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Abstract

Background:

In the present study, thirty six male Sprague Dawley rats were randomly divided into six groups and were injected with varying doses of alloxan (Ax) and nicotinamide (NA). The serum levels of glucose, insulin, and adiponectin were measured weekly up to 4 weeks.

Results:

Elevated levels of glucose were observed in all groups on days 7, 14, 21, and 28, except in groups a and f (control). The serum insulin levels were significantly elevated in groups b and c on day 7, when compared with that in group f, whereas a decrease in the serum insulin levels was observed in groups d and e on days 21 and 28. The adiponectin levels showed inconsistencies on days 7 and 14. However, significant decrease in the adiponectin levels was observed on days 21 and 28. Histological section of the pancreas showed mild (group a), moderate (group b) to severe (groups c, d, and e) degenerative changes. Concomitant fatty changes in the liver and inflammatory infiltration of the kidney were markedly observed in all the treated groups, when compared to control.

Conclusion:

These results suggested that the use of selective combination of Ax120 + NA50 injection demonstrated type II diabetes mellitus in rats.

Keywords

Diabetes adiponectin inflammatory alloxan nicotinamide

Introduction

International Diabetes Federation reported that million people suffering from diabetes worldwide, with the majority suffering from type II diabetes mellitus (DM). This condition, if left unmanaged, has the potential to lead to various complications, including cardiovascular diseases, blindness, kidney failure, liver damage, and lower limb amputations as a result of poor healing ability in diabetic patients.¹ Although various models of inducing type II DM have been reported and discussed in the literature, streptozotocin (STZ) and alloxan (Ax) are noted to be the most commonly used chemical induction models.^2

–7

Among these two, STZ is more widely promoted for use in combination with nicotinamide (NA) to induce type II DM in experimental models.^8

–11 Administration of NA has been proven to be advantageous in preventing β-cell apoptosis. In several studies, administration of high fat diet (HFD) at multiple time point with the use of STZ has also been used to induce type II DM. It has been demonstrated that this model takes at least 10–12 weeks to produce a consistent diabetic model, which would be useful for analyses.^12
–14 Moreover, the adjustment of dosage is very important, which would otherwise induce mortality in HFD-treated rats. Although much faster than STZ, HFD and combination of STZ model still demands a minimum of 5–6 weeks to create a consistent and stable diabetic model.

The major limitation of using Ax is that it produces a certain level of mortality in the animal models. In fact, it has been shown that Ax produced relatively higher mortality, when compared with STZ.¹⁵ As a thiol reagent, Ax selectively inhibits glucose-induced insulin secretion through its ability to specifically inhibit the glucokinase through oxidation of functionally essential thiol groups in glucokinase, thereby impairing oxidative metabolism and glucose sensor function of this signaling enzyme of the β-cell.¹⁶ Alternatively, NA has been shown to protect the β-cell from this effect, and in several studies, its use as a posttreatment drug in DM-induced model has been reported to terminate the progressive damage effects of several chemicals.^17,18 Therefore, it also paramount that a model must be able to mimic varying degrees of insulin resistance similar to that observed in the DM patient population. Studies have reported that adiponectin is a major cytokine secreted by adipocyte. It plays major role in glucose metabolism. Adiponectin improves insulin sensitivity and inflammation a major mechanism in the development of type 2 diabetes, hence quantification of adiponectin in this study would be useful when validating type 2 diabetes model.¹⁹ The present study was thus conducted to determine the appropriate dose combination of coadministered Ax and NA, which is required to produce a consistent and stable type II DM model in rats with the lowest mortality possible.

Material and methods

Animals

Thirty-six (N = 36) male Sprague Dawley (SD) rats aged between 8 and 10 weeks and weighing between 250 and 300 g were maintained on rodent chow at the Animal Experimental Unit (an AAALAC accredited center) of the University of Malaya. All the animals were exposed to 12-h dark/12-h light cycle and were randomly assigned to six experimental groups according to dosage combination to induce type II DM. The study was approved by the University of Malaya Ethics Review Committee for Animal Research (IACUC).

Ax-NA administration

Blood samples were collected from the tail veins of the rats by using 1-mL syringe and 26½ gauge needle to measure the baseline blood glucose (fasting), insulin, and adiponectin levels before the induction of type II DM. The animals were then allowed to fast for 12 h before collecting the blood samples to measure fasting blood glucose level at variable time points such as days 0, 7, 14, 21, and 28. The glucose levels were determined by using FreeStyle Optium Blood Glucose Monitoring System. Type II DM was induced by single intraperitoneal injection of monohydrated Ax (Sigma, St. Louis, Missouri, USA), followed by single intraperitoneal injection of NA, that is, a mode of coadministration (Sigma). The dosage combinations of monohydrated Ax and NA were dissolved in sterile 0.9% saline. The group details are shown in Table 1.

