Grp78 heterozygosity regulates chaperone balance in exocrine pancreas with differential response to cerulein-induced acute pancreatitis

Abstract

The endoplasmic reticulum (ER) is abundant in the acinar cells of the exocrine pancreas. To test the role of ER homeostasis in acute pancreatitis, we manipulated GRP78 levels, a major ER chaperone, in mice. Grp78(+/+) and (+/-) littermates were fed either a regular diet (RD) or a high-fat diet. Acinar cells were examined for ER structure by electron microscopy, and ER chaperone levels were assessed by immunoblotting. Pancreatitis was induced by cerulein injection, and multiple pathological parameters were analyzed. Grp78(+/-) mice showed decreased GRP78 expression in acinar cells. Exocrine pancreata of RD-fed Grp78(+/-) mice in an outbred C57BL/6 × 129/sv genetic background exhibited ER lumen dilation, a reduction in chaperones calnexin (CNX) and calreticulin (CRT), and exacerbated pancreatitis associated with high CHOP induction. With the high-fat diet regimen, Grp78 heterozygosity triggered GRP94 up-regulation and restoration of GRP78, CNX, and CRT to wild-type levels, corresponding with mitigated pancreatitis on cerulein insult. Interestingly, after backcrossing into the C57BL/6 background, RD-fed Grp78(+/-) mice exhibited an increase in GRP94 and levels of CNX and CRT equivalent to wild type, associated with decreased experimental pancreatitis severity. Administration of a chemical chaperone, 4-phenolbutyrate, was protective against cerulein-induced death. Thus, in exocrine pancreata, Grp78 heterozygosity regulates ER chaperone balance, in dietary- and genetic background-dependent manners, and improved ER protein folding capacity might be protective against pancreatitis.

Figure 1

Reduction of GRP78 protein levels in pancreatic acinar cells of adult Grp78^+/− mice. A: Representative H&E staining of pancreatic sections from 7-month-old Grp78^+/+ and ^+/− mice fed regular diet (RD, n ≥ 3 for each genotype). B: Confocal microscopy of GRP78 (green) immunofluorescence (IF) on pancreatic acinar cells. N, nucleus. n = 2 (^+/+) or 4 (^+/−).

Figure 2

Dilated ER lumen and ER chaperone reduction in Grp78^+/− exocrine pancreas. A: Representative transmission electron micrographs (TEM) of pancreatic acinar cells of 6-month-old Grp78^+/+ and ^+/− mice fed RD (n = 2 for each genotype). Lower panels demonstrate higher magnification of the boxed area of the corresponding upper panels. Arrowheads indicate endoplasmic reticulum (ER). N, nucleus; G, secretory granules. B: Protein levels of ER chaperones GRP78, GRP94, calnexin (CNX), and calreticulin (CRT) were examined in pancreas from 7-month-old Grp78^+/+ and ^+/− mice fed RD (n ≥ 3 for each genotype) by Western blotting. Left panel: representative blots. Right panel: quantitative levels normalized against β-actin. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01.

Figure 3

HFD-fed Grp78^+/− mice gained similar weight as wild types. A: Scheme of high-fat diet (HFD) feeding. Cohorts of Grp78^+/+ and ^+/− male littermates were fed a RD after weaned at 3 weeks of age, and switched to HFD feeding from 10 weeks of age. B: Fasting body weight. n ≥ 5 mice per condition. *P < 0.05, **P < 0.01 for HFD versus RD. C: Food intake measurement during the 10th week of HFD. n = 9 mice for each genotype. Data are presented as the mean ± SEM. D: Oil Red O staining of stool smear from mice during the 20^th week of HFD. Negative control, dH₂O; positive control, white adipose extract. n = 3 (+/+) or 4 (+/−).

