MATERIALS AND METHODS

Chemical Agents
All chemicals unless otherwise stated were purchased from Sigma Chemical Company (St Louis, MO) USA. Retinyl palmitate 1,600,000 usp units per gram.

Animals
The experiments were performed on male Sprague Dawley Albino rats (approx 180 g) obtained from Physiology Department Faculty of Medicine, Bayero University Kano, Nigeria. All aspects of animal care complied with the ethical guidelines and technical requirements approved by the Institutional Animal Ethics Committee.

Animals were housed individually in cages in an environmentally controlled animal facility (room temperature, 12 h light: 12 h dark cycle) with free access to a standard commercial diet and water ad libitum. The weight gain, food and water intake were determined daily in the morning. The experiment was conducted for a period of 5 weeks.

Induction and Treatment of Diabetes
All animals were fed on normal diet for 7 days of acclimatization. Diabetes was included by an intraperitoneal (IP) injection of 100 mg alloxan kg^-1 b.wt.

Diabetes was induced by a single injection of alloxan 100 mg alloxan kg^-1 b.wt. by IP to rats fasting for at least 16 h in freshly prepared 10 mmol L^-1 sodium citrate, pH 4.5. Blood glucose levels were measured daily 3 days prior and 7 days after alloxan administration. Development of diabetes mellitus was proven by sustained hyperglycaemia and glycosuria [diabetic rats had glycaemia >11.11 mmol and glucosuria. This was based on the observation that only diabetic rats that had a fasting glucose greater than 200 mg dL^-1 would be included in the study (Stanley and Venogopal, 2001).

Experimental Design
The rats were randomly divided into 4 groups (n = 5) as follows:

Glucose Estimation
Animals were starved for 16 h before blood collection. Fasting blood glucose was estimated by glucose oxidase method according manufacturer’s procedure (Randox laboratories Ltd. Ardmore, United Kingdom). Urine was collected in cage urine separator bottle containing 1 mL of 10% thymol and glucose determined using Combostik (DFI Co. Ltd., Gimhae, Gyuang-Nam, Korea).

Histochemical Analysis
Histochemical analysis was done according to Bancroft and Stevens (1982).

Sacrificing Procedure
The Rats were made unconscious with carbon (IV) oxide before sacrifice by surgical dislocation of neck.

Statistical Analysis
Mean±SD was used to describe variables. Two-Sample Independent student t-test as well ANOVA using OpenEpi Version 2 program was used in the comparison of group means to determine the significance of the result.

RESULTS

Table 1 shows the fasting blood glucose one week before and one week after induction of diabetic mellitus. Blood glucose level of vitamin A group was compared with diabetic control. The effect of vitamin A on glucose level in the serum showed significant difference compared to diabetic control (p<0.0001). Histochemical analysis suggested that vitamin A protected the integrity of pancreas tissue against allaxon induced damage. Glucosuria increased significantly in diabetic animals compared with vitamin A treated group where there was no glucosuria. Though treatment with vitamin A one week after alloxan induced diabetes did not have any significant effect compared with result before treatment with Vitamin A (16.81±2.88 vs. 19.60±2.95 mmol L^-1, p<0.96, respectively), it however reduced the level of glucose by 3 mmol L^-1, suggesting that the normal level of serum glucose seem in vitamin A treated group before induction was achieved by the protection of the pancreatic tissue against alloxan induced damage rather than reversing the effect by vitamin A (Table 1).

Table 1:	Fasting blood sugar (mmol L-1) of rats
Result Mean±SD for n = 5, ap<0.0001 for comparing diabetic control and non diabetic control after induction of diabetes bp<0.001 for comparing diabetic control (Group II) and vitamin A (III) after alloxan treatment, cp<0.0001 for comparing non-diabetic control and group (IV) before vitamin A supplementation

Fig. 1:	Histochemical structure of pancreatic cells of albino rats stained with haemtoxylin, (a) Allaxon treated showing cell necrosis in which beta cell are severely a ffected, (b) vitamin A treated in which the cells integrity is not affected and (c) control non diabetic fed on normal diet ad libitum

Table 2:	Weekly mean body weight of rats (g)
Result Mean±SD for n = 5

Histochemical study of pancreatic tissue is shown in Fig. 1a-c. Vitamin A group were compared to both controls (diabetic and normal control).

The diabetic control group II showed necrotic beta cells, suggesting that the integrity of pancreatic tissue was compromised following diabetes induction (Fig. 1a). Vitamin A group revealed normal and remarkable pancreatic cells, indicating that the integrity of pancreatic tissues was protected by vitamin A against alloxan induced damage (Fig. 1b).

The normal control group showed normal pancreatic tissue architecture (Fig. 1c). Table 2 shows the weekly body weights one week before and 4 weeks after induction of diabetes mellitus. The weekly mean body weights are in grams. Group 1 and Group 3 appeared to have weekly increase in weight but Group 2 has reduction in weight at week four this indicates that the antioxidant do not retard growth of the animals. In the case of diabetes incidence there could be lost of weight due to mobilization of protein store.

