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 5

PKD2 mediates IRBIT phosphorylation and NHE3 activation.

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PKD2 mediates IRBIT phosphorylation and NHE3 activation. (A) Caco-2bbe/N...
(A) Caco-2bbe/NHE3V cells expressing WT or S68A mutant HA-IRBIT were treated with or without insulin for 15 minutes. HA-IRBIT was immunoprecipitated with anti-HA antibody, and phosphorylation of IRBIT was determined using anti–phospho-Ser antibody. Representative blots of 3 independent experiments are shown. (B) Caco-2bbe cells were pretreated with 20 μM LY294002 (LY) or 5 μM Gӧ6983 (6983). Phosphorylation of PKD2 (p-PKD2) was examined in response to insulin treatment for 15 minutes. n = 3. (C) Cellular localization of p-PKD2 (green) and IRBIT (red) in Caco-2bbe/NHE3V/HA-IRBIT cells was analyzed by confocal microscopy. F-actin (blue) was used as a marker of the apical compartment of the cells. Scale bar: 10 μm. n = 3. (D) Caco-2bbe/NHE3V/HA-IRBIT cells were pretreated with 20 μM LY294002 or 2 μM Gӧ6976. HA-IRBIT was immunoprecipitated, and phosphorylation of IRBIT was determined. The amount of NHE3 coimmunoprecipitated was determined with anti-VSVG antibody. n = 3. (E) Phosphorylation of IRBIT in response to insulin was determined in PKD2 knockdown (shPKD2) and control cells. Expression of PKD2 and β-tubulin in cell lysates is shown. n = 3. (F) The IRBIT-NHE3 interaction was examined in Caco-2bbe/NHE3V/HA-IRBIT cells transfected with shPKD2 or shCon. n = 3. (G) NHE3 activity was determined in Caco-2bbe/NHE3V/HA-IRBIT cells with stable knockdown of PKD2. n = 6. *P < 0.05, 2-tailed Student’s t test. Error bars indicate mean ± SEM.