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Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis
Olga A. Mareninova, Kip Hermann, Samuel W. French, Mark S. O’Konski, Stephen J. Pandol, Paul Webster, Ann H. Erickson, Nobuhiko Katunuma, Fred S. Gorelick, Ilya Gukovsky, Anna S. Gukovskaya
Olga A. Mareninova, Kip Hermann, Samuel W. French, Mark S. O’Konski, Stephen J. Pandol, Paul Webster, Ann H. Erickson, Nobuhiko Katunuma, Fred S. Gorelick, Ilya Gukovsky, Anna S. Gukovskaya
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Research Article Gastroenterology

Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis

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Abstract

The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.

Authors

Olga A. Mareninova, Kip Hermann, Samuel W. French, Mark S. O’Konski, Stephen J. Pandol, Paul Webster, Ann H. Erickson, Nobuhiko Katunuma, Fred S. Gorelick, Ilya Gukovsky, Anna S. Gukovskaya

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Figure 8

Autophagy impairment mediates intra-acinar trypsin accumulation in pancreatitis.

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Autophagy impairment mediates intra-acinar trypsin accumulation in pancr...
(A) Rat pancreatic acinar cells were incubated with or without 100 nM CCK and 10 mM 3-MA. Trypsin activity after 30-minute incubation was measured in cell homogenates by a fluorogenic assay. LC3 dots were visualized, as illustrated in Figure 2B, and counted using ImageJ software. (B) Mouse acinar cells were transfected with Atg5 siRNA or control siRNA, as described in Methods. The transfection efficiency is illustrated in Figure 1F. Trypsin activity was measured under fluorescence microscope in live cells loaded with a trypsin substrate BZiPAR and incubated for 30 minutes with and without 100 nM CCK (see Supplemental Figure 6). The fluorescence from cleaved BZiPAR was measured per cross-sectioned cell area using ImageJ software, and 150–200 acinar cells were assessed for each condition. (C) Rats were subjected to CR pancreatitis and killed 30 minutes after the first CR injection. Pancreatic tissue sections were double immunostained for LC3 and TAP or LAMP-2 and TAP (colocalization shown by arrowheads). (D–F) Rats were subjected to fasting and CR or Arg pancreatitis (see Methods), and pancreatic subcellular fractions were obtained as in Figure 4. (D and E) Trypsin activity was measured in whole tissue homogenates or subcellular fractions by a fluorogenic assay. (F) TAP levels in fractions Z and L were measured by ELISA and in each fraction were normalized to those for the feeding conditions. Values in A, B, and D–F are mean ± SEM (n = 3).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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