AMPK Deletion in the Intestinal Epithelium Reveals Region‐Specific Roles in Health and Disease

Abstract

AMP‐Activated Protein Kinase (AMPK) is a conserved energy sensor that has been suggested to modulate intestinal barrier function. Intestinal barrier function contributes to partitioning of the gut lumen and lamina propria and is primarily mediated by intestinal epithelial cell (IEC) control of permeability, ion transport, and regulation of cell turnover. Dysfunction of the intestinal barrier is a feature of pathologies such as Inflammatory Bowel Disease (IBD) and diarrhea. The aim of this study was to identify the impact of AMPK activity on intestinal barrier function by utilizing a tissue‐specific knockout mouse model lacking both AMPK‐α catalytic isoforms.MethodsIntestinal epithelial‐specific AMPK knockout mice (AMPKαΔIEC KO) were created by crossing Prkaa1 and Prkaa2 floxed mice (AMPKαfl/fl) with Villin‐Cre mice. Large intestine (cecum, proximal, and distal colon) was stripped of the seromuscular layer and mounted in Ussing chambers containing Kreb’s Ringer’s buffer. Transepithelial electrical resistance (TER), 4 kDa FITC‐dextran (FD4) permeability and ion transport responses to Forskolin (20 μM, bilateral) and Carbachol (300 μM, serosal) were measured. Protein expression was analyzed in isolated IECs or whole tissue by immunoblotting or by immunostaining of whole intestinal tissue. Colitis was induced by 5% Dextran Sodium Sulfate (DSS) in drinking water (ad libitum for 5 days) followed by 3 days of water.ResultsAMPKαΔIEC KO mice had decreased TER only in the proximal colon vs. AMPKαfl/fl mice (p = 0.057, n = 6–7), with no change in FD4 permeability. Decreased TER in the proximal colon was associated with reduced Claudin‐4 expression. Claudin‐4 in the distal colon was significantly decreased in AMPKαΔIEC KO mice (48% decrease, p = 0.0136, n = 5). Ion transport responses to Carbachol were increased in the cecum of AMPKαΔIEC KO mice vs. AMPKαfl/fl mice (p = 0.012, n = 3), while Forskolin responses were unchanged. Expression of the AMPK‐regulated ion transport protein, Na+‐K+‐Cl− Cotransporter 1 (NKCC1) was increased in the cecum and distal colon of AMPKαΔIEC KOvs. AMPKαfl/fl mice, thus indicating that AMPK repressed NKCC1 expression in these regions (p<0.05 ‐ p<0.001). Inflammatory challenge (DSS colitis) caused increased cell death (TUNEL staining) in cecum and distal colon but TUNEL staining was reduced in the cecum of AMPKαΔIEC KO mice vs. AMPKαfl/fl mice (6.2‐fold, p = 0.0241, n = 2–3). Consistent with unaltered TUNEL staining in the proximal colon, the apoptosis markers cleaved caspase‐3 and PARP‐1 were also reduced in both mouse genotypes. However, despite increased TUNEL staining, these cleavage events were not increased in the cecum or distal colon of DSS‐treated mice suggesting cell death occurred by a mechanism independent of caspase‐3 and PARP cleavage.ConclusionsEpithelial AMPK regulation of intestinal barrier permeability, ion transport, and inflammation‐induced cell death displays regional heterogeneity along the large intestine.Support or Funding InformationNIH 2R01DK091281 (DFM)