Subha Arthur

Na-glutamine co-transporters B0AT1 in villus and SN2 in crypts are differentially altered in chronically inflamed rabbit intestine

Glutamine is a major nutrient utilized by the intestinal epithelium and is primarily assimilated via Na-glutamine co-transport (NGcT) on the brush border membrane (BBM) of enterocytes. Recently we reported that B(0)AT1 (SLC6A19) mediates glutamine absorption in villus while SN2 (SLC38A5) does the same in crypt cells. However, how B(0)AT1 and SN2 are affected during intestinal inflammation is unknown. In the present study it was shown that during chronic enteritis NGcT was inhibited in villus cells, however, it was stimulated in crypt cells. Our studies also demonstrated that the mechanism of inhibition of NGcT during chronic enteritis was secondary to a reduction in the number of B(0)AT1 co-transporters in the villus cell BBM without a change in the affinity of the co-transporter. In contrast, stimulation of NGcT in crypt cells was secondary to an increase in the affinity of SN2 for glutamine without an alteration in the number of co-transporters. Thus, glutamine assimilation which occurs via distinct transporters in crypt and villus cells is altered in the chronically inflamed intestine.

Regulation of sodium-glutamine cotransport in villus and crypt cells by glucocorticoids during chronic enteritis

Background: Assimilation of the preferred nutrient of enterocytes is mediated primarily by sodium (Na)‐dependent cotransport (NGct) in the intestine. The predominant NGcT in villus cells, B0AT1, is inhibited secondary to a decrease in cotransporter numbers during chronic intestinal inflammation. In contrast, NGcT mediated by SN2 in crypt cells is stimulated secondary to increased affinity of the cotransporter for glutamine during chronic ileitis. Glucocorticoid is a mainstay of treatment for inflammatory bowel disease. However, its effect on NGcT is not known. Methods: The inhibition of B0AT1 in villus cells during chronic intestinal inflammation was reversed back to normal by methylprednisolone (MP). This was secondary to the restoration of the cotransporter numbers in the brush border membrane rather than an alteration in the affinity. The stimulation of NGcT in crypt cells during chronic ileitis was also restored back to its normal levels by MP treatment. This reversal was secondary to the restoration of the altered affinity of the cotransporter SN2 for glutamine. Results: Kinetic studies and western blot analysis were consistent with functional studies for both cotransporters. Thus, glucocorticoids restore two uniquely altered Na‐glutamine cotransporters, B0AT1 in villus and SN2 in crypt cells during chronic enteritis. Conclusions: These data indicate that glucocorticoids function as an upstream broad spectrum immune modulator in the chronically inflamed intestine. (Inflamm Bowel Dis 2012;)

Unique regulation of Na-glutamine cotransