Transgenic expression of GFP-LC3 perturbs autophagy in exocrine pancreas and acute pancreatitis responses in mice.

Pancreatitis is a common, sometimes fatal, disease of exocrine pancreas, initiated by damaged acinar cells. Recent studies implicate disordered macroautophagy/autophagy in pancreatitis pathogenesis. ATG8/LC3 protein is critical for autophagosome formation and a widely used marker of autophagic vacuoles. Transgenic GFP-LC3 mice are a valuable tool to investigate autophagy ; however, comparison of homeostatic and disease responses between GFP-LC3 and wild-type (WT) mice has not been done. We examined the effects of GFP-LC3 expression on autophagy, acinar cell function, and experimental pancreatitis. Unexpectedly, GFP-LC3 expression markedly increased endogenous LC3-II level in pancreas, caused by downregulation of ATG4B, the protease that deconjugates/delipidates LC3-II. By contrast, GFP-LC3 expression had lesser or no effect on autophagy in liver, lung and spleen. Autophagic flux analysis showed that autophagosome formation in GFP-LC3 acinar cells increased 3-fold but was not fully counterbalanced by increased autophagic degradation. Acinar cell (ex vivo) pancreatitis inhibited autophagic flux in WT and essentially blocked it in GFP-LC3 cells. In vivo pancreatitis caused autophagy impairment in WT mice, manifest by upregulation of LC3-II and SQSTM1/p62, increased number and size of autophagic vacuoles, and decreased level of TFEB, all of which were exacerbated in GFP-LC3 mice. GFP-LC3 expression affected key pancreatitis responses; most dramatically, it worsened increases in serum AMY (amylase), a diagnostic marker of acute pancreatitis, in several mouse models. The results emphasize physiological importance of autophagy for acinar cell function, demonstrate organ-specific effects of GFP-LC3 expression, and indicate that application of GFP-LC3 mice in disease models should be done with caution.Abbreviations: AP: acute pancreatitis; Arg-AP: L-arginine-induced acute pancreatitis; ATG: autophagy-related (protein); AVs: autophagic vacuoles; CCK: cholecystokinin-8; CDE: choline-deficient, D,L-ethionine supplemented diet; CER: caerulein (ortholog of CCK); CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; ER: endoplasmic reticulum; LAMP: lysosomal-associated membrane protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; TEM: transmission electron microscopy; TFEB: transcription factor EB; ZG: zymogen granule(s).

Mechanism of mitochondrial permeability transition pore induction and damage in the pancreas: inhibition prevents acute pancreatitis by protecting production of ATP.

OBJECTIVE: Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established. DESIGN: We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis. RESULTS: MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished. CONCLUSIONS: This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.