Monosodium glutamate-induced diabetic mice are ... - Carcinogenesis

Carcinogenesis Advance Access published January 4, 2012 

© The Author 201 2 . Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@ oup.com 

Monosodium glutamate-induced diabetic mice are susceptible to 

azoxymethane-induced colon tumorigenesis 

Kazuya Hata 1,2,* , Masaya Kubota 3 , Masahito Shimizu 3 , Hisataka 

Moriwaki 3 , Toshiya Kuno 1 , Takuji Tanaka 1,4,5* , Akira Hara 1 , and 

Yoshinobu Hirose 1 

1 Department of Tumor Pathology, Gifu University Graduate School of 

Medicine, 1-1 Yanagido, Gifu 501-1194, Japan. E-mails: 

k-hata-sun@hhc.eisai.co.jp (K.H.); tkuno@gifu-u.ac.jp (T.K.); 

takutt@toukaisaibou.co.jp (T.T.); ahara@gifu-u.ac.jp (A.H.); 

yhirose@gifu-u.ac.jp (Y. H.) 

2 Kamiishidu Division, Sunplanet Co., Gifu 503-1602, Japan. E-mail: 

3 Department of Medicine, Gifu University Graduate School of Medicine, 

Gifu 501-1194, Japan. E-mails: samurai0201@yahoo.co.jp (M.K.); 

shimim-gif@umin.ac.jp (M.S.); hmori@gifu-u.ac.jp (H.M.) 

4 Department of Oncologic Pathology, Kanazawa Medical University, 

Ishikawa 920-0293, Japan. 

5 Tohkai Cytopathology Institute: Cancer Research and Prevention 

(TCI-CaRP), Gify 500-8285, Japan. 

*Corresponding author. K.H., Tel: +81-584-46-3241, Fax. 

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+81-584-48-0011. E-mail: k-hata-sun@hhc.eisai.co.jp; and T.T., Tel: 

+81-50-273-4399, Fax: +81-58-273-4392, E-mail: 

takutt@toukaisaibou.co.jp 

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by guest on March 29, 2013

Abstract 

Obese people and diabetic patients are known to be high risk of 

colorectal cancer (CRC), suggesting need of a new preclinical animal model, 

by which to extensively study the diverse mechanisms, therapy, and 

prevention. The present study aimed to determine whether experimental 

obese and diabetic mice produced by monosodium glutamate (MSG) 

treatment are susceptible to azoxymethane (AOM)-induced colon 

tumorigenesis using early biomarkers, aberrant crypts foci (ACF) and 

β-catenin-accumulated crypts (BCAC), of colorectal carcinogenesis. Male 

Crj:CD-1 (ICR) newborns were daily given four subcutaneous injections of 

MSG (2 mg/g body weight) to induce diabetes and obesity. They were then 

given four intraperitoneal injections of AOM (15 mg/kg body weight) or 

saline (0.1 ml saline/10 g body weight). Ten weeks after the last injection of 

AOM, the MSG-AOM mice had a significant increase in the multiplicity of 

BCAC (13.83±7.44, p

highly susceptible to AOM-induced colorectal carcinogenesis, suggesting 

potential utility of our MSG-AOM mice for further investigation of the 

possible underlying events that affect the positive association between 

obese/diabetes and CRC. 

(274 words). 

Key words: type 2 diabetes mellitus; monosodium glutamate; azoxymethane, 

pre-cancerous colonic lesions; Crj:CD-1 (ICR) mice. 

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Introduction 

Epidemiological studies have shown that obesity and diabetes mellitus 

may be one of the risk factors for colorectal cancer (CRC) development 

[1-7]. At present, hyperinsulinemia [8,9], hypercholesterolemia [10,11], 

hyperglycemia [9,12], hyperlipidemia [7] are considered to be possible risk 

factors of CRC. In addition, insulin-like growth factor (IGF) pathway is 

involved in colorectal carcinoagenesis [13-16] and the signaling pathway 

is .reported to be a potential target of CRC treatment [17-19] and CRC 

chemoprevention [20,21]. Thus, importance of the growth hormone/IGF-1 

axis [22] and IGF/IGF-1 receptor (IGF-1R) axis [15,23,24] is postulated in 

carcinogenesis in CRC development. In fact, our experimental studies 

indicated that the IGF/IGF-1R axis is altered during carcinogenesis in 

colorectum [25,26] and other tissue [27,28] and the axis is a good target for 

cancer chemoprevention [25-28]. However, the underlying mechanisms of 

how these chronic diseases promote colon carcinogenesis still remain 

unknown [19]. On this context new research animal models are needed to 

investigate the diverse aspects of the mechanisms. 

