Table 1.
Comparison of LDC-HF and LDC-E diets to Breeder Chow.
Figure 1.
WT and Mist1−/− mice show no overt response to ethanol feeding.
Caloric intake (A) and weight gain (B) was monitored over six weeks of feeding with a Lieber-DeCarli Ethanol (LDC-E), LDC high fat (LDC-HF) or breeding chow (BC) diet. Letters represent statistically significant differences between groups (see text for details). Groups were compared using Two-way ANOVA and Bonferroni post-hoc test. *P<0.05, **P<0.01, ***P<0.001. n = 7 (BC), n = 11 (LDC-HF), n = 12 (LDC-E). The LDC-HF diet resulted in increased weight gain in both genotypes. At six weeks, mice were killed and serum amylase (C) and pancreatic edema (D) calculated. No significant differences were observed between genotypes or treatments. n = 11 (LDC-HF), n = 12 (LDC-E).
Figure 2.
Mist1−/− pancreata develop periductal accumulations of inflammatory cells in response to ethanol feeding.
Representative photomicrographs of Gomori's trichrome stained pancreatic sections from WT (A, B) and Mist1−/− mice (C, D) fed a LDC-HF (A, C) or LDC-E (B, D) diet for 6 weeks. Higher magnification images from the same sections (Ai–Di) were used to highlight specific morphological events. Cellular accumulations (arrowhead) surrounding ducts (*) and adipose accumulations (arrow) were observed only in LDC-E fed Mist1−/− mice. (E, F) Immunofluorescent analysis for the T-lymphocyte marker CD4 (E) indicated that these accumulations consisted of lymphocytes (arrowhead). Sections were co-stained with DAPI (F) to reveal all cells. Magnification bars = 40 µm.
Figure 3.
Mist1−/− pancreatic tissue exhibits increased activation of the UPR.
(A) Western blot analysis of key UPR markers in 2 month old WT and Mist1−/− whole pancreatic lysates. Mist1−/− extracts show significantly increased accumulations of BiP/GRP78 (B), GADD34 (C) and sXBP1 (D), but not peIF2α (p = 0.743) or uXBP1 (p = 0.532) relative to WT pancreatic tissue. *P<0.05. n = 3.
Figure 4.
Ethanol feeding leads to increased accumulation of spliced (s) Xbp1 only in WT pancreatic tissue.
RT-PCR analysis of Xbp1 splicing in WT (A) and Mist1−/− (B) mice fed breeder chow (BC), LDC-E or LDC-HF diets revealed accumulation of spliced (s) Xbp1 mRNA in response to both LDC-E and LDC-HF diets in WT pancreatic tissue. Mist1−/− mRNA samples showed sXbp1 accumulating in BC fed mice and decreased Xbp1 accumulation in LDC-HF and LDC-E mice. (C) Quantification of the ratio of spliced (s) Xbp1 to unspliced (u) Xbp1 as determined by densitometric analysis of images in A. Mist1−/− mice showed increased amounts of sXbp1 relative to WT mice when fed the BC diet. LDC-HF and LDC-E diets led to a significant increase in Xbp1 splicing while pancreatic tissue from LDC-E fed Mist1−/− mice showed decreased amounts of splicing. Groups were compared using One-way ANOVA and a Tukey post-hoc test: *P<0.05, **P<0.01. n = 3 (BC), n = 6 (LDC-HF & E).
Figure 5.
High fat and ethanol diets activate PERK signaling in pancreatic acinar cells.
(A) Western blotting analysis for peIF2α, BiP/GRP78 (upper band) or β-actin (as a control) in pancreatic tissue of WT or Mist1−/− mice fed breeder's chow (BC) or LDC-E or LDC-HF diets for 6 weeks. Quantitative analysis of peIF2α (B) or BiP/GRP78 (C) accumulation from (A) relative to β-actin accumulation. LDC-E and LDC-HF diets lead to significant increases in peIF2α compared to BC diets in WT mice but not in Mist1−/− tissue. Conversely, while BiP/GRP78 levels are unchanged in WT mice, they decrease upon exposure to LDC diets in Mist1−/− mice. Groups were compared using One way ANOVA and a Tukey post-hoc tests: *P<0.05, **P<0.01. n = 3 (BC); n = 6 (LDC-HF & E).
Figure 6.
Mist1 expression is not altered in response to ethanol feeding.
Quantitative RT-PCR shows no difference in Mist1 levels in response to either LDC-E or LDC-HF diets. Error bars = mean ± SE (n = 3).
Figure 7.
Ethanol-fed Mist1−/− mice show limited increases in autophagy and apoptosis.
(A) Quantitative analysis of LC3-II puncta, shown as the area of LC3 dots per area of DAPI stained nuclei. Groups were compared using One-way ANOVA and a Tukey post- hoc test: *P<0.05. n = 3 (BC), n = 5 (LDC-HF & LDC-E). Quantification of the LC3-II to LC3-I ratio by densitometric analysis of western blots for WT (B) and Mist1−/− mice fed breeder's chow (BC), or diets high in fat (LDC-HF) or ethanol (LDC-E). *P<0.5, n = 3. (D–G) Images from TUNEL analysis performed on pancreatic tissue from WT and Mist1−/− mice showed minimal (<<1%) TUNEL+ nuclei (arrows) under all dietary conditions.