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 10

Deranged cholesterol metabolism causes acinar cell damage, while its normalization ameliorates experimental pancreatitis.

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Deranged cholesterol metabolism causes acinar cell damage, while its nor...
(A–D) Cholesterol content and trypsin activity were measured (per mg protein) in WT mouse acinar cells incubated for 1 hour without and with methyl-β-cyclodextrin (MCD; 10 mM) or cholesterol-loaded MCD (MCD-Chol; 200 μg/mL), in the presence or absence of 100 nM CCK. Values are expressed relative to those in cells without MCD/MCD-Chol. (E–J) WT mice received daily i.p. injections of the LXR agonist T0901317 (T09), simvastatin (Sim; 20 mg/kg each), or vehicle. On day 4, mice were subjected to CER-AP and pancreata collected for analyses (see also Supplemental Figure 9). Cholesterol content (E), trypsin activity (F), and pancreas histopathology (H&E staining; G) were measured. (H, I, L, and N) IB analysis of markers/mediators of autophagy (LC3, p62), ER stress (CHOP, GRP-78), and mitophagy (Parkin). Ub, ubiquitylated proteins. (J) Mitochondrial fragmentation was assessed by immunostaining for Tom20, as detailed in Figure 7G. (K and L) Mouse acinar cells were treated for 16 hours with LDL (30 μg/mL) or the combination of LDL and 10 μM U18666A. (M and N) Mice received injections of U18666A as detailed in Figure 9B. Pancreas histopathology (H&E staining; M) and levels of autophagy markers/mediators (N) were measured. Each symbol in A–F and I represents an individual mouse or cell preparation. Values are mean ± SEM from 3 to 7 mice or cell preparations per condition. §P < 0.05, §§P < 0.01 vs. control cells (without MCD/MCD-Chol). ***P < 0.001 vs. saline-treated mice. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CER-AP alone (no inhibitors). Significance was determined by 2-tailed Student’s t test (A–D) or 1-way ANOVA followed by Tukey’s multiple comparison test (E, F, and I). Scale bars: 10 μm.