We have previously reported that development of AOM-induced 

pre-cancerous lesions is enhanced in C57BL/KsJ-db/db mice with 

hyperleptinemia and hyperinsulinemia [29]. Such an animal model may give 

important implications for further exploration of the possible underlying 

events that affect the positive association between CRC and obesity and/or 

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diabetes [30-32]. A number of animal models for diabetes and/or obesity 

have been reported. One such model is produced by injection of 

monosodium glutamate (MSG). When MSG is applied to Crj:CD-1 (ICR) 

newborn mice (MSG mice), they develop diabetic condition 

(hyperinsulinemia, hyperglycemia and hyperplastic islets) without 

polyphagia [33,34]. 

It is believed that colorectal carcinogenesis is a representative 

multi-step tumorigenesis with events of genetic alterations, Several small 

lesions, including aberrant crypt foci (ACF) [35,36], mucin-depleted foci 

(MDF) [37], and β-catenin-accumulated crypts (BCAC) [38], are proposed 

as early-appearing pre-neoplastic lesions [37]. While ACF and MDF are 

recognized on the surface of cancer-predisposed colons of rodents and 

human [37], BCAC are identified in colonic mucosa at the early stages of 

colon carcinogenesis [39]. Accumulating evidence suggests that BCAC are 

independent small dysplastic lesions and/or microadenomas and progressed 

pre-cancerous lesions [40] in colon carcinogenesis when compared ACF and 

MDF [39]. These early lesions are widely used for investigating 

pathobiology of colorectal carcinogenesis [37]. 

In the current study, new born Crj:CD-1 (ICR) mice were treated with 

MSG to produce diabetes and obesity, and subsequently they received a 

colonic carcinogen, azoxymethane (AOM). Our results indicated that the 

MSG mice are highly susceptible to AOM-induced colorectal carcinogenesis 

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y counting the number of BCAC, but not ACF, and possible involvement of 

the IGF/IGF-1R axis in colorectal tumorigenesis of diabetic and obese mice 

induced by MSG and AOM. Our main goal is to assess the involvement of 

obesity/diabetes-associated events, such as hyperinsulinemia, in colorectal 

carcinogenesis in vivo. 

Materials and Methods 

Animals and chemicals 

The pregnant Crj:CD-1 (ICR) mice were purchased from Charles River 

Japan, Inc. (Kanagawa, Japan) and their newborns were used in the study. 

MSG was obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, 

Japan) and AOM from Sigma Chemical Co. (St. Louis, MO, USA). Mice 

used for the experiment were maintained in the well-controlled room with a 

high-efficiency particulate air (HEPA) filter, a 12h lighting (7:00-19:00), 

25±2°C room temperature, and 55±15% humidity. Mice (3-6 mice/cage) 

were housed in polycarbonate cages measuring W225 x D338 x H140 mm 

(Japan CLEA, Inc. (Tokyo, Japan) with the floor covered with a sheet of roll 

paper (Japan SLC). MF (Oriental Yeast Co., Ltd., Tokyo, Japan) was used as 

a basal diet throughout the study. Groundwater that was chlorine-treated and 

subjected to ultraviolet disinfection was used as drinking water in a bottle. 

We fully complied with the “Guidelines Concerning Experimental Animals” 

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issued by the Japanese Association for Laboratory Animal Science and 

exercised due consideration so as not to cause any ethical problem. 

Experimental procedure 

The newborns were divided into two groups according to the treatments. 

The birth date was the beginning of four daily subcutaneous injections of 

MSG (2 mg/g body weight, MSG mice) and physiological saline (Saline 

mice). Among these mice, males were subjected to the study. They were 

divided into four groups at 4 weeks of age: groups 1 (12 males) and 2 (6 

males) of the MSG mice received four weekly intraperitoneal injections of 

AOM (15 mg/kg body weight, the MSG-AOM mice) and physiological 

saline (0.1 ml/10g body weight, the MSG-Saline mice), respectively. 

Similarly, two groups of the ICR-Saline mice were given AOM or saline, 

belonging to groups 3 (11 males, the Saline-AOM mice) and 4 (5 males, the 

Saline-Saline). At the termination of the experiment (10 weeks after the last 

injection of AOM and 17 weeks of age of mice), all animals were sacrificed 

to analyze the number of colonic ACF and BCAC, clinical serum chemistry, 

and mRNA expression of IGF1, IGF2, and IGF-1R in the colonic mucosa. 

