Mitochondrial Dysfunction, Through Impaired Autophagy, Leads to Endoplasmic Reticulum Stress, Deregulated Lipid Metabolism, and Pancreatitis in Animal Models

Abstract

Background & aims: Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca2+, mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. We analyzed the mechanisms of these dysfunctions and their relationships, and how these contribute to development of AP in mice and rats.

Methods: Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence.

Results: Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca2+ overload or through a Ca2+ overload-independent pathway that involved reduced activity of ATP synthase (80% inhibition in pancreatic mitochondria isolated from rats or mice given L-arginine). Both pathways were mediated by cyclophilin D and led to mitochondrial depolarization and fragmentation. Mitochondrial dysfunction caused pancreatic ER stress, impaired autophagy, and deregulation of lipid metabolism. These pathologic responses were abrogated in cyclophilin D-knockout mice. Administration of trehalose largely prevented trypsinogen activation, necrosis, and other parameters of pancreatic injury in mice with L-arginine AP. Tissues from patients with pancreatitis had markers of mitochondrial damage and impaired autophagy, compared with normal pancreas.

Conclusions: In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with ATP synthase mediates L-arginine-induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP.

Keywords: Acinar Cell; Inflammatory Response; Lamellar Bodies; Pancreas.

Conflict of interest statement

Conflicts of interest

The authors disclose no conflicts.

Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.

Mitochondrial dysfunction occurs early in Arg-AP; it is mediated by cyclophilin D but not by Ca2+ overload. Rats (A,B) and wild-type (wt) or cyclophilin D knockout (CypD KO) mice (C-E) were subjected to Arg-AP, as detailed in Methods, and killed at indicated times. Pancreatic mitochondria were isolated and assayed either in Ca2+-free medium containing 1 mmol/L EGTA (A-D) or at indicated free Ca2+ concentrations maintained with Ca2+/EGTA buffers (E). ΔΨm was measured with tetraphenyl phosphonium ion (TPP+) electrode and quantified as the difference between TPP+ levels in mitochondria suspension before and after addition of 5 μmol/L DNP, a mitochondrial uncoupler [illustrated in (A)]. The rate of ΔΨm recovery after ADP (20 μmol/L) -induced drop was measured as a slope of ΔΨm increase back to steady-state level [shown in (A) in red]. Both parameters were normalized to mitochondria from control animals. Shown are the individual values and means ± SEM (n = 6–10 per group). *P < .05 vs control (saline-treated) animals; #P < .05 vs CypD KO mice with the same treatment. (F). Wt mouse acinar cells were loaded with 25 μmol/L BAPTA-AM (where noted) and incubated with 20 μmol/L Arg for indicated times. ΔΨm was measured using the dye TMRM (1 μmol/L). The difference between TMRM fluorescence in control cells before and after addition of the mitochondrial uncoupler FCCP (10 μmol/L) was taken as 100% ΔΨm. Values are mean ± SEM from 3–4 cell preparations. *P < .05 vs control cells.

Figure 2.

Mitochondrial dysfunction in Arg-AP is caused by cyclophilin D-mediated perturbation of ATP synthase. Mice (A,C-H) and rats (B,H) were subjected to Arg-AP or CER-AP. Pancreatic levels of free Arg (A) were measured with LC-MS in whole tissue and isolated mitochondria; ATP synthase enzymatic activity (B,C), in isolated mitochondria; and ATP levels (D), in tissue homogenates. Shown are the individual values and means ± SEM (n = 3–9 per group). *P < .05 vs control animals; #P < .05 vs CypD KO mice. (E-G). ATP synthase and cyclophilin D were immunoprecipitated (IP) from pancreatic mitochondria isolated from mice at indicated conditions, and their levels in the immunoprecipitates were measured by IB. (H). Pathologic alterations in mitochondrial ultrastructure in pancreas of rats and mice with Arg-AP (EM). Scale bars: 0.5 μm. Larger fields from these micrographs are shown in Supplementary Figure 5.

Figure 3.

