Dysregulation of mannose-6-phosphate–dependent cholesterol homeostasis in acinar cells mediates pancreatitis
Olga A. Mareninova, Eszter T. Vegh, Natalia Shalbueva, Carli J.M. Wightman, Dustin L. Dillon, Sudarshan Malla, Yan Xie, Toshimasa Takahashi, Zoltan Rakonczay Jr., Samuel W. French, Herbert Y. Gaisano, Fred S. Gorelick, Stephen J. Pandol, Steven J. Bensinger, Nicholas O. Davidson, David W. Dawson, Ilya Gukovsky, Anna S. Gukovskaya
Olga A. Mareninova, Eszter T. Vegh, Natalia Shalbueva, Carli J.M. Wightman, Dustin L. Dillon, Sudarshan Malla, Yan Xie, Toshimasa Takahashi, Zoltan Rakonczay Jr., Samuel W. French, Herbert Y. Gaisano, Fred S. Gorelick, Stephen J. Pandol, Steven J. Bensinger, Nicholas O. Davidson, David W. Dawson, Ilya Gukovsky, Anna S. Gukovskaya
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Research Article Cell biology Gastroenterology

Dysregulation of mannose-6-phosphate–dependent cholesterol homeostasis in acinar cells mediates pancreatitis

Abstract

Disordered lysosomal/autophagy pathways initiate and drive pancreatitis, but the underlying mechanisms and links to disease pathology are poorly understood. Here, we show that the mannose-6-phosphate (M6P) pathway of hydrolase delivery to lysosomes critically regulates pancreatic acinar cell cholesterol metabolism. Ablation of the Gnptab gene encoding a key enzyme in the M6P pathway disrupted acinar cell cholesterol turnover, causing accumulation of nonesterified cholesterol in lysosomes/autolysosomes, its depletion in the plasma membrane, and upregulation of cholesterol synthesis and uptake. We found similar dysregulation of acinar cell cholesterol, and a decrease in GNPTAB levels, in both WT experimental pancreatitis and human disease. The mechanisms mediating pancreatic cholesterol dyshomeostasis in Gnptab–/– and experimental models involve a disordered endolysosomal system, resulting in impaired cholesterol transport through lysosomes and blockage of autophagic flux. By contrast, in Gnptab–/– liver the endolysosomal system and cholesterol homeostasis were largely unaffected. Gnptab–/– mice developed spontaneous pancreatitis. Normalization of cholesterol metabolism by pharmacologic means alleviated responses of experimental pancreatitis, particularly trypsinogen activation, the disease hallmark. The results reveal the essential role of the M6P pathway in maintaining exocrine pancreas homeostasis and function, and implicate cholesterol disordering in the pathogenesis of pancreatitis.

Authors

Olga A. Mareninova, Eszter T. Vegh, Natalia Shalbueva, Carli J.M. Wightman, Dustin L. Dillon, Sudarshan Malla, Yan Xie, Toshimasa Takahashi, Zoltan Rakonczay Jr., Samuel W. French, Herbert Y. Gaisano, Fred S. Gorelick, Stephen J. Pandol, Steven J. Bensinger, Nicholas O. Davidson, David W. Dawson, Ilya Gukovsky, Anna S. Gukovskaya

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Figure 8

Derangement of cholesterol metabolism in experimental pancreatitis.

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Derangement of cholesterol metabolism in experimental pancreatitis. Char...
Characteristics of cholesterol metabolism were measured, as detailed in Figure 4, in pancreas of WT mice subjected to pancreatitis induced with cerulein (CER-AP), L-arginine (Arg-AP), or CDE diet (CDE-AP) (A, B, and E–G); ex vivo pancreatitis induced in WT mouse (C and I–M) or human acinar cells (D) with 100 nM CCK (CCK-AP) or 1 mM carbachol; and isolated pancreatic mitochondria (H). (B) Cholesterol ester (CE) heatmap; each column represents an individual mouse. (F) IB analysis of the indicated proteins. (G) LAL activity was measured by a fluorogenic assay (n = 6 mice per condition). (H) Cholesterol content was measured by enzymatic assay. (I–M) Filipin colocalization with indicated proteins was quantified as detailed in Figure 2. Each symbol in A, C–E, and H represents an individual mouse, or cell or mitochondria preparation (n = 3–15 per condition). Each symbol in J, K, and M corresponds to 20–30 cells in a different field (n = 10–20 fields from 3 cell preparations per condition). Values are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the same parameter in control/untreated pancreas, acinar cells or isolated mitochondria. Significance was determined by 2-tailed Student’s t test (B, E, H, J, K, and M) or 1-way ANOVA followed by Tukey’s multiple comparison test (A, C, and D). Scale bars: 10 μm.