Counting the numbers of ACF and BCAC 

The ACF and BCAC were determined according to the standard 

procedures described previously [30,31,41]. ACF are defined as single or 

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multiple crypts that have altered luminal openings, exhibit thickened 

epithelia and are larger than adjacent normal crypts [35]. BCAC, which 

have high frequency mutations in β-catenin gene, demonstrate histological 

dysplasia with a disruption of the cellular morphology and an accumulation 

of this protein (Fig. 2B) [39]. BCAC do not have a typical ACF-like 

appearance because the lesion is not recognized on the mucosal surface like 

ACF, and is only identified in the histological sections of en face 

preparations. Both of these lesions are utilized as biomarkers to evaluate a 

number of agents for their potential chemopreventive [42-44] and 

tumor-promotion [45] properties. After the colons were fixed flat in 10% 

buffered formalin for 24 hours, the mucosal surface of the colons were 

stained with methylene blue (0.5% in distilled water) and then the number 

of ACF were counted under a light microscope. Thereafter, the distal parts 

(5 cm from anus) of the colon were cut to count the number of BCAC. To 

identify BCAC intramucosal lesions, the distal part of the colon (mean area: 

0.7 cm 2 /colon) was embedded in paraffin and then a total of 20 serial 

sections (4-μ m thick each) per colon were made by an en face preparation 

[30,31,41]. For each case, 2 serial sections were used to analyze BCAC. 

Histopathology and immunohistochemical analyses for β-catenin and 

IGF-1R 

Three serial sections were made from paraffin-embedded tissue blocks. 

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Two sections were subjected to hematoxylin and eosin (H & E) staining for 

histopathology and β-catenin immunohistochemistry to count the number of 

BCAC. Immunohistochemistry for β-catenin and IGF-1R was performed 

using the labeled streptavidin-biotin method (LSAB kit; DAKO, Glostrup, 

Denmark), as previously described [30,31]. Primary antibodies of 

anti- β-catenin antibody (1:1000 final dilution) and anti-IGF-1R antibody 

(1:100 final dilution) obtained from Transduction Laboratories (catalog no. 

610154; San Jose, CA) and Santa Cruz Biotechnology, Inc. (sc-7907; Santa 

Cruz, CA), respectively, were applied on the sections. Negative control 

sections were immunostained without the primary antibody. 

Blood chemistry 

At 17 weeks of age, blood samples (0.5-1.0 ml/mouse) were collected 

for determination of total cholesterol, triglyceride and glucose by a simple 

measurement device (DRICHEM Fujifilm Medical Co., Ltd., Tokyo). The 

concentration of blood insulin was measured by a LBIS insulin measuring 

kit for mice (Shibayagi Co., Ltd., Gunma). 

RNA extraction and quantitative real-time RT-PCR analysis 

A quantitative real-time RT-PCR analysis was carried out in the scraped 

colonic mucosa of the MSG-AOM mice and the Salin-AOM mice. Total RNA 

was isolated from the the scraped colon mucosa of the mice using the 

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RNAqueous-4PCR kit (Ambion Applied Biosystems, Austin, TX, USA). The 

cDNA was synthesized from 0.2 μg total RNA using the SuperScript III 

First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). The primers 

used for the amplification of IGF-1, IGF-2, and IGF-1R specific genes were 

as follows: IGF-1 forward, 5-CTGGACCAGAGACCCTTTGC-3 and reverse, 

5-GGACGGGGACTTCTGAGTCTT-3; IGF-2 forward, 

5-GTGCTGCATCGCTGCTTAC-3 and reverse, 

5-ACGTCCCTCTCGGACTTGG-3; and IGF-1R forward, 

5-GTGGGGGCTCGTGTTTCTC-3 and reverse, 

5-GATCACCGTGCAGTTTTCCA-3. Real-time PCR was done in a 

LightCycler (Roche Diagnostics Co., Indianapolis, IN, USA) with SYBR 

Premix Ex Taq (TaKaRa Bio, Shiga, Japan). The expression levels of the 

IGF-1, IGF-2, and IGF-1R genes were normalized to the β-actin gene 

expression level. 