Table 1.

Dosage combination of monohydrated Ax and NA for the induction of type II DM.

Group	Dosage (mg/kg body weight)
Group	Monohydrated Ax	NA
a	100	50
b	100	150
c	120	50
d	120	100
e	120	150
f (Control)		–

NA: nicotinamide; Ax: alloxan; DM: diabetes mellitus.

Serum adiponectin and insulin levels

Whole blood was collected from rats’ tail vein into empty 2.0-mL serum tubes at variable time points such as days 0, 7, 14, 21, and 28. The serum was separated from whole blood by centrifugation at 3000g for 5 min and stored at −80°C for further analysis. The serum adiponectin (#KA1026, Abnova, Taipei, Taiwan) and insulin (#10-1250-01, Mecordia, Uppsala, Sweden) levels were measured by using commercially available sandwich enzyme-linked immunosorbent assay kit, according to the manufacturer’s instructions.

Histological analysis

Pancreas, liver, and kidney tissue samples were harvested upon killing the animals and fixed in 10% formaldehyde. The tissues were processed and embedded in paraffin before sectioning to a size of 4.5 µm using microtome. The samples were then stained by employing hematoxylin and eosin staining.

Results and discussion

To date, many diabetic preclinical models have revealed that type II DM can be induced either (i) spontaneously, (ii) using chemicals, (iii) through diet, (iv) by surgical manipulations, and/or (v) through a combination of any of these methods. Several studies have also suggested that type II DM can be achieved by administering HFD followed by an injection of STZ to produce insulin resistance and glucose intolerance.^13,10 In addition, significant adiponectin reduction has also been noted to play an important role in the development of insulin resistance and metabolic syndrome.^20,21 The abovementioned induction models have also been reported to exhibit pathological conditions that are similar to those of type II diabetes in humans; however, it has been shown that such attempts have failed to reproduce the complexity of human diabetes.²² Nevertheless, these models may still prove useful to provide further insights into the various aspects of diabetes progression and, therefore, hold certain merits.²³ Currently, reports on using STZ and NA to induce non-insulin–dependent diabetes mellitus (NIDDM) are becoming more common. It is more likely that this choice reflects the preference of researchers because there is a lack of reports suggesting that one method is superior over the other.^24

–27 In these studies, rats are usually administered with NA 15 min before STZ injection because STZ causes pancreatic β-cell destruction, while the administration of NA prior to STZ can partially protect β-cell against STZ, resulting in partial loss of glucose metabolism and only moderate insulin deficiency.²⁸ Several reports have demonstrated that this DM model is useful to explore the efficacy of various diabetogenic drugs. However, there have been concerns raised about the stability of the diabetic condition, which is characterized by the moderate decline in glucose tolerance with loss of early phase of insulin secretion and reduced pancreatic insulin storage.²⁸

To the best of the authors’ knowledge, no optimization studies have been carried out using different concentrations of Ax and NA at different time points to generate conditions that more appropriately mimic those of type II diabetes in patients. In the present study, we observed a significant increase in the levels of blood glucose (Figure 1) in all the treatment groups on days 7 14, 21, and 28 following the coadministration of Ax-NA, when compared with the baseline glucose level (4 mmol/L). Group c demonstrated the highest average increase (p < 0.05) of glucose level, which was approximately fivefold (20 mmol/L) on days 7, 14, and 21, when compared with the baseline glucose level. In groups b, d, and e, there was approximately threefold increase in the glucose level (15 mmol/L) on days 7, 14, and 21, when compared with the baseline level. On day 28, although all the groups showed some decrease in the glucose levels, a twofold (10 mmol/L) increase was maintained, when compared with the baseline level. These results are summarized in Figure 1.

Figure 1.

Blood samples were collected from the tail veins of the rats at variable time points (days 0, 7, 14, 21, and 28) by using 1-mL syringe and 26½ gauge needle to measure the blood glucose (mmol/L) in control and experimental group induced with combinations of Ax + NA. *p < 0.05: when compared with control group. NA: nicotinamide; Ax: alloxan.

It has been reported that the induction of hyperglycemic condition strongly depends on the pre- or posttreatment of NA against Ax injection. A previous study demonstrated that the increase in the glucose levels was higher following the treatment with Ax, while administration of Ax followed by NA protected β-cells, thereby considerably reducing the diabetic condition. However, the major limitations of that study were the use of a single concentration in the mice model and lack of histological evidences.²⁹

In the present study, the changes in the insulin level at different time points are shown in Figure 2. When compared with the control group f, in groups b and c, an increase in the insulin level of approximately 1.6 fold was observed (p < 0.05) on day 7, whereas in groups d (Ax 120 mg/kg + NA 100 mg/kg) and e (Ax 120 mg/kg + NA 150 mg/kg), a decreasing trend of approximately 0.6 fold was observed on day 28 (p < 0.05). This may be owing to the fact that the administration of NA may have inhibited the poly ADP ribose synthetase that protects against decrease in islets proinsulin biosynthesis induced by chemical agents such as Ax, as suggested in many literatures. This generally occurs when NA is administered before induction of diabetes in animal models. However, these changes were not observed when the NA was administered after Ax was injected, suggesting that the damaging effects can be rather quick and that NA is protective but will not reverse the damaged cells.²⁹

Figure 2.

