Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss
Peijian He, … , Shanthi Srinivasan, C. Chris Yun
Peijian He, … , Shanthi Srinivasan, C. Chris Yun
Published August 10, 2015
Citation Information: J Clin Invest. 2015;125(9):3519-3531. https://doi.org/10.1172/JCI79552.
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Research Article Endocrinology

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss

Abstract

Diarrhea is one of the troublesome complications of diabetes, and the underlying causes of this problem are complex. Here, we investigated whether altered electrolyte transport contributes to diabetic diarrhea. We found that the expression of Na+/H+ exchanger NHE3 and several scaffold proteins, including NHE3 regulatory factors (NHERFs), inositol trisphosphate (IP3) receptor-binding protein released with IP3 (IRBIT), and ezrin, was decreased in the intestinal brush border membrane (BBM) of mice with streptozotocin-induced diabetes. Treatment of diabetic mice with insulin restored intestinal NHE3 activity and fluid absorption. Molecular analysis revealed that NHE3, NHERF1, IRBIT, and ezrin form macrocomplexes, which are perturbed under diabetic conditions, and insulin administration reconstituted these macrocomplexes and restored NHE3 expression in the BBM. Silencing of NHERF1 or IRBIT prevented NHE3 trafficking to the BBM and insulin-dependent NHE3 activation. IRBIT facilitated the interaction of NHE3 with NHERF1 via protein kinase D2–dependent phosphorylation. Insulin stimulated ezrin phosphorylation, which enhanced the interaction of ezrin with NHERF1, IRBIT, and NHE3. Additionally, oral administration of lysophosphatidic acid (LPA) increased NHE3 activity and fluid absorption in diabetic mice via an insulin-independent pathway. Together, these findings indicate the importance of NHE3 in diabetic diarrhea and suggest LPA administration as a potential therapeutic strategy for management of diabetic diarrhea.

Authors

Peijian He, Luqing Zhao, Lixin Zhu, Edward J. Weinman, Roberto De Giorgio, Michael Koval, Shanthi Srinivasan, C. Chris Yun

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Figure 6

Ezrin assembles with NHERF1, IRBIT, and NHE3 in macromolecular complexes and mediates NHE3 activation by insulin.

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Ezrin assembles with NHERF1, IRBIT, and NHE3 in macromolecular complexes...
(A) Phosphorylation of ezrin at Thr567 (p-Ezrin) was examined in Caco-2bbe/NHE3V/HA-IRBIT cells. Cells were pretreated with or without 20 μM LY294002 or 5 μM Gö6983, followed by insulin treatment for 15 minutes. n = 4. (B) Subcellular localization of NHE3 (green) and ezrin (red) in the apical zone (3 μm) of Caco-2bbe/NHE3V/HA-IRBIT cells. Scale bar: 10 μm. Representative images of 3 independent experiments are shown. (C) The interaction of ezrin with NHE3 and IRBIT was examined in Caco-2bbe/NHE3V/HA-IRBIT cells in the presence of a cross-linker, dithiobis(succinimidyl propionate) (DSP). n = 3. (D) The ezrin-NHERF1 interaction was examined in Caco-2bbe/NHE3V/HA-IRBIT cells expressing CFP-NHERF1. n = 4. (E) The effects of NHERF1 and NHERF2 knockdown on the ezrin-NHE3 interaction were determined in Caco-2bbe/NHE3V/HA-IRBIT cells. n = 4. (F) NHE3 activity (mean ± SEM) was determined in Caco-2bbe/NHE3V/HA-IRBIT cells expressing WT or mutant (T567A) CFP-ezrin. n = 6. *P < 0.05; **P < 0.01, 2-tailed Student’s t test. Error bars indicate mean ± SEM. (G) BBMVs of control and DM mice treated with or without insulin for 30 minutes were resolved by BN-PAGE. Expression of NHE3, IRBIT, NHERF1, and ezrin is shown. Villin was used as a marker of BBM. Arrows indicate the macrocomplexes containing NHE3. Representative blots of 3 independent experiments are shown.