, Ltd., Pune, Maharashtra, India) and water ad libitum throughout the experimental period. The experiments were carried out in accordance with CPCSEA guidelines and the protocol was approved by the Institutional Animal Ethics Committee (regd. no. 438/01/a/CPCSEA/ dt.17.07.2001, in its resolution number 9/IAEC/SVU/2001/dt. 4.03.2002). Chemicals Streptozotocin (STZ) was obtained from Sigma Chemicals (USA). All the other chemicals used were of analytical grade. Induction of Diabetes The animals were fasted overnight and diabetes was induced by a single intraperitoneal injection of a freshly prepared solution of STZ (50 mg/kg body weight) in a 0.1 M cold citrate buffer (pH 4.5). The animals were considered as diabetic, if their blood glucose values were above 250 mg/dL on the third day after STZ injection. Grouping of Animals The rats were divided into five groups of six rats in each group and treated as follows:
Biochemical Assays The superoxide dismutase (SOD) activity was assayed in the liver and kidney homogenates by the method of Misra and Fridovich [14] at 480 nm for 4 min on a Hitachi U-2000 spectrophotometer. The activity was expressed as the amount of enzyme that inhibits the oxidation of epinephrine by 50%, which is equal to 1 U per milligram of protein. The catalase (CAT) activity was determined at room temperature by using the method of Aebi, [15] and the absorbance of the sample was measured at 240 nm for 1 min by a UV spectrophotometer. The extent of lipid peroxidation was estimated as the concentration of thiobarbituric acid-reactive malondialdehyde (MDA) by using the method of Ohkawa et al. [16] The blood glucose levels were measured by using an Accucheck glucometer (Roche, Germany). Statistical Analysis Analysis of variance (ANOVA) and Duncan′s multiple comparison tests among data were carried out using SPSS (version 13.5; SPSS Inc., Chicago, IL, USA) and MS Office, Excel, software for the significance of the main effects (factors), and treatments along with their interactions. Statistical significance was set at P < 0.05. Results Effect of Alcohol on Blood Glucose and Body Weight Changes In alcohol-treated rats, there was increase in blood glucose levels. In diabetic rats with alcohol treatment, blood glucose levels were significantly (P < 0.001) higher than those in normal control, alcohol-treated and diabetic rats [Table - 1]. In the alcohol-treated group, the body weight was increased. However, in the diabetic group, the body weight decreased significantly (P<0.001) whereas in alcohol-treated diabetic rats, the body weights were decreased [Table - 2]. Effect of Alcohol on Antioxidant Enzymes in Liver and Kidney Tissues of Diabetic Rats A significant (P < 0.001) reduction in SOD and CAT activities are observed in alcohol-treated and diabetic control rats as compared to normal control rats. MDA levels were increased significantly (P < 0.001) in alcohol-treated and diabetic rats Furthermore in alcohol treated diabetic rats, we observed significantly (P < 0.001) low activities of SOD and CAT and a very high level of MDA [Figure - 1]. Impact of Alcohol on Histopathological Changes in Liver and Kidney Tissues of Diabetic Rats In STZ-induced diabetic control rats, severe tubular degeneration, degeneration of glomeruli, focal necrosis of tubules, cystic dilatation of tubules, and fatty infiltration in the kidney tissue were observed. The above pathological changes were enhanced in alcohol-treated diabetic rats and dilatation of Bowman′s capsule and hyaline casts were also observed [Figure - 2]. In the alcohol-treated group and diabetic group, hepatocytes, central vein, and sinusoids of liver were damaged, whereas in the diabetic group treated with alcohol, the hepatocytes, sinusoids, and central vein were more severely damaged [Figure - 3]. Discussion The present study was designed to investigate the effect of alcohol on blood glucose levels and antioxidant enzymes in STZ-induced diabetic rats. In the present investigation, diabetic rats showed the elevation in blood glucose levels, confirming abnormalities of glucose metabolism [17] and also due to the destruction of pancreatic beta cells by STZ reinforcing the view that STZ induces diabetes probably through the generation of free radicals [18] whereas in alcohol treatment (At) alone and combination treatment (D + At) groups, the blood glucose levels were increased. A number of researchers have reported that alcohol administration to diabetic rats elevates the blood glucose levels. Alcohol treatment has been implicated to induce beta cell dysfunction, increase insulin secretary responses, and enhance glucose disposal rates in subjects with diabetes. [19] In this study, alcohol treatment in the rats increased the body weight. This may be due to the deposition of lipids in adipose tissue and fluid accumulation in the organs. This finding is in agreement with previous findings of Rajakrishnan et al. [20] In the diabetic group, the body weight was decreased. Weight loss during diabetes is mainly related to urinary glucose excretion because cells use glucose for many functions. Another factor could be the osmotic diuresis resulting in hyperosmotic dehydration. [21] With alcohol treatment in diabetic rats, we observed significantly (P < 0.001) lower body weights compared to diabetic rats. This was due to the dehydration, excess utilization of proteins for the energy. So, in alcohol-treated