Dysregulation of mannose-6-phosphate–dependent cholesterol homeostasis in acinar cells mediates pancreatitis
Olga A. Mareninova, … , Ilya Gukovsky, Anna S. Gukovskaya
Olga A. Mareninova, … , Ilya Gukovsky, Anna S. Gukovskaya
Published June 15, 2021
Citation Information: J Clin Invest. 2021;131(15):e146870. https://doi.org/10.1172/JCI146870.
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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 3

Gnptab ablation impairs early-to-late endosome maturation and endocytic recycling.

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Gnptab ablation impairs early-to-late endosome maturation and endocytic...
Immunofluorescence analysis of markers of early (EEA1, TfR, Rab5), recycling (Rab11), and late (LAMP2, Rab7) endosomes in pancreatic (A and C–H) tissue and (B, I, and J) acinar cells (see also images in the Supplemental Figure 1). In I and J, acinar cells were transduced with both CellLight Rab5-GFP and Rab7-RFP. Quantitative colocalization analysis for indicated proteins was done as detailed in Figure 2. Values are mean ± SEM; each symbol corresponds to 20 to 30 cells in a different field (n = 7–14 fields from 3 to 4 mice or cell preparations per condition). ***P < 0.001 vs. WT; 2-tailed Student’s t test. (K and L) To measure transferrin (Tf) endocytosis, acinar cells were loaded with Tf-594 at 0°C, transferred to 37°C, incubated for the indicated times, and visualized with confocal microscopy. Internalized Tf-594 was quantified using Volocity software. Values are mean ± SEM; each symbol corresponds to 10 to 15 cells in a different field (n = 6–11 fields from 3 WT and 3 KO cell preparations). ***P < 0.001 vs. corresponding WT condition; 1-way ANOVA followed by Tukey’s multiple comparison test. Scale bars: 10 μm.