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

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 3

Starvation increases, but CCK hyperstimulation decreases autophagy-mediated protein degradation in pancreatic acinar cells.

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Starvation increases, but CCK hyperstimulation decreases autophagy-media...
(A) Autophagy-mediated degradation of long-lived proteins was measured in mouse pancreatic acinar cells by pulse-chase assay, as described in Methods. Briefly, the culture medium was supplemented for the first 6 hours with [14C]-valine to label proteins, after which cells were chased for 16 hours in fresh medium containing cold valine. Cells were then switched to either (ii–iv) medium 199 containing amino acids or (i) nutrient-free (i.e., free of amino acids) medium, and further cultured for 4 hours with 0.1 nM (ii) or 100 nM (iv) CCK, or without CCK (i and iii), both in the presence and absence of 10 mM 3-MA. Protein degradation was measured as the net release of TCA-soluble radioactivity; values obtained in the presence of 3-MA were subtracted as nonautophagic background. The measurements were in duplicates, and the experiment was repeated with similar results. Data represent mean ± SEM. (B) Quantification of LC3 dots in acinar cells incubated in conditions iii and iv described in A (that is, control versus 100 nM CCK). LC3 dots were visualized under confocal microscope as illustrated in Figure 2B and counted, with the use of ImageJ software, in at least 1,000 acinar cells for each condition. Values are mean ± SEM (n = 4).