Dataset Information

D-glucose acts via sodium/glucose cotransporter 1 to increase NHE3 in mouse jejunal brush border by a Na+/H+ exchange regulatory factor 2-dependent process.

ABSTRACT:

Background & aims

Oral rehydration solutions reduce diarrhea-associated mortality. Stimulated sodium absorption by these solutions is mediated by the Na(+)/H(+) hydrogen exchanger NHE3 and is increased by Na(+)-glucose co-transport in vitro, but the mechanisms of this up-regulated process are only partially understood.

Methods

Intracellular pH was measured in jejunal enterocytes of wild-type mice and mice with disrupted Na+/H+ exchange regulatory co-factor 2 (NHERF2-/- mice) by multiphoton microscopy. Diarrhea was induced by cholera toxin. Caco-2BBe cells that express NHE3 and the sodium/glucose cotransporter 1 (SGLT1) were studied by fluorometry, before and after siRNA-mediated knockdown of NHERF1 or NHERF2. NHE3 distribution was assessed by cell-surface biotinylation and confocal microscopy. Brush-border mobility was determined by fluorescence recovery after photobleaching and confocal microscopy.

Results

The nonmetabolized SGLT1 substrate ?-methyl-D-Glu (?-MD-G) activated jejunal NHE3; this process required Akt and NHERF2. ?-MD-G normalized NHE3 activity after cholera toxin-induced diarrhea. ?-MD-G-stimulated jejunal NHE3 activity was defective in NHERF2-/- mice and cells with NHERF2 knockdown, but occurred normally with NHERF1 knockdown; was associated with increased NHE3 surface expression in Caco-2 cells, which also was NHERF2-dependent; was associated with dissociation of NHE3 from NHERF2 and an increase in the NHE3 mobile fraction from the brush border; and was accompanied by a NHERF2 ezrin-radixin-moesin-binding domain-dependent increase in co-precipitation of ezrin with NHE3.

Conclusions

SGLT1-mediated Na-glucose co-transport stimulates NHE3 activity in vivo by an Akt- and NHERF2-dependent signaling pathway. It is associated with increased brush-border NHE3 and association between ezrin and NHE3. Activation of NHE3 corrects cholera toxin-induced defects in Na absorption and might contribute to the efficacy of oral rehydration solutions.

SUBMITTER: Lin R

PROVIDER: S-EPMC3031713 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Publications

D-glucose acts via sodium/glucose cotransporter 1 to increase NHE3 in mouse jejunal brush border by a Na+/H+ exchange regulatory factor 2-dependent process.

Lin Rong R Murtazina Rakhilya R Cha Boyoung B Chakraborty Molee M Sarker Rafiquel R Chen Tian-E TE Lin Zhihong Z Hogema Boris M BM de Jonge Hugo R HR Seidler Ursula U Turner Jerrold R JR Li Xuhang X Kovbasnjuk Olga O Donowitz Mark M

Gastroenterology 20101023 2

<h4>Background & aims</h4>Oral rehydration solutions reduce diarrhea-associated mortality. Stimulated sodium absorption by these solutions is mediated by the Na(+)/H(+) hydrogen exchanger NHE3 and is increased by Na(+)-glucose co-transport in vitro, but the mechanisms of this up-regulated process are only partially understood.<h4>Methods</h4>Intracellular pH was measured in jejunal enterocytes of wild-type mice and mice with disrupted Na+/H+ exchange regulatory co-factor 2 (NHERF2-/- mice) by mu ...[more]

PMID: 20977906

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Project description:Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

Project description:To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

Dataset Information

D-glucose acts via sodium/glucose cotransporter 1 to increase NHE3 in mouse jejunal brush border by a Na+/H+ exchange regulatory factor 2-dependent process.

Background & aims

Methods

Results

Conclusions

Publications

D-glucose acts via sodium/glucose cotransporter 1 to increase NHE3 in mouse jejunal brush border by a Na+/H+ exchange regulatory factor 2-dependent process.

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