Genetic and Diet-Induced Obesity Increased Intestinal Tumorigenesis in the Double Mutant Mouse Model Multiple Intestinal Neoplasia X Obese via Disturbed Glucose Regulation and Inflammation

Abstract

We have studied how spontaneous or carcinogen-induced intestinal tumorigenesis was affected by genetic or diet-induced obesity in C57BL/6J-Apc (Min/+) X C57BL/6J-Lep (ob/+) mice. Obesity was induced by the obese (ob) mutation in the lep gene coding for the hormone leptin, or by a 45% fat diet. The effects of obesity were examined on spontaneous intestinal tumors caused by the multiple intestinal neoplasia (Min) mutation in the adenomatous polyposis coli (Apc) gene and on tumors induced by the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). F1 ob/ob (homozygous mutated) mice had increased body weight (bw) and number of spontaneous and PhIP-induced small intestinal tumors (in Apc (Min/+) mice), versus ob/wt (heterozygous mutated) and wt/wt mice (homozygous wild-type). A 45% fat diet exacerbated bw and spontaneous tumor numbers versus 10% fat, but not PhIP-induced tumors. Except for bw, ob/wt and wt/wt were not significantly different. The obesity caused hyperglucosemia and insulinemia in ob/ob mice. A 45% fat diet further increased glucose, but not insulin. Inflammation was seen as increased TNFα levels in ob/ob mice. Thus the results implicate disturbed glucose regulation and inflammation as mechanisms involved in the association between obesity and intestinal tumorigenesis. Ob/ob mice had shorter lifespan than ob/wt and wt/wt mice.

Figure 1

Body weight as area under the curve (AUC). Body weight was recorded weekly from weaning at week 3 until termination at week 11 and is presented for Apc ^Min/+ (a) females and (b) males, and Apc ^+/+ (c) females and (d) males (mean ± SD). The mice were exposed to 0.9% NaCl and given a 10% fat diet (white columns) or 0.9% NaCl and a 45% fat diet (light grey columns), or they were exposed to PhIP and given a 10% fat diet (dark grey columns) or PhIP and a 45% fat diet (black columns). n = 10–18 mice. a.u. = arbitrary units.

Figure 2

Number of small intestinal tumors. The number of small intestinal tumors (mean ± SD) is shown for (a) female and (b) male Apc ^Min/+ mice with the three ob genotypes, terminated at 11 weeks of age. The mice were exposed to 0.9% NaCl and given a 10% fat diet (white columns) or 0.9% NaCl and a 45% fat diet (light grey columns), or they were exposed to PhIP and given a 10% fat diet (dark grey columns) or PhIP and a 45% fat diet (black columns). n = 10–17 mice.

Figure 3

The size of the small intestinal tumors. This is illustrated by curves of distributions of tumors size classes (of 0.25 mm tumor diameter intervals) calculated as mean number of tumors in each tumor size class for each treatment group. The effect of a 45% fat diet compared with a 10% fat diet is shown for female Apc ^Min/+ X Lep ^ob/ob mice (○), Apc ^Min/+ X Lep ^ob/wt (∆) and Apc ^Min/+ X Lep ^wt/wt mice (□) on a 10% fat diet, and the same genotypes on a 45% fat diet (filled symbols) exposed to (a) 0.9% NaCl, or (b) PhIP. The effect of exposure to PhIP compared with 0.9% NaCl is shown for male Apc ^Min/+ X Lep ^ob/ob mice (○), Apc ^Min/+ X Lep ^ob/wt (∆) and Apc ^Min/+ X Lep ^wt/wt mice (□) exposed to 0.9% NaCl, or the same genotypes exposed to PhIP (filled symbols) on (c) a 10% fat diet, or (d) a 45% fat diet. n = 10–17 mice.

Figure 4

Localization of tumors along the small intestine and colon. This is shown for pooled female and male Apc ^Min/+ X Lep ^ob/ob mice (○), Apc ^Min/+ X Lep ^ob/wt (∆) and Apc ^Min/+ X Lep ^wt/wt mice (□) on a 10% fat diet (open symbols) or a 45% fat diet (filled symbols) treated with (a) 0.9% NaCl or (b) PhIP. The tumor position is given as distance from the stomach measured in cm. Mean number of tumors/cm intestine for the mice in each experimental group was scored. n = 10–17 mice.

Figure 5

Blood glucose levels as area under the curve (AUC) from a glucose tolerance test (GTT). The GTT was performed on mice fasted for 6 h at 6 weeks of age. Blood glucose was measured 5 min before and 15, 30, 60 and 120 min after an i.p. injection of 2 g/kg bw glucose. The data were pooled for Apc ^Min/+ and Apc ^+/+ mice. The mice were treated with either 0.9% NaCl, (a) females and (b) males, or with PhIP, (c) females and (d) males. The mice had ob genotype ob/ob (○), ob/wt (∆) or wt/wt (□) and were given a 10% fat diet (open symbols) or a 45% fat diet (filled symbols). n = 4–9 mice.

Figure 6

Levels of insulin and the proinflammatory cytokines IL-6 and TNFα in plasma. Insulin levels (ng/mL) were measured with ELISA in plasma obtained from the mice at termination, for (a) females and (b) males (median and individual values are shown as columns and dots, resp.). IL-6 (c) and TNFα (d) levels (both in pg/mL) were measured with bead-based immunoassays and analysed by flow cytometer in plasma obtained from the mice at termination. The data shown are from females and males combined (median and individual values are shown as columns and dots, resp.). n = 6 mice (3 of each gender).

Figure 7

Survival of mice. Untreated pooled female and male Apc ^Min/+ and Apc ^+/+ mice of all three ob genotypes were kept under regular observation until euthanized. Survival of each genotype of mice was depicted as decreasing % of surviving mice compared with the number of mice present at the start of the experiment (n = 26, 30 and 31 for groups of Apc ^Min/+ X Lep ^ob/ob (○), Apc ^Min/+ X Lep ^ob/wt (∆) and Apc ^Min/+ X Lep ^wt/wt (□) mice, respectively, and n = 35, 28 and 24 for Apc ^+/+ X Lep ^ob/ob (●), Apc ^+/+ X Lep ^ob/wt (▲) and Apc ^+/+ X Lep ^wt/wt (■) mice, respectively.