Normalizing mitochondrial function by cyclophilin D genetic ablation improves Arg-AP. Mice were subjected to Arg-AP, killed at 72 hours (A), 24 hours (C,E-G), or as indicated (B); and histopathologic changes (A; H&E staining) and pancreatitis responses (B-H) were measured. (C,D). Inflammatory infiltration was measured on pancreatic tissue immunostained for the neutrophil marker myeloperoxidase (MPO) and macrophage marker F4/80 (see also Supplementary Figure 7). (E,F). Pancreatic levels of phosphorylated and total p65/RelA (E) were measured by IB, and NF-κB DNA binding activity (F), by EMSA. In this and other figures, ERK1/2 or GAPDH are loading controls, and each lane on IB represents an individual animal. The narrow white space on EMSA gel indicates the lanes are on the same gel but not contiguous. (G,H). Subcellular localization of HMGB1 in pancreas was analyzed by IF using Volocity software. Scale bars: 10 μm. Values are mean ± SEM (n = 3–4). *P < .05 vs wt saline-treated mice; #P < .05 vs same treatment in CypD KO mice; $P < .05 vs saline-treated CypD KO mice.

Figure 4.

Arg-AP profoundly disturbs pancreatic lipid metabolism, which is prevented by cyclophilin D genetic ablation. Mice were subjected to 24 hours Arg-AP. H&E staining (A) shows multiple small vacuoles in pancreas of wt animals with Arg-AP, identified by EM as lamellar bodies (B,C) and lipid droplets (D). Boxed area in (B), showing accumulation of lamellar bodies, is enlarged in (C). Scale bars: 10 μm (A), 6.7 μm (B), and 1.3 μm (C,D). (E). IB analysis of perilipin 2 (PLIN2), a marker of lipid droplets. The narrow white space indicates the lanes are on the same blot but not contiguous. (F-L). Pancreatic tissue triacylglycerols (TAG) and free fatty acids (FFA) were measured with LC-MS and GC-MS as detailed in Methods. Total, saturated, and unsaturated TAG and FFA levels were quantified based on their detailed profiles (see Supplementary Figure 8C,D), normalized per mg protein, and further normalized to wt control mice. (I-L). Effect of Arg-AP on individual FFAs (the numbers of carbons and of double bonds are indicated). Values are mean ± SEM (n = 3). *P < .05 vs wt saline-treated mice; #P < .05 vs same treatment in CypD KO mice.

Figure 5.

Cyclophilin D genetic ablation alleviates ER stress and normalizes autophagy in Arg-AP. Mice were subjected to Arg-AP and killed at indicated times (A,B) or at 24 hours (C-I). Markers/mediators of (A,B) ER stress, (D-F) autophagic/lysosomal pathways, and (G) mitophagy were analyzed in pancreatic tissue by IB. Ub, ubiquitin; CatB, cathepsin B. The IB in (F) shows the intermediate/single-chain (sc) CatB form and the heavy chain of mature/double-chain (dc) form. (C). EM showing abnormally large autolysosome with poorly degraded material in pancreas of Arg-AP mouse. The boxed area is enlarged in Supplementary Figure 10. Scale bar: 1.7 μm. (H,I). Pancreatic acinar cells were isolated from mice of indicated groups, incubated for 2 hours in the presence or absence of lysosomal protease inhibitors, E64D + pepstatin A (20 μmol/L each), and LC3-I/II levels measured by IB. In (B) and (I), densitometric band intensities for specified proteins were normalized to that of ERK in the same sample, and the mean ratios further normalized to that in wt control group. Values are mean ± SEM (n = 3). *P < .05 vs wt saline-treated mice; #P < .05 vs same treatment in CypD KO mice; $P < .05 vs saline-treated CypD KO mice.

Figure 6.

Enhancement of autophagic activity with trehalose improves Arg-AP. Wt mice received daily i.p. injections of trehalose (or vehicle) for 2 weeks, then subjected to Arg-AP and killed 24 hours later. (A,B,F). Effects of trehalose on markers/mediators of autophagic and lysosomal pathways (LC3, p62, ubiquitin, CatB), ER stress (CHOP, sXBP1), lipid droplets (PLIN2), and inflammation (phospho-p65/RelA) were analyzed by IB. The IB in (B) shows the intermediate, single-chain (sc) and the mature, double-chain (dc) forms of CatB. (C,D). H&E staining and EM demonstrate the absence of abnormally large autolysosomes and lamellar bodies in trehalose-treated mice with Arg-AP. Scale bars: 10 μm (C), 2 μm (D). (E,G). Effects of trehalose on Arg-AP responses (E) and pancreatic FFA levels (G), measured as in Figure 5. *P < .05 vs control (saline-treated) mice without trehalose; #P < .05 vs trehalose-treated mice with Arg-AP; $P < .05 vs control (saline-treated) mice with trehalose. (H). Schematic illustrating the pathways of mitochondrial dysfunction and their links to pancreatitis.