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 3

Activation of NHE3 by insulin requires NHERF1 and IRBIT.

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Activation of NHE3 by insulin requires NHERF1 and IRBIT. (A) WT, Nherf1–...
(A) WT, Nherf1–/–, and Nherf2–/– mice were treated with or without insulin for 30 minutes. NHE3 activity was determined in isolated villi. n = 4 mice for each genotype. **P < 0.01; #P < 0.01 compared with WT without insulin. (B) Expression of NHE3-binding proteins in Caco-2bbe and SK-CO15 cells. β-Actin was used as an internal control. n = 3. (C) NHE3 activity was measured in SK-CO15 cells transfected with control shRNA (shCon) or shRNA for IRBIT (shIRBIT). n = 8. **P < 0.01. (D) NHE3 activity was measured in Caco-2bbe/NHE3V cells stably transfected with an empty vector pCDH or pCDH/HA-IRBIT (HA-IRBIT). n = 8. **P < 0.01. NHE3 activity was determined in (E) SK-CO15 and (F) Caco-2bbe/NHE3V/HA-IRBIT cells transduced with shCon or shNHERF1. n = 8. **P < 0.01. (G) Surface expression of NHE3 was determined in Caco-2bbe/NHE3V/HA-IRBIT cells with NHERF1 knockdown. The amount of surface (S) NHE3 was normalized to total (T) NHE3, and the relative changes are shown. n = 3. *P < 0.05 compared with untreated control. (H) NHE3 surface expression was determined in SK-CO15 cells with IRBIT knockdown. n = 3. *P < 0.05 compared with untreated control. Statistical analysis was performed using 2-tailed Student’s t test. Error bars indicate mean ± SEM.