Biology subjects

Lin, R.-X.

Publications and source records attributed to Lin, R.-X..

5 recordsLinked to original sources

bioRxiv2025

Differential Regulation of NHE3 Expression in Type 1 and Type 2 Diabetic Intestine: Impaired Endosomal Regulation of NHE3 Expression in Type 1 Diabetes

Chronic diarrhea is a frequent gastrointestinal complication in both type 1 (T1D) and type 2 diabetes (T2D), though the underlying mechanisms differ: T1D is linked to autonomic neuropathy and disrupted transporter regulation, while T2D is often linked to medications and intestinal inflammation. Using streptozotocin-induced mouse models of T1D and T2D, we observed increased luminal fluid in the small intestine of both. Given the role of Na+/H+ exchanger 3 (NHE3) in fluid absorption and its loss in most diarrheal diseases, we examined NHE3 expression across intestinal segments. In T1D, NHE3 protein was significantly reduced in the duodenum and jejunum without changes in mRNA, suggesting post-transcriptional regulation. In contrast, T2D mice exhibited reduced NHE3 protein and mRNA, restricted to the proximal colon. To investigate mechanisms underlying NHE3 loss in T1D, we evaluated endosomal scaffolding proteins involved in NHE3 trafficking. While our previous work showed that the Sorting Nexin-27 (SNX27)-retromer complex does not regulate NHE3 protein stability, we found that SNX17 was significantly decreased in the small intestine of T1D mice but unchanged in T2D. SNX17 knockdown in SK-CO15 cells reduced NHE3 activity and stability. A GST pull-down assay showed that SNX17 interacts with the C-terminus of NHE3. Mutation of the NHE3 distal NPxY motif disrupted this interaction, leading to reduced NHE3 expression and increased degradation. These findings reveal segment-specific and mechanistically distinct causes of diabetic diarrhea in T1D versus T2D, and identify SNX17 loss as a contributor to reduced NHE3 stability and activity in T1D, likely promoting diabetic diarrhea. New and NoteworthyThis study identifies distinct mechanisms of impaired sodium absorption contributing to diabetic diarrhea in type-1 and type-2 diabetes. We identify SNX17 as a novel regulator of NHE3 in the small intestine, showing that SNX17 loss in T1D contributes to post-translational NHE3 destabilization. In contrast, T2D-associated NHE3 downregulation is transcriptional and confined to the colon. These findings reveal disease- and region-specific regulatory pathways that drive impaired fluid absorption in diabetes, with direct implications for the development of targeted therapies. Graphical abstract Schematic overview of differential NHE3 regulation in diabetic diarrhea.In Type 1 Diabetes (T1D), reduced SNX17 expression in the small intestine causes increased degradation of NHE3 and impaired sodium absorption. In Type 2 Diabetes (T2D), SNX17 levels remain unchanged in the small intestine, but NHE3 expression is transcriptionally downregulated in the proximal colon. These distinct mechanisms contribute to segment-specific differential regulation of NHE3 in T1 and T2D diabetes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/675364v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@eabf0aorg.highwire.dtl.DTLVardef@1a46891org.highwire.dtl.DTLVardef@cd2d5corg.highwire.dtl.DTLVardef@5d185b_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Partially differentiated enterocytes in ileal and distal-colonic human F508del-CF-enteroids 1 secrete fluid in response to forskolin and linaclotide.

Constipation causes significant morbidity in Cystic Fibrosis (CF) patients. Using CF patient (F508del) derived ex vivo ileal and distal colonic/rectal enteroids as a model and the Forskolin Induced Swelling Assay (FIS), we compared CFTR mediated fluid secretion in human enterocytes across the crypt-villus axis. CFTR expression and FIS decreased as enterocytes differentiated from crypt to become partially differentiated and then mature villus cells. While there was no FIS response in undifferentiated (crypt enterocytes) F508del-CF enteroids, partially differentiated F508del-CF enteroids had a swelling response to forskolin (cAMP) and linaclotide (cGMP) which was [~]48%, and [~]67% of the response in healthy enteroids, respectively and was prevented by a CFTR inhibitor. Also, linaclotide and a general PDE inhibitor independently enhanced combined CFTR-modulator-induced FIS response from partially differentiated F508del-CF enteroids. These findings demonstrate that partially differentiated ileal and distal colonic F508del-CFTR enteroids can be stimulated to secrete fluid by cAMP and cGMP.

physiology↗

bioRxiv2022

SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+ dependent manner by cAMP AND ATP in intestinal epithelial cells

