Fig 1.
HFCD leads to greater weight gain in male and female KC mice.
(A) A schematic view of the study design. (B) Weight gain of male (left panel) and female (right panel) KC mice fed the CD or HFCD. The values are means ± SD. *P<0.05, Student’s t-tests. For the male mice collected at 3, 6, and 9 months, n = 12 (5 on CD and 7 on HFCD), 12 (6 on CD and 6 on HFCD), and 11 (5 on CD and 6 on HFCD), respectively. For the female mice collected at 3, 6, and 9 months, n = 11 (6 on CD and 5 on HFCD), 12 (5 on CD and 7 on HFCD), and 11 (5 on CD and 6 on HFCD), respectively. (C) Plasma levels of insulin, leptin, cholesterol, glucose, and triglycerides in 9-month-old KC mice fed the CD or HFCD. Data are depicted as means ± SD. *P<0.05 vs. control, Student’s t-tests.
Fig 2.
Histological analysis of the pancreas.
Pancreas histology of male and female KC mice fed the CD or HFCD for 3, 6, and 9 months. For each group, images (10x) from two different mice were shown. CA, cancer.
Fig 3.
HFCD leads to increased cancer incidence in male and female KC mice.
(A) The cancer incidence (%) for CD- and HFCD-fed KC mice at indicated ages. (B) Gender-specific analyses of the PDAC incidence (%) for CD- and HFCD-fed KC mice at indicated ages. The numbers of mice included in the analyses are: n = 34 for 3-month-old mice including 13 on CD (6 males and 7 females) and 21 on HFCD (12 males and 9 females), n = 31 for 6-month-old mice including 12 on CD (6 males and 6 females) and 19 on HFCD (9 males and 10 females), and n = 36 for 9-month-old mice including 17 on CD (9 males and 8 females) and 19 on HFCD (10 males and 9 females).
Fig 4.
HFCD leads to accelerated pancreatic neoplasia in male and female KC mice.
Staged PanIN distribution in cancer-free male and female, CD- and HFCD-fed KC mice at indicated ages. The values are means ± SD.
Fig 5.
HFCD promotes inflammation in the pancreas of KC mice.
Inflammatory parameters in the histological sections of pancreas were evaluated quantitatively. Specifically, acinar loss scores, inflammatory scores, fibrosis scores, and pancreatitis indices were determined for male and female KC mice fed the CD or HFCD for different time periods. The values are means ± SD. *P<0.05, Student’s t-tests.
Fig 6.
HFCD markedly accelerates stroma formation, extracellular matrix deposition and exocrine atrophy in KC mice.
(A) The extent of pancreatic collagen deposition was evaluated by Sirius red staining. Graph shows percentage of Sirius red-stained area in pancreas tissue sections at the indicated ages. Data represent mean ± SEM; 8–10 random pancreatic sections were evaluated per mouse; 3–4 mice per group. *P<0.05 vs. CD. (B) Pictures illustrate Sirius red staining in pancreatic tissue sections of KC mice fed the CD or HFCD for 6 months, the time-point displaying the highest differences in collagen deposition between CD-fed and HFCD-fed mice. (C) Pancreatic levels of fibrosis-related proteins were analyzed by Western blotting in pancreas lysates from KC mice fed the CD or HFCD for 9 months. Picture shows representative immunoblots of fibronectin; prolyl-4-hydroxylase (P4HA2), a key collagen processing enzyme; cadherin 11, a mesenchymal marker expressed by activated myofibroblasts; α-SMA, a myofibroblast marker; and p-STAT3 (Y705)/ total STAT3. Picture also shows protein levels of pancreatic amylase, a digestive enzyme produced by acinar cells and GAPDH used as loading control. Each lane represents an individual mouse; three mice per group are shown. (D) Graphs show optical density of immunoblots depicted in panel D. Data in graphs represent mean ± SEM, n = 3. *P<0.05 vs. CD.
Fig 7.
HFCD leads to an accumulation of autophagic vacuoles and p62/SQSTM1.
(A,D,F) Representative IF images of LC3-II (A,F), p62/SQSTM1 (p62) (D,F) in PanIN lesions (A,D), and histologically normal exocrine pancreas (F) of KC mice of indicated age fed CD and high fat calorie diet HFCD. DAPI was used to stain nuclei, and DIC to visualize tissue structure (upper lines). Scale bar: 20 μm. (B) Immunoblot analysis of LC3 in pancreas from 3- and 9-month-old KC mice. GAPDH is a loading control. (C,E,G) Quantification of LC3-II (C,G) and p62 (E,G) integral intensity, average size of puncta and % positively stained area was performed with ImageJ software. The values are means ± SEM (3 mice were analyzed for each strain, age and diet). * P< 0.05 vs. 3 months (mo) CD, # p < 0.0001 vs. 3 mo HFCD, $ p < 0.05 vs. 9 mo CD, ƒ p< 0.05 vs. 3 months (mo) CD.
Table 1.
Enriched pathways of genes with variants unique to HFCD (pooled analysis).
Table 2.
List of genes and variant counts unique to HFCD in individual mice.
Table 3.
Enriched pathways and genes (≥10 genes) with variants common to each HFCD-fed mouse.