Serum separated from blood samples collected from the tail veins of the rats at variable time points (days 0, 7, 14, 21, and 28). The serum insulin levels were measured using commercially available sandwich ELISA kit (μg/ml) in control and experimental group induced with combinations of Ax + NA. *p < 0.05: when compared with control group. NA: nicotinamide; Ax: alloxan.

In the present study, the levels of adiponectin were monitored at different time points to evaluate the possibility of whether type II DM occurred in NA-Ax–induced rats at different doses (Figure 3). The groups a and b showed considerable increase (p < 0.05) in the levels of adiponectin on day 7, when compared with the control. However, the groups b, c, d, and e showed approximately twofold decrease in the levels of adiponectin on day 28, when compared with the control group f. The circulating adiponectin levels are considered as a biological marker, and decreasing levels of circulating adiponectin may act as a mediator for the pathological changes in type II DM. Adiponectin is produced predominantly by adipocytes and plays an important role in metabolic homeostasis. It has been shown that adiponectin has insulin-sensitizing, anti-inflammatory, and antioxidant effects.³⁰ These properties may help explain the inverse associations between circulating adiponectin levels and diseases, which, in several studies, have including the reversal of cardiovascular disease and type II DM.³¹

Figure 3.

Serum separated from blood samples collected from the tail veins of the rats at variable time points (days 0, 7, 14, 21, and 28). The serum insulin was measured using commercially available sandwich ELISA kit (μg/ml) in control and experimental group induced with combinations of Ax + NA. *p < 0.05 when compared with control group. NA: nicotinamide; Ax: alloxan.

It is interesting to note that a combination of Ax and NA has been shown to produce DM very rapidly and that in these models, the serum results mimic those of type II DM profile, that is, an increase in serum glucose, increase or near-normal insulin level, and a decrease in adiponectin level. In addition, the number of mortality in this DM-induced model is reduced, the reason for which remains elusive. It has therefore been suggested, based on the deduction made from the results of several published works, that a combination of Ax and NA may be useful in creating type II DM in animal models while reducing the rate of mortality. However, despite the potential benefit of using this model, a coadministration of Ax and NA at different concentrations in rats has not been reported. This is important because it would allow researchers to determine the minimal dose needed to create a type II DM model effectively.

It has been reported that low dose of STZ (35 mg/kg) could mildly induce diabetic syndrome with a moderate pancreatic injury. A further decrease in the STZ dose (30 mg/mg) has been demonstrated to exhibit no significant decrease in the insulin content in 40% of the rats examined. However, 30% of the animals showed moderate decrease in insulin. Altogether, the ability to secrete insulin varied significantly among the rats.²⁸ Thus, while the dose-dependent effect of STZ-NA diabetic model has been established, models using Ax-NA is relatively scarce but theoretically possible. The present study proved that Ax-NA could be used to establish diabetic model. It was clearly demonstrated that a combination dose of 120 mg/kg of AZ and 50 mg/kg of NA provided the best outcome and mimics the DM model well. More importantly, in the present study, the morality of rats was relatively low in all the groups. Only one animal death was noted in groups d and e, respectively, demonstrating that the dosages used were well tolerated by the animals.

The photomicrograph (Figure 4(f)) of the pancreas in normal rats demonstrated the features of normal acini and islets of Langerhans, while in the group of rats that received different dosages of NA and Ax, it was apparent that moderate to severe changes had occurred in the islets cells. Almost all the groups treated with different combinations of different dosages of NA and Ax showed mild (group a; Figure 4(a)), moderate (group b; Figure 4(b)) to severe (groups c, d, and e) pyknotic nuclei and acidophilic cytoplasmic appearance with vacuolar changes and cellular swelling, while the changes in the islets cells were not severe (Figure 4(c), to (e)). These changes were concordant with the changes in the decreased levels of adiponectin and increased glucose levels without alterations in the levels of insulin. The degenerative changes but not complete destruction in the islets with β-cell is an important reason for the onset of insulin resistance. The degenerative changes observed in the islet cells, as evidenced by the loss of β-cells and a thick layer of peripheral non-β-cells, are strongly suggestive of a correlation to the incidence of type II DM.³² Although the nature of degenerative changes resulting from the use of different combinations or concentrations of NA and Ax was significant on day 28, the induction of diabetes by using this combination also depended on the rat strain used, because some strains may be resistant to such changes.²⁸

Figure 4.