DISCUSSION

Several lines of evidence have shown that diabetes mellitus and complication thereof from the disorder can arise due to oxidative stress. Similarly increased lipid peroxides have been seen in many animal models of diabetes mellitus (Anjaneyulu and Chopra, 2004).

Since the understanding that the selective destruction by alloxan of pancreatic beta cells is mediated via generation of oxidative stress (Dunn and McLetchie, 1943), interest has been stimulated in the use of antioxidant to prevent chemically induced damage of pancreatic cells. Toxicity of alloxan is elicited through its reduction by glutathione to dialuric acid, in which redox recycling process generates ROS that damages the beta cells (Malaisse, 1982). Furthermore, transition metals such as iron and copper, which are potentially involved in the generation of hydroxyl free radical are also involved in alloxan mediated killing of beta cells (Malaisse, 1982; Wolff, 1993; Wilson et al., 1984). It has been known that vitamin E prevented alloxan induced diabetes in rats (Fatemi et al., 2008). This study suggested that vitamin A consumption has beneficial effect and dose used in this study was effectively potent to inhibit the toxicity of alloxan on β-cells of pancreas. In the recent past, it has been demonstrated that intravenous administration of retinyl paltimate prior to that of alloxan administration, protected against alloxan induced diabetic in rats (Chertow et al., 1989). However, there is paucity of information as to whether vitamin A supplementation in the diet can protect against alloxan induced damage of beta cells. Though Siefter et al. (1981) have demonstrated the efficacy of using twice the recommended daily allowance of vitamin A in rodent with streptozotocin induced diabetes to improve wound healing process caused by steptozotocin, the work did not look at the potential of vitamin A dietary supplementation in the prevention of chemically induced diabetes. Hence, this work provided insight as to the potential of vitamin A dietary supplementation at human RDA in the prevention of chemically induced diabetes mellitus in rodent. Similarly, the study corroborated with the Siefter et al. (1981) finding, that once beta cells are destroyed, vitamin A does not alter, carbohydrate metabolism, but has provided a reserviour of information about protection of chemical induced diabetes mellitus using food supplemented with vitamin A. This was in variance with Chertow et al. (1989) study, where administration of Vitamin A at twice human RDA was used some hours prior to alloxan administration. Treatment with dietary vitamin in this study did not alter hyperglacaemia and glucosuria in animals with alloxan induced diabetes. It is thought possible that since vitamin E has been demonstrated to protect against chemically induced destruction of beta cell, that vitamin A which shares the same mechanism of free radical scavenging may have similar effect. In this study, we were able to show that vitamin A supplementation at daily recommended dose given to human can protect against alloxan induced diabetes in rats. The addition of vitamin A to the diet is computed based on the amount of diet taken by the rats over a period of 7 days. The daily recommended allowance of vitamin A for adult is 700 μg (considering the average body weight is 77 kg; (Olson, 1987). Hence base on that, 824 IU per 100 g diet was given to the experimental animals. It is also interesting to note that the histological architecture of the pancreatic cells was highly protected by vitamin A. This study suggested that vitamin A supplementation may have an effect on chemical induced diabetes mellitus. Furthermore in view of its antioxidant property it may as well delay complication arising from the disease. Future work should look at the potential of combination of vitamin A and E at lower dose in preventing oxidative stress induced diabetes. This would be more therapeutically valuable in view of the low income of the mass of Nigerians. Diabetes mellitus is becoming a major global health (Anonymous, 1993) and societal problem, costing the United States approximately $132 billion annually (Hogan et al., 2003). In Sub-Saharan Africa, there are no enough financial resources to adequately carter for this disease, while urban migration and increasing sedentary lifestyle in sub-Saharan Africa are increasing the incidence of the condition here Nigeria. While the war on substandard and fake drugs, including antidiabetics is yet to be won, the collapse of the power sector in Nigeria has made sustained potency of hypoglycemics including insulin near impossible in most cases. These negative factors collectively have escalated the development of diabetic complications which are known to be difficult and costly to manage even in the developed world. Hence, since, complication arising from diabetes mellitus is strongly associated with generation of reactive oxygen species, it is thought possible that use of sustain antioxidants at concentration that may not be toxic, may provide a better way in augmenting the managing of the condition as well as protecting against oxidative stress induced diabetes mellitus. The pathogenesis of type I diabetes mellitus is invariably associated with oxidative stress, hence alloxan induced diabetic condition is elicited through generation of free radical, it is likely dietary supplementation with Vitamin at RDA dosage may likely attenuate the effect of free radicals in the induction of type I diabetes mellitus.

ACKNOWLEDGMENTS

Authors wish to acknowledge the assistance of Dr. M.S. Mijinyawa, a veterinarian with Kano State Ministry of Agriculture, Nigeria for teaching how to inject alloxan to the rats. Their gratitude to Mallam Yakubu for taking care of the animals in the animal house of the Department of Biological Science, Bayero University Kano, Nigeria and Mr Sunday Okoro of Physiology Department Faculty of Medicine Bayero University, Kano for helping to collect blood and supervision of the animals.