Chronic pancreatitis (CP) is characterized by repeated episodes of acute pancreatitis (AP) leading to chronic inflammation, atrophy and fibrosis. In AP early zymogen activation is a critical event, caused by a co-localization of zymogens with lysosomol proteases in the autophagy pathway. Here, we investigated chymotrypsinogen activation in ATG5-deficient mice showing an impaired autophagy.
Chronic pancreatitis was induced by repetitive caerulein hyperstimulation over a time of 6 weeks. Male ATG5 (flox/flox) and pancreas-specific ATG5-KO mice were sacrificed after 1, 2 and 6 weeks. We performed density gradient centrifugation of homogenized pancreas to separate organelles in which marker proteins and enzymes were determined by western blotting and as enzyme activities.
CP induced atrophy of exocrine tissue was characterized by a decrease in pancreatic weight and contents of amylase, lipase and trypsinogen. However this loss of exocrine tissue was not different in KO mice. Unstimulated ATGKO-mice showed an intrinsic chymotrypsin activity in dense granular fractions that decreased in the time-course of CP. In ATG5f/f mice, but not in KO-mice, chymotrypsin activity was found in autophagosomal compartments declining in further course of CP.
Atg5-deficiency results in the appearance of a intrinsic chymotrypsin activity in dense vesicular fractions. However, chymotrypsinogen activation is significantly decreased in autophagosomal vesicles of ATG-KO-mice indicating an impaired autophagic flux.