coadministration of alloxan and nicotinamide in rats produces biochemical changes in blood and...

Article

Coadministration of alloxanand nicotinamide in rats producesbiochemical changes in bloodand pathological alterationscomparable to the changes intype II diabetes mellitus

KK Vattam1, HRB Raghavendran1, MR Murali1,H Savatey1 and T Kamarul1,2

AbstractBackground: In the present study, thirty six male Sprague Dawley rats were randomly divided into six groupsand were injected with varying doses of alloxan (Ax) and nicotinamide (NA). The serum levels of glucose, insu-lin, 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 andf (control). The serum insulin levels were significantly elevated in groups b and c on day 7, when compared withthat in group f, whereas a decrease in the serum insulin levels was observed in groups d and e on days 21 and28. The adiponectin levels showed inconsistencies on days 7 and 14. However, significant decrease in the adi-ponectin 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 liverand inflammatory infiltration of the kidney were markedly observed in all the treated groups, when comparedto control.Conclusion: These results suggested that the use of selective combination of Ax120 þ NA50 injectiondemonstrated type II diabetes mellitus in rats.

KeywordsDiabetes, adiponectin, inflammatory, alloxan, nicotinamide

Introduction

International Diabetes Federation reported that mil-

lion 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 Admin-

istration of NA has been proven to be advantageous in

1Tissue Engineering Group, Department of Orthopaedic Surgery,NOCERAL, University of Malaya, Kuala Lumpur, Malaysia2Clinical Investigative Centre (CIC), University Malaya MedicalCentre, Kuala Lumpur, Malaysia

Corresponding authors:T Kamarul and HRB Raghavendran, Tissue Engineering Group,Department of Orthopaedic Surgery, NOCERAL, University ofMalaya, 50603 Kuala Lumpur, Malaysia.Emails: [email protected] and [email protected]

Human and Experimental Toxicology1–9

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preventing �-cell apoptosis. In several studies, admin-

istration 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 More-

over, 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 rela-

tively higher mortality, when compared with STZ.15

As a thiol reagent, Ax selectively inhibits glucose-

induced insulin secretion through its ability to specif-

ically 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

�-cell.16 Alternatively, NA has been shown to protect

the �-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 vary-

ing 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 metabo-

lism. 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 con-

ducted 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 ¼ 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), insu-

lin, 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 mono-

hydrated Ax (Sigma, St. Louis, Missouri, USA), fol-

lowed by single intraperitoneal injection of NA, that

is, a mode of coadministration (Sigma). The dosage

combinations of monohydrated Ax and NA were dis-

solved in sterile 0.9% saline. The group details are

shown in Table 1.

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 anal-

ysis. The serum adiponectin (#KA1026, Abnova,

Taipei, Taiwan) and insulin (#10-1250-01, Mecor-

dia, Uppsala, Sweden) levels were measured by

using commercially available sandwich enzyme-

linked immunosorbent assay kit, according to the

manufacturer’s instructions.

Table 1. Dosage combination of monohydrated Ax andNA for the induction of type II DM.

Group

Dosage (mg/kg body weight)

Monohydrated Ax NA

a 100 50b 100 150c 120 50d 120 100e 120 150f (Control) –

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

2 Human and Experimental Toxicology



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 mm 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. Sev-

eral 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 glu-

cose intolerance.13,10 In addition, significant adipo-

nectin reduction has also been noted to play an

important role in the development of insulin resis-

tance and metabolic syndrome.20,21 The abovemen-

tioned 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 repro-

duce the complexity of human diabetes.22 Neverthe-

less, these models may still prove useful to provide

further insights into the various aspects of diabetes

progression and, therefore, hold certain merits.23 Cur-

rently, 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 �-cell

destruction, while the administration of NA prior to

STZ can partially protect �-cell against STZ, resulting

in partial loss of glucose metabolism and only moder-

ate insulin deficiency.28 Several reports have demon-

strated 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 pan-

creatic insulin storage.28

To the best of the authors’ knowledge, no optimiza-

tion studies have been carried out using different con-

centrations 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 three-

fold 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

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

Fasting day 0 day 7 day 14 day 21 day 28

Glu

cose

(mm

ol/L

)Ax100 + NA50

Ax100 + NA150

Ax120 + NA50

Ax120 + NA100

Ax120 + NA150

Control

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

Vattam et al. 3



with the baseline level. These results are summarized

in Figure 1.