In polarized intestinal epithelial cells, DRA is a brush border (BB) Cl-/HCO3- exchanger that is part of neutral NaCl absorption under baseline conditions but in cAMP driven diarrheas it is stimulated and contributes to increased anion secretion. To further understand regulation of DRA in conditions mimicking some diarrheal diseases, differentiated Caco-2/BBE cells were exposed to forskolin and ATP. Forskolin and ATP both acutely stimulated DRA in a concentration dependent manner. Forskolin at 1{micro}M and ATP at 0.25 {micro}M had minimal to no effect on DRA activity given individually; however, together, they stimulated DRA activity to levels seen with maximum concentrations of forskolin and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP alone increased Ca2+ in a concentration dependent manner, while forskolin (1 {micro}M), that by itself did not significantly alter Ca2+, followed by 0.25 {micro}M ATP produced a large increase in Ca2+ that was [~]equal to the elevation caused by 1 {micro}M ATP. BAPTA-AM pretreatment prevented the ATP and forskolin/ATP synergistic increased DRA activity and the increase in intracellular Ca2+ caused by ATP/forskolin. Conclusion: In Caco-2/BBE cells subthreshold concentrations of forskolin (cAMP) and ATP (Ca2+) synergistically increased intracellular Ca2+ and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, while separation of DRA from NHE3 contributes to reduced NaCl absorption.

physiology↗

bioRxiv2021

CHRONIC INFLAMMATION IN ULCERATIVE COLITIS CAUSES LONG TERM CHANGES IN GOBLETCELL FUNCTION

ObjectiveOne of the features of ulcerative colitis (UC) is a defect in the protective mucus layer. This has been attributed to a reduced number of goblet cells (GC). However, it is not known whether abnormal GC mucus secretion also contributes to the reduced mucus layer. Our aims were to test the hypothesis that GC secretion was abnormal in UC with the changes persistent in colonoids even in the absence of immune cells. DesignColonoids were established from intestinal stem cells of healthy subjects (HS) and from patients with UC (inactive and active sites). Colonoids were maintained as undifferentiated (UD) or induced to differentiate (DF) and studied as 3D or monolayers on Transwell filters. Total RNA was extracted for quantitative real-time PCR analysis. Carbachol and PGE2 mediated stimulation followed by examination of mucus layer by MUC2 IF/confocal microscopy and TEM were performed. ResultsColonoids derived from patients with UC can be propagated over many passages; however, they exhibit a reduced rate of growth and TEER compared with colonoids from HS. Differentiated UC colonoid monolayers form a thin and non-continuous mucus layer. UC colonoids have increased expression of secretory lineage markers: ATOH1 and SPDEF, including MUC2 positive GCs and ChgA positive enteroendocrine cells but failed to secrete mucin when exposed to the cholinergic agonist carbachol and PGE2, which caused increased secretion in HS. Exposure to TNF- (5days), reduced the number of GC with a greater percentage decrease in UC colonoids compared to HS. ConclusionsAbnormal mucus layer in UC is due to long term changes in epithelial cells that lead to abnormal mucus secretion as well as effects of the inflammatory environment to reduce the number of GC. This continued defect in GC mucus secretion may be involved in UC recurrence.

physiology↗

bioRxiv2020

Chronic exposure of ErbB TKIs inhibits DRA expression and activity through an ERK/Elk-1/CREB/AP-1 dependent pathway

Diarrhea is the major side effect of first- and second-generation ErbB tyrosine kinase inhibitors (TKI), the mechanism of which remains incompletely understood. The current studies were carried out over the time frame that ErbB TKIs usually initiate diarrhea. We report in Caco-2/bbe cells that exposure of ErbB TKIs, but not non-ErbB TKIs for six days at clinically-relevant concentrations significantly reduced the expression of DRA and inhibited apical Cl-/HCO3-exchange activity. The ErbB TKIs decreased DRA expression through an ERK/Elk-1/CREB/AP-1 dependent pathway. The blockade of ERK phosphorylation by ErbB TKIs decreased the phosphorylation of Elk-1 and the amount of total and p-CREB, and reduced the expression of C-Fos, which is part of the AP-1 complex that maintain DRA expression. Altogether, our studies demonstrate that ErbB TKIs decrease expression and activity of DRA, which occurs over the time frame that these drugs clinically cause diarrhea, and since DRA is part of the intestinal neutral NaCl absorptive process, the reduced absorption is likely to represent a major contributor to the ErbB TKI-associated diarrhea.

pharmacology and toxicology↗