Statistical analysis 

Measurements are expressed as mean ± SD, and differences if present 

were compared by one-way analysis of ANOVA (Tukey-Kramer’s multiple 

comparison’s test) or two-tailed unpaired t-test. The incidences of intestinal 

tumors were compared by Fisher’s exact probability test. The results were 

considered statistically significant if the p values were

Results 

General observations 

As shown in Fig. 1, the mean body weight of MSG-Saline mice (group 2) 

was much greater than Saline-Saline mice (group 4) during the study. The 

average body weights of the AOM-injected groups belonging to groups 1 

(MSG-AOM) and 3 (Saline-AOM) were smaller than that of saline-injected 

groups, groups 2 (MSG-Saline) and 4 (Saline-Saline), during the study. At 

the termination of the experiment the mean body weights of groups 1 and 3 

were significantly lower than groups 2 and 4, respectively (p

Serum levels of glucose, total cholesterol, triglyceride and insulin 

The serum concentrations of glucose, total cholesterol, triglyceride, and 

insulin at 17 weeks of age are shown in Table 2. MSG treatment 

significantly elevated all measures regardless of the AOM exposure (group 

1 vs. group 3, p

81-fold increase) and IRF-1R (2.43-fold increase), when compared to the 

Saline-AOM mice. The increase of IGF-1R was statistically significant 

(p

gene mutations, are demonstrated in human ACF [46-48]. BCAC, which 

accumulate β-catenin protein in the nucleus and cytoplasm, are also 

regarded as putative precursors to colorectal adenomas [37]. Several rodent 

studies have shown that both of these lesions are useful as biomarkers to 

evaluate the chemopreventive properties of specific agents [49,50]. In 

human colorectum, increased plasma IGF-1 levels are associated with the 

number of dysplastic ACF [51], suggesting that IGF-1 may be a promoter of 

the growth of dysplastic ACF and an independent risk factor of CRC. It may 

be possible that the number of dysplastic ACF, but not hyperplastic ACF, 

may be increased in the MSG-AOM mice, although we did not analyze two 

types of ACF. 

Neonatal injections of MSG to mice or rats cause hyperthalamic damage 

[52,53], and as a consequence, these animals present several 

neuroendocrine and metabolic alterations, which lead central obesity, type 2 

diabetes, insulin resistance, hyperinsulinemia, hypertriglyceridemia, and 

hyperlipidemia [33]. These abnormalities are risks for the development of 

CRC [9]. The pancreatic islets in the mice subcutaneously injected MSG in 

neonatal period are hyperplastic up to 54 weeks of age [33]. Therefore, .our 

model described here may be suitable to study the pathobiology of diabetes- 

and obesity-asosciated colorectal carcinogenesis. 

In this study, blood insulin level of the MSG-Saline mice (group 2) was 

the highest among the groups. There is accumulating evidence suggesting 

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that hyperinsulinemia is involved in colon carcinogenesis, obesity, and 

diabetes [3,8,9,12,54,55]. Several epidemiological studies indicate that type 

2 diabetic patients with hyperinsulinemia increases risk for CRC [3,12]. 

Additionally, continuous injections of insulin promote AOM-induced colon 

carcinogenesis in rats [56,57]. Hence, it seems likely that hyperinsulinemia 

in the MSG-AOM mice enhanced the development of AOM-induced lesions 

in the present study. Hyperglycemia and hypercholesterolemia observed in 

the MSG-AOM mice also contribute the development of BCAC and ACF in 

the colorectum, since these conditions are positively associated with CRC 

occurrence [9,11,12]. Hyperinsulinemia, hyperglycemia and 

hypercholesterolemia may singly or synergistically promote the 

development of preneoplastic and neoplastic colonic lesions. Although 

insulin resistance in colorectal carcinogenesis of obese people and/or type 2 

diabetic patients is reported [7,55,58,59], we did not investigate presence or 

absence of insulin resistance in this study, Corpet et al. reportted that diet 

that increase some indirect insulin resistance markers does not promote 

colon carcinogenesis in female rats when ACF are used as a biomarker [60]. 

Regarding the mode of action, the current consensus assumes that the 

IGF-1 pathway plays a role in insulin-related tumor promotion in the colon 

[20,61]. IGF-1 binds to the IGF-1R, activates a signal cascade and triggers 

cell proliferation in several tissues, including colon [62]. Insulin at 

supra-physiological levels also binds to and activates the IGF-1R because of 

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its homology with the insulin receptor [62]. Furthermore, hyperinsulinemia 

was shown to indirectly increase bioavailability of IGF-1 by regulating 

levels of IGF-binding proteins [63]. In this study, IGF-1R 

immunohistochemical expression of IGF-1 was strongly positive in the 

cytoplasm of BCAC developed in the MSG-AOM mice. Indeed, 

over-expression of IGF-1R was also reported in human CRC [64]. 