diabetic rats, decreased body weight was observed. SOD scavenges the superoxide radical by converting it to H 2 O 2 and oxygen. In the present study, the SOD activity was decreased in the diabetic group. Pari and Latha [22] also reported that the SOD activity was decreased in diabetic rats. The decrease in the SOD activity in diabetic rats could result from the inefficient scavenging of ROS, which might be implicated in the oxidative inactivation of enzymes and especially the deleterious effects due to the accumulation of superoxide radicals, or by glycosylation of the enzymes, which have been reported to occur in diabetes. [23] In the current investigation, we observed a decreased activity of SOD in the alcohol-treated group. Shanmugam et al. [24] and Mallikarjuna et al. [13] reported that alcohol administration depleted the SOD activity in the kidney and liver tissues of albino rats. Several studies have reported that alcohol consumption decreases the SOD activity in the liver, heart, brain, kidney muscle, and serum. [25] The reduced activity of SOD in the presence of alcohol may cause the accumulation of O2·¯, H 2 O 2 , or the products of its decomposition. However, in the combination treatment group, i.e., alcohol-treated diabetic rats (D + At), the SOD activity was significantly (P < 0.001) decreased compared to that of the alcohol-treated group and diabetic control group. This may be due to the excessive production of free radicals and superoxide radicals, so the SOD activity was decreased to counter the same. In the present study, the CAT activity was decreased in diabetic rats. The reduced activity of CAT in kidney and liver tissues may result in a number of deleterious effects due to the accumulation of H 2 O 2 . [26] The CAT activity was also decreased in alcohol-treated (At) rats in the present study. The reduced activity of CAT in the presence of alcohol causes the accumulation of free radicals which are toxic in nature. Balasubramaniyan et al. [27] and Mallikarjuna et al. [13] reported that the CAT activity in kidney and liver was decreased in the alcohol-treated group. CAT helps to scavenge hydroxyl ions, so the CAT activity was lowered in alcohol-treated rats [28] whereas in the combination treatment (D + At) group, the CAT activity was significantly decreased (P < 0.001) compared to the alcohol-treated or diabetic group. This may be due to the excess production and accumulation of hydroxyl radicals. CAT helps in neutralizing these toxic hydroxyl radicals, so the CAT activity was decreased. The additive effects of alcohol are observed in diabetic animals causing a greater reduction in these two enzymes. In the current study, MDA levels were increased in diabetic rats. Previous studies have reported an increase in lipid peroxidation in the liver, kidney, and brain of diabetic rats. [29] Lipid peroxide-mediated tissue damage has been observed in type I and type II diabetes. Higher levels of lipid peroxides in plasma urine and renal proximal tubules were observed in diabetic rats. Kakkar et al. [30] also reported the same. Ostrowska et al. [31] reported a threefold higher concentration of lipid hydroperoxides in alcohol-treated rats compared to the control groups. During alcohol metabolism, potentially dangerous byproducts are generated including ROS which react with membrane lipids and cause lipid peroxidation leading to cell death. [32] In alcohol treated diabetic rats, we observed significantly (P < 0.001) higher levels of MDA compared to alcohol and diabetic groups. During combination treatment, more free radicals are produced, hence the MDA level was increased in combination treatment. The histopathological studies revealed that in the kidney tissue of alcohol-treated rats, tubular degeneration, necrosis of renal cells, and degeneration of Bowman′s capsule were observed. In diabetic control rats, severe tubular degeneration, degeneration of glomeruli, focal necrosis of tubules, cystic dilatation of tubules, and fatty infiltration were observed. This might be associated with increased diuresis and renal hypertrophy in diabetic rats. The above-mentioned pathological changes were more severe in alcohol-treated diabetic rats. The dilatation of Bowmen′s capsule and hyaline casts were also observed. The histopathological studies of the alcoholic liver showed the disruption of hepatocytes, sinusoids, and central vein. This proves that free radical production might cause hepatic damage. In the diabetic group, greater damage of hepatocytes, sinusoids, and central vein was observed Whereas in the alcohol-treated diabetic group, hepatocytes, sinusoids, and central vein were more severely degenerated. The histological evidence of alcohol-treated diabetic rats suggests that structural alterations at the end of 30 days are due to diabetic stress and alcoholic stress. Thus, in addition to elevated levels of blood glucose, histopathological observations also support the concept that alcohol produced additive effects, and hence renal, hepatic tissue damage in diabetic rats. Conclusion From this study, it was concluded that alcohol consumption appears as an aggravating factor in diabetes. To our knowledge, this is the first report on the effect of alcohol on blood glucose levels and antioxidant enzymes in diabetic rats. However, further studies are necessary to confirm these effects. References
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