It has been reported that the induction of hypergly-

cemic condition strongly depends on the pre- or post-

treatment 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

�-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 þ NA 100 mg/kg) and e

(Ax 120 mg/kg þ 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. How-

ever, 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

In the present study, the levels of adiponectin were

monitored at different time points to evaluate the pos-

sibility 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 adi-

ponectin on day 28, when compared with the control

group f. The circulating adiponectin levels are consid-

ered 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 proper-

ties may help explain the inverse associations between

circulating adiponectin levels and diseases, which, in

several studies, have including the reversal of cardio-

vascular disease and type II DM.31

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

0

0.1

0.2

0.3

0.4

0.5

0.6

day 0 day 7 day 14 day 21 day 28

Insu

lin (µ

g/m

L)Ax100 + NA50Ax100 + NA150Ax120 + NA50Ax120 + NA100Ax120 + NA150Control

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 (mg/ml) in controland experimental group induced with combinations of Ax þ NA. *p < 0.05: when compared with control group. NA:nicotinamide; Ax: alloxan.




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 creat-

ing type II DM in animal models while reducing the

rate of mortality. However, despite the potential ben-

efit 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 moder-

ate 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 possi-

ble. The present study proved that Ax-NA could be used

to establish diabetic model. It was clearly demon-

strated 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 degen-

erative changes but not complete destruction in the

islets with �-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 �-cells and a thick layer of peripheral non-�-cells,

are strongly suggestive of a correlation to the inci-

dence of type II DM.32 Although the nature of degen-

erative 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

Although the present study suggested that DM was

induced owing to the effect on �-cell, it is not clear

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

day 0 day 7 day 14 day 21 day 28

Adi

pone

ctin

(µg/

mL) Ax100 + NA50

Ax100 + NA150

Ax120 + NA50

Ax120 + NA100

Ax120 + NA150

Control

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 (mg/ml) in control andexperimental group induced with combinations of Ax þ NA. *p < 0.05 when compared with control group. NA: nico-tinamide; Ax: alloxan.

Vattam et al. 5



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 acido-

philic 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 cer-

tain combinations may be less detrimental to the

liver. It is a well-known fact that fatty changes are

linked to the impairment of mitochondrial �-oxida-

tion of fatty acids. This leads to the esterification

of fatty acids in the cytoplasmic region, character-

ized by the presence of lipid droplets within the

hepatocytes.33 In general, major metabolic diseases

such as NIDDM and obesity are inflammatory condi-

tions 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

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 character-

ized 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 stud-

ies have shown that uncontrolled hyperglycemia and

hyperlipidemia are factors responsible for diabetic

nephropathy progression, which triggers vascular oxi-

dative 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.

In conclusion, this study demonstrated that the use

of Ax-NA combination is effective for the development

Figure 4. Pancreas tissue samples were harvested upon killing the animals and fixed in 10% formaldehyde. The tissueswere processed and embedded in paraffin before sectioning to a size of 4.5 mm using microtome. The samples were thenstained by employing hematoxylin and eosin staining. (a) Ax100 þ NA50, (b) Ax100 þ NA150, (c) Ax120 þ NA50, (d)Ax100þNA100, (e) Ax100þNA150), and (f) control. White arrows indicate pyknotic nuclei and acidophilic cytoplasmicappearance with vacuolar changes and cellular swelling, while the changes in the islets cells were not severe. NA: nico-tinamide; Ax: alloxan.




Figure 5. Liver tissue samples were harvested upon killing the animals and fixed in 10% formaldehyde. The tissues wereprocessed and embedded in paraffin before sectioning to a size of 4.5 mm using microtome. The samples were then stainedby employing hematoxylin and eosin staining. (a) Ax100 þ NA50, (b) Ax100 þ NA150, (c) Ax120 þ NA50, (d) Ax100 þNA100, (e) Ax100 þNA150, and (f) control. Black arrow shows the fatty infiltration, while the white arrows indicate thevacuolization of the hepatocyte in the groups a to e treated with different combination of Ax and NA. NA: nicotinamide;Ax: alloxan.

Figure 6. Kidney tissue samples were harvested upon killing the animals and fixed in 10% formaldehyde. The tissues wereprocessed and embedded in paraffin before sectioning to a size of 4.5 mm using microtome. The samples were then stainedby employing hematoxylin and eosin staining. (a) Ax100 þ NA50, (b) Ax100 þ NA150, (c) Ax120 þ NA50, (d) Ax100 þNA100, (e) Ax100 þ NA150, and (f) control. The black arrows indicate cellular swelling, glomerular infiltration, andcongestion of capillaries and necrosis. NA: nicotinamide; Ax: alloxan.

Vattam et al. 7



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 pre-

ferable, resulting in hyperglycemia with near-normal

insulin and reduced adiponectin levels. Furthermore,

the use of this combination also produced degenera-

tive changes in the main organs, as expected in DM

conditions, but did not result in early animal death.

Conflict of interest

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...

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