Pancreas tissue samples were harvested upon killing the animals and fixed in 10% formaldehyde. The tissues were processed and embedded in paraffin before sectioning to a size of 4.5 µm using microtome. The samples were then stained by employing hematoxylin and eosin staining. (a) Ax100 + NA50, (b) Ax100 + NA150, (c) Ax120 + NA50, (d) Ax100 + NA100, (e) Ax100 + NA150), and (f) control. White arrows indicate pyknotic nuclei and acidophilic cytoplasmic appearance with vacuolar changes and cellular swelling, while the changes in the islets cells were not severe. NA: nicotinamide; Ax: alloxan.

Although the present study suggested that DM was induced owing to the effect on β-cell, it is not clear whether this is the only mechanism responsible for the induction of DM. We were able to show that the livers of rats treated with NA and Ax exhibited degenerative changes (Figure 5(a) to (e)) and that the hepatocytes presented fat deposits, pyknotic nucleus, and acidophilic cytoplasm. In addition, disarrangement of hepatic chords, vacuolization, and necrotic cells was also observed in all groups, with mild and moderate changes noted in groups a and b, respectively. It is interesting to note that significant changes were less obvious in groups c, d, and e, when compared with the control group f (Figure 5(f)), suggesting that certain combinations may be less detrimental to the liver. It is a well-known fact that fatty changes are linked to the impairment of mitochondrial β-oxidation of fatty acids. This leads to the esterification of fatty acids in the cytoplasmic region, characterized by the presence of lipid droplets within the hepatocytes.³³ In general, major metabolic diseases such as NIDDM and obesity are inflammatory conditions and that the responses to these conditions are generally mediated through Kupffer cells attached to the endothelial lining located at the periportal sinusoids.³³ It has been reported that these Kupffer cells are activated during HFD that will induce insulin insensitivity, leading to disorders in lipid metabolism and hepatic insulin resistance.³⁴

Figure 5.

Liver tissue samples were harvested upon killing the animals and fixed in 10% formaldehyde. The tissues were processed and embedded in paraffin before sectioning to a size of 4.5 µm using microtome. The samples were then stained by employing hematoxylin and eosin staining. (a) Ax100 + NA50, (b) Ax100 + NA150, (c) Ax120 + NA50, (d) Ax100 + NA100, (e) Ax100 + NA150, and (f) control. Black arrow shows the fatty infiltration, while the white arrows indicate the vacuolization of the hepatocyte in the groups a to e treated with different combination of Ax and NA. NA: nicotinamide; Ax: alloxan.

Another organ that is commonly affected as the result of DM is kidney. It has been reported that degenerative changes in the glomerular basement membrane, hypertrophy, glomerular hyperfiltration, and accumulation of extracellular matrix in the tubules are common findings in DM.³⁵ In the present study, administration of NA- and Ax-induced NIDDM in rats, leading to acute changes in the renal tissue characterized by cellular swelling, glomerular infiltration, and congestion of capillaries and necrosis (Figure 6(a) to (e)). The inflammatory changes in the kidney were mild and moderate in groups a and b (Figure 6(a) and (b)), whereas the degenerative changes were dominant in groups c, e, and d (Figure 6(c) to (e)). Previous studies have shown that uncontrolled hyperglycemia and hyperlipidemia are factors responsible for diabetic nephropathy progression, which triggers vascular oxidative stress³⁶. Although this study has shown the type II DM model using chemical induction, further studies are ongoing to track the mechanism of action involved in this model.

Figure 6.

Kidney tissue samples were harvested upon killing the animals and fixed in 10% formaldehyde. The tissues were processed and embedded in paraffin before sectioning to a size of 4.5 µm using microtome. The samples were then stained by employing hematoxylin and eosin staining. (a) Ax100 + NA50, (b) Ax100 + NA150, (c) Ax120 + NA50, (d) Ax100 + NA100, (e) Ax100 + NA150, and (f) control. The black arrows indicate cellular swelling, glomerular infiltration, and congestion of capillaries and necrosis. NA: nicotinamide; Ax: alloxan.

In conclusion, this study demonstrated that the use of Ax-NA combination is effective for the development of type II DM model in SD rats and that a combination of 120 mg/kg of Ax and 50 mg/kg of NA is most preferable, resulting in hyperglycemia with near-normal insulin and reduced adiponectin levels. Furthermore, the use of this combination also produced degenerative changes in the main organs, as expected in DM conditions, but did not result in early animal death.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research is supported by HIR-MoE Grant (reference number—UM.C/625/1/HIR/ MOHE/CHAN/03, account number—A000003-50001).

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Coadministration of alloxan and nicotinamide in rats produces biochemical changes in blood and pathological alterations comparable to the changes in type II diabetes mellitus