Figure 4

Recovery of ER morphology and chaperone levels in exocrine pancreas of Grp78^+/− mice after HFD. A: Representative TEM of pancreatic acinar cells from mice after 12 weeks of HFD (n = 2 for each genotype). Lower panels demonstrate higher magnification of the boxed area of the corresponding upper panels. Arrowheads indicate ER. N, nucleus; G, secretory granules. B and C: Protein levels of ER chaperones GRP78, GRP94, calnexin (CNX), and calreticulin (CRT) were examined in whole pancreas (B) and isolated islets of Langerhans (C) from Grp78^+/+ and ^+/− mice after 20 weeks of HFD (n ≥ 3 for each genotype) by Western blotting. Left panels: representative blots. Right panels: quantitative levels normalized against β-actin. Data are presented as the mean ± SEM. **P < 0.01.

Figure 5

Diet-induced differential response to experimental pancreatitis in Grp78^+/− mice. A: Scheme of cerulein-induced acute pancreatitis. Thirty-week-old Grp78^+/+ and ^+/− male littermates, fed RD or HFD, were subjected to seven hourly IP injections of cerulein (50 μg/kg body weight), followed by sacrifice and analysis. B: Representative H&E staining of pancreatic sections from mice after cerulein injection (n ≥ 3 for each condition). Lower panels demonstrate higher magnification of the boxed area of the corresponding upper panels. C and D: H&E-stained pancreas sections were counted for neutrophils (C) or quantitated for area of edema represented by non-parenchymal space (D). E and F: Serum samples were assayed for amylase (E) and lipase (F). Data are presented as the mean ± SEM. n ≥ 3 mice for each condition. *P < 0.05, **P < 0.01.

Figure 6

Differential cell death in exocrine pancreas of Grp78^+/− mice with experimental pancreatitis. The Grp78^+/+ and ^+/− mice were fed with either RD or HFD and treated with either PBS or cerulein as indicated. A: TUNEL assay on pancreas sections. Representative fields showing TUNEL signal alone (upper panels) or merged with DAPI (lower panels). B: Apoptotic cells (TUNEL merged with DAPI) were counted as percentage of total cells (DAPI). C: Necrotic cells were counted on H&E-stained pancreas sections as percentage of total cells. Data are presented as the mean ± SEM. n ≥ 3 mice for each condition. **P < 0.01.

Figure 7

Specific regulation of pancreatic ER chaperones and response to experimental pancreatitis in Grp78^+/− mice backcrossed into a C57BL/6 background. A: Protein levels of ER chaperones were examined in pancreas from RD-fed 7-month-old Grp78^+/+ and ^+/− littermates backcrossed into C57BL/6 background for seven generations (n ≥ 3 for each genotype) by Western blotting. Left panel: representative blots. Right panel: quantitative levels normalized against GAPDH. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01. B: Representative H&E staining of pancreatic sections from mice after cerulein injection (n ≥ 3 for each genotype).

Figure 8

Modulation of ER stress response by cerulein and diet in pancreas of Grp78^+/− mice. Whole cell lysates of pancreas from Grp78^+/+ and ^+/− mice, fed RD or HFD, after seven hourly PBS or cerulein injections (n ≥ 3 for each condition), were subjected to immunoblotting of the indicated ER stress response proteins: (A) pSer51- and total eIF2α, (B) CHOP, GADD34, spliced form of XBP-1 (XBP-1s), and EDEM, with GAPDH serving as loading control. NIH3T3 cells, either nontreated (−) or treated with 300 nmol/L thapsigargin for 16 hours (Tg) served as negative and positive controls, respectively. Left panels: representative Western blots. Lanes for the NIH3T3 samples were run on the same gel as the tissue samples but were noncontiguous. Right panels: quantitation of relative protein levels after normalization against GADPH levels. Data are presented as the mean ± SD. **P < 0.01.

Figure 9

4-PBA protected against cerulein-induced AR42J cell death. Trypan blue exclusion assay was performed on AR42J cells after 72-hour treatment of 4-PBA at the indicated doses, with (+) or without (−) simultaneous treatment of 1 μmol/L cerulein during the last 24 hours. 120–500 cells were counted for each sample. Percentage of dead cells is presented as the mean ± SEM from triplicate samples. *P < 0.05, **P < 0.01.