Diet supplemented with citrus unshiu segment membrane suppresses chemically induced colonic preneoplastic lesions and fatty liver in male db/db mice

Abstract

The modulatory effects of dietary citrus unshiu segment membrane (CUSM) on the occurrence of aberrant crypt foci (ACF) and b-catenin accumulated crypts (BCACs) were determined in male C57BL/KsJ-db/db (db/db) mice initiated with azoxymethane (AOM). Male db/db, db/þ and þ/þ mice were given 5 weekly subcutaneous injections of AOM (15 mg/kg body weight), and then they were fed the diet containing 0.02%, 0.1% or 0.5% CUSM for 7 weeks. At Week 12, a significant increase in the numbers of ACF and BCAC was noted in the db/db mice in comparison with the db/ þ and þ/þ mice. Feeding with CUSM caused reduction in the frequency of ACF in all genotypes of mice and the potency was high in order of the db/db mice, db/þ mice and þ/þ mice. The number of BCACs was also reduced by feeding with CUSM, thus resulting in a 28-61% reduction in the db/db mice, possibly due to suppression of cell proliferation activity in the lesions by feeding with CUSM-containing diet. Clinical chemistry revealed a low serum level of triglyceride in mice fed CUSM. In addition, CUSM feeding inhibited fatty metamorphosis and fibrosis in the liver of db/db mice. Our findings show that CUSM in the diet has a chemopreventive ability against the early phase of AOM-induced colon carcinogenesis in the db/db as well as db/þ and þ/þ mice, indicating potential use of CUSM in cancer chemoprevention in obese people. ' 2006 Wiley-Liss, Inc.Key words: citrus unshiu segment membrane; ACF; BCAC; colon carcinogenesis; db/db mice The modern Western lifestyle, including a high caloric intake, high-fat diets and physical inactivity, results in a positive energy balance, diabetes and obesity. These lifestyle patterns might also be risk factors for the development of colorectal cancer (CRC), 1 which is one of the major causes of morbidity and mortality in the Western world. 2 This malignancy has also increased in Asia owing to the changes in lifestyle, such as the dietary habit of increased meat consumption. 2,3 Several prospective and case-control studies have addressed the relationship between obesity/diabetes and CRC. 1,4,5 C57BL/KsJ-db/db (db/db) mice are used as a genetically altered animal model with genotypes of obesity and diabetes mellitus. 6 A disruption of the leptin receptor (Ob-R) gene in these mice leads to an over-expression of leptin in the adipose tissue and a concomitantly high serum concentration of leptin. 7,8 The synthesis of leptin in adipocytes is influenced by insulin, 9 tumor necrosis factor-a, 10 glucocorticoids, 11 reproductive hormones 12 and prostaglandins 13 that may be involved in the neoplastic processes. 14 In addition, leptin can act as a growth factor in colonic epithelial cells 15 while modulating the proliferation of colonic cryptal cells. 16 In contrast, more leptin in the blood clearly decreased colon carcinogenesis in 3 different animal models. 17,18 Thus, leptin might be one of the biological factors involved in the development of CRC associated with obesity/diabetes. The db/db mouse, therefore, is a very useful model for elucidating ...

Supplementation with Branched-chain Amino Acids Inhibits Azoxymethane-induced Colonic Preneoplastic Lesions in Male C57BL/KsJ-db/db Mice

“…The ACF and BCAC were determined according to the standard procedures described previously (20,21,25). ACF are defined as single or multiple crypts that have altered luminal openings, exhibit thickened epithelia, and are larger than adjacent normal crypts (22).…”

“…Thereafter, the distal parts (5 cm from the 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 per colon) was embedded in paraffin, and then a total of 20 serial sections (4-Am thick each) per colon were made by an en face preparation (20,21,25). For each case, 2 serial sections were used to analyze BCAC.…”

“…The other section was used for the proliferating cell nuclear antigen (PCNA), a G 1 -to-S phase marker, immunohistochemistry to estimate the cell proliferative activity in the colonic mucosa. Immunohistochemical analyses for h-catenin and PCNA were done with the labeled streptavidin-biotin method (LSAB kit; DAKO) as previously described (20,21). Anti -h-catenin antibody (1:1,000 final dilution) was obtained from Transduction Laboratories (catalogue no.…”

“…At sacrifice, blood samples were collected from the AOM-treated mice (groups 1-3) to measure the serum concentrations of insulin, leptin, triglyceride, total cholesterol, IGF-I, IGF-II, and BCAA. The serum triglyceride, total cholesterol, and BCAA levels were assayed as described previously (20,29). The serum insulin, leptin, IGF-I, and IGF-II were determined by an enzyme immunoassay according to the manufacturer's protocol (R&D Systems).…”

“…The mice are susceptible to the colonic carcinogen azoxymethane (AOM) because the development of AOMinduced aberrant crypt foci (ACF) and h-catenin -accumulated crypts (BCAC), both of which are putative precursor lesions for colonic adenocarcinoma (22,23), is enhanced in db/db mice compared with db/+ or +/+ mice (19,20). In the present study, we investigated in detail the effects of BCAA on the development of colonic premalignant lesions, ACF and BCAC, in db/db mice initiated with AOM, focusing on the improvement of hyperinsulinemia, hyperlipidemia, and hyperleptinemia.…”