High-fat, high-calorie diet promotes early pancreatic neoplasia in the conditional KrasG12D mouse model

Abstract

There is epidemiologic evidence that obesity increases the risk of cancers. Several underlying mechanisms, including inflammation and insulin resistance, are proposed. However, the driving mechanisms in pancreatic cancer are poorly understood. The goal of the present study was to develop a model of diet-induced obesity and pancreatic cancer development in a state-of-the-art mouse model, which resembles important clinical features of human obesity, for example, weight gain and metabolic disturbances. Offspring of Pdx-1-Cre and LSL-KrasG12D mice were allocated to either a high-fat, high-calorie diet (HFCD; ∼4,535 kcal/kg; 40% of calories from fats) or control diet (∼3,725 kcal/kg; 12% of calories from fats) for 3 months. Compared with control animals, mice fed with the HFCD significantly gained more weight and developed hyperinsulinemia, hyperglycemia, hyperleptinemia, and elevated levels of insulin-like growth factor I (IGF-I). The pancreas of HFCD-fed animals showed robust signs of inflammation with increased numbers of infiltrating inflammatory cells (macrophages and T cells), elevated levels of several cytokines and chemokines, increased stromal fibrosis, and more advanced PanIN lesions. Our results show that a diet high in fats and calories leads to obesity and metabolic disturbances similar to humans and accelerates early pancreatic neoplasia in the conditional KrasG12D mouse model. This model and findings will provide the basis for more robust studies attempting to unravel the mechanisms underlying the cancer-promoting properties of obesity, as well as to evaluate dietary- and chemopreventive strategies targeting obesity-associated pancreatic cancer development.

Figure 1

A) Weekly measurements of weight (in grams) of mice fed the control or high fat, high calorie diet (HFCD). Data are depicted as average ± SD. B) Weight gain (in grams) after 14 weeks of mice fed the control diet or HFCD. Data are depicted as average ± SD. *; p<0.05 vs. control. C) Weight gain (in grams) after 14 weeks of female and male mice fed the control diet or HFCD. Data are depicted as average ± SD. *; p<0.05 vs. control. D) Representative micro-CT images of a lean mouse (left panel) and obese mouse (right panel).

Figure 2

Serum levels of insulin, glucose, insulin-like growth factor-1 (IGF-1), leptin, cholesterol, and triglycerides at 14 weeks of mice fed the control diet or HFCD. Data are depicted as average ± SD. *; p<0.05 vs. control.

Figure 3

Semi-quantitative analyses of inflammatory parameters in the pancreas of mice fed the control diet or HFCD. A) Percentage (%) of intact acini, percentage (%) of acinar cell loss, number of inflammatory cells per high power field (hpf), and percentage (%) of stromal fibrosis are analyzed separately for wildtype and mutant mice fed the control diet or HFCD. *; p<0.05. B) Acinar loss score, inflammatory score, fibrosis score, and pancreatitis index in wildtype and mutant mice fed the control diet or HFCD. *; p<0.05.

Figure 4

Immunohistochemistry of infiltrating immune cells into the pancreas of conditional KrasG12D mice fed the HFCD. Tissue sections were stained with antibodies against B220 (B-cell marker), CD-3 (T-cell marker), and F4/80 (macrophage marker). Upper row is 200× magnification and lower row is higher 400× magnification.

Figure 5

Cytokine and chemokine levels were measured in the pancreas of wildtype (WT) and conditional KrasG12D (Kras) mice fed either the control diet (CD) or high fat, high calorie diet (HFCD). Data are depicted as average ± SD. *; p<0.05 vs. CD.

Figure 6

Immunofluorescence staining of α-smooth muscle actin (a-SMA, marker of active pancreatic stellate cells; red staining) and fibronectin (FN, green staining) in conditional KrasG12D mice (MUT) fed the control diet (CD) or a high-fat, high-calorie (HFCD) diet for 3 months. Blue staining represents DAPI-positive nuclei. Magnification, ×400. B) Graph shows quantification of and α-SMA and FN staining in pancreatic tissue sections. Data are depicted as average ± SD.

Figure 7

A) Distribution (in %) of normal pancreatic ducts and mPanIN-1a, mPanIN-1b, mPanIN-2 and mPanIN-3 lesions in the pancreas of mutant mice fed the control diet or HFCD. *; p<0.05 vs. mutant CD. B) Representative H.E. staining of pancreatic tissue sections at 14 weeks: Wildtype mice fed the control diet; wildtype mice fed the HFCD; mutant mice fed the control diet; mutant mice fed the HFCD.