Effect of inflammatory cytokines and high fat diet on inositol-1,4,5-trisphosphate (IP3) receptors binding protein released with IP3 (IRBIT) expression in intestinal cells

IP3, upon binding to the IP3 receptor (IP3R), causes the release of intracellular calcium from the endoplasmic reticulum, which drives many cellular responses (e.g., cell spreading, exocytosis). In addition to releasing calcium, IP3 also causes the release of IRBIT from the IP3R. Over the past decade, IRBIT has been described as a protein that regulates calcium release, due to interaction with the IP3R, the activity of the Na-HCO3 cotransporter, the cystic fibrosis transmembrane regulator, and the Na/H exchanger (NHE3). Lack of reabsorption of Na+ by NHE3 in the intestine is responsible for diarrhea. Recently it was shown that IRBIT and NHE3 expression was decreased in a mouse model of diabetes, and the loss of NHE3 expression induced diarrhea in this model. Insulin treatment restored IRBIT and NHE3 expression, resulting in a decrease of diarrhea. Besides insulin, very little is known about factors regulating IRBIT expression in intestinal epithelial cells. In this work, we set to study the effect of inflammatory cytokines and high calorie diet on IRBIT expression, due to the fact that diabetes is associated with chronic inflammation and high caloric intake. To test the effect of inflammatory cytokines, we used the human colonic crypt cells T84. Exposing T84 cells to interleukin 13 or tumor necrosis factor alpha for 72 hours decreased IRBIT expression by 36% (P \u3c 0.001, n = 5) and 44% (P \u3c 0.001, n = 3) respectively. Finally, we compared the expression of IRBIT in mice fed with low fat milk (control) versus high milk fat (37%). We found that in the duodenum of 3 mice with a high fat diet, a substantial increase of IRBIT expression compared to the control. Our work is the first to demonstrate that inflammatory cytokines and dietary fat can alter IRBIT expression

Elucidating internalization mechanism of the Na-K-2Cl cotransporter 1 and its fate in the endocytotic pathway during protein kinase C activation in epithelial cells

Gut clearance (i.e., fluid secretion) is an important mechanism for host defense. Fluid secretion flushes luminal toxins and prevents bacterial attachment to intestinal epithelial cells, which otherwise would harm the host. In the colon, transepithelial chloride fluid secretion drives fluid secretion. The basolateral Na-K-2Cl cotransporter 1 (NKCC1) is the main protein pumping chloride inside the cell for its secretion by apical chloride channels. Previous studies have demonstrated that activation of the protein kinase C (PKC) causes a rapid internalization of NKCC1, thus decreasing chloride secretion. To date, the protein kinase C downstream targets involved in NKCC1 internalization and the fate of NKCC1 in the endocytic pathway is unknown. Using the human colonic crypt cells T84, we demonstrate that T84 cells express α-adducin and Myristoylated, Alanine-Rich C Kinase Substrate, two substrates of the PKC involved in protein internalization in other cells. In presence of phorbol 12-myristate 13-acetate (PMA), an activator of the conventional and novel PKC, we demonstrate that α-adducin is strongly phosphorylated in T84 cells. Next, we hypothesized that upon activation by PKC, α-adducin binds to NKCC1. In T84 cells subjected to PMA, we show that phospho α-adducin co-immunoprecipitates with NKCC1. Next, we used Mardin Darby Canine Kidney (MDCK) cells stably expressing eGFP-NKCC1. In this model, using immunocytochemistry, we show that NKCC1 colocalizes with α-adducin at the plasma membrane during PKC activation. Finally, we tested the fate of NKCC1 in the endocytic pathway. In MDCK cells exposed to PMA, we found that NKCC1 colocalizes with LAMP1, a marker of the lysosome. In conclusion, our data suggest that α-adducin participates to NKCC1 internalization during PKC activation and NKCC1 is targeted for degradation