Intestinal Epithelial AMPK Does Not Protect Against High Dose DSS‐Induced Colitis in Mice
Authors: King Stephanie J. , Girgis Kyrillos , Alvarez Rocio , Jones Russell G. , McCole Declan F. 
Published: June 2021

AMP‐Activated Protein Kinase (AMPK) is a master regulator of cellular energy status whose activity requires α1 and α2 isoforms of the catalytic subunit. Recent evidence suggested AMPK protects against DSS‐induced colitis using villin‐Cre AMPKα1 knockout mice and nonspecific pharmacological activators. However, previous studies have not tested if both AMPKα isoforms are required for protection as α1/α2 double‐knockout is embryonic lethal in mice. Moreover, we previously showed AMPKα1 protein is increased in AMPKα2−/− mice, potentially confounding conclusions. Furthermore, the role of intestinal epithelial AMPK in inflammatory responses remains poorly understood. The goals of this study were to determine if AMPK is necessary for intestinal homeostasis and to confirm if intestinal epithelial AMPK is required for protection against colitis in a mouse model.

Methods

Intestinal epithelial‐specific deletion of both AMPK catalytic isoforms (AMPKαΔIEC KO) was generated by crossing villin‐Cre recombinase mice with Prkaa1 and Prkaa2 floxed mice (AMPKαfl/fl). Barrier integrity and function were assessed histologically, by 40 kDa FITC‐dextran (FD4) intestinal permeability assay and by agonist stimulation of chloride secretion ex vivo in Ussing chambers. Colitis was induced with 5% dextran sulfate sodium (DSS) in drinking water for 5 days, followed by 3 days of water. Body weight, stool consistency and hemoccult were recorded daily to determine disease activity index scores. Intestinal permeability during colitis was determined by FD4 permeability. Mice were euthanized according to institutional IACUC protocols. Tissues were fixed in 4% paraformaldehyde for imaging or flash frozen for protein and RNA analysis.

Results

Unchallenged AMPKαΔIEC KO mice showed no histologic changes in intestinal integrity, nor any significant change in intestinal permeability or agonist‐stimulated chloride secretion. Mice administered 5% DSS developed severe colitis indicated by dramatic weight loss and increased disease activity index score compared to untreated controls. Colitis induced similar levels of colonic shortening and increased intestinal permeability in both floxed and AMPKαΔIEC KO mice, however AMPK‐regulated pro‐inflammatory gene expression was unchanged compared to untreated controls. Despite the lack of an overt phenotypic difference in AMPKαΔIEC KO mice compared to AMPKαfl/fl mice, we did observe significant differences in protein expression. The tight junction protein claudin 2 was significantly decreased in proximal colon of AMPKαΔIEC KO mice compared to AMPKαfl/fl mice without DSS challenge. Untreated and DSS‐treated AMPKαΔIEC KO mice had decreased levels of the apoptosis markers cleaved caspase 3 in ileum, and PARP in proximal colon respectively, while cleaved caspase 3 was increased in distal colon compared to matched AMPKαfl/fl mice.

Conclusion

Loss of AMPK alone altered levels of claudin 2 and apoptotic markers in intestinal epithelial cells but did not alter DSS‐induced colitis severity.

Support or Funding Information

NIH 2R01DK091281 (DFM)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.