Accordingly, it may be possible that hyperinsulinemia in MSG-AOM mice 

activates the signaling cascades involving the IGF-IR, resulting in a 

proliferative response [19,59,61]. Another interesting findings regarding 

IGF-1R immunohistochemistry is that inflammatory cells infiltrated around 

BCAC were positively reacted against the IGF-1R antibody. Significance of 

the findings is not known, but similar findings have been reported in human 

Crohn’s disease [65]. IGF-1 and IGF2 are potentially relevant mediators in 

the chronic inflammation [27] and mediate the majority of their biological 

action through IGF-1R [66]. Thus, our findings on the IGF-1R 

immunohistochemical positivity in inflammatory cells suggest that 

inflammation in the microenvironment of pre-cancerous lesions for CRC 

may .contribute to the growth of the lesions [67,68]. 

In conclusion, our data indicate that the MSG-AOM mice with 

hyperinsulinemia hypercholesterolemia, hyperglycemia, and 

hypercholesterolemia are highly susceptible to colorectal carcinogenesis 

and the MSG-AOM mouse model could be useful for investigating the 

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mechanisms of obesity/diabetes-associated events involving in colorectal 

carcinogenesis and the therapeutic and chemopreventive strategies of CRC 

in obese people and/or type 2 diabetic patients. 

Acknowledgments 

We wish to thank Kyoko Takahashi and Ayako Suga for their technical 

assistance, and Yoshitaka Kinjo for care of the animals. This work was 

supported in part by a Grant-in-Aid from the Ministry of Health, Labour, and 

Welfare of Japan (to Y.H.); a Grant-in-Aid for the 3rd Term Comprehensive 

10-Year Strategy for Cancer Control from the Ministry of Health, Labour and 

Welfare of Japan (to T.T.); a Grant-in-Aid for Cancer Research from the 

Ministry of Health, Labour and Welfare of Japan; and Grants-in-Aid for 

Scientific Research (No. 23501324) from the Ministry of Education, Culture, 

Sports, Science and Technology of Japan (to T.T.); 

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Figure legend 

Fig. 1. Body weight gains of all groups during the study. 

Fig. 2. Histopathology (A) of a BCAC developed in a MSG-AOM mouse. 

Immunohistochemistry of (B) β-catenin and (C) IGF-1R in the BCAC shows 

positive reaction of β-catenin in their cell membrane and cytoplasm and 

IGF-1R in their cytoplasm. Bars=50 μm. (A) H & E stain, (B) β-catenin 

immunohistochemistry, and (C) IGF-1R immunohistochemistry. 

Fig. 3. mRNA expression of IGF-1, IGF-2, and IGF-1R in the colonic mucosa of the 

MSG-AOM mice and the Saline-AOM mice 

Hata 30 



Hata 1 

Table 1. Body weights and numbers of ACF and BCAC per colon of mice treated with AOM and/or MSG at the end of study (17 

weeks of age) 

Group no. 

1 

(MSG-AOM) 

2 

(MSG-Saline) 

3 

(Salin-AOM) 

4 

(Saline-Saline) 

a Mean±SD. 

No. of mice 

examined 

12 

6 

11 

5 

MSG 

(2 mg/kg bw) 

+ 

(4 times daily) 

+ 


- 

(saline) 

- 

(saline) 

Treatments 

AOM 

(15 mg/kg bw) 

+ 

(4 times weekly) 

- 

(saline) 

+ 


- 

(saline) 

Body weight 

b,c,d Significantly different from group 2 ( b p

Table 2. Serum levels of total cholesterol, triglycerides, glucose, and insulin of mice treated with AOM and/or MSG at the end of 

study (17 weeks of age) 

Group 

no. 

1 

(MSG- 

AOM) 

2 

(MSG- 

Saline) 

3 

(Salin- 

AOM) 

4 

(Saline- 

Saline) 

a Mean±SD. 

No. of 

mice 

examined 

12 

6 

11 

5 

MSG 

(2 mg/kg bw) 

+ 


+ 


- 

(saline) 

- 

(saline) 

Treatments 

b Significantly different from group 3 (p

y guest on March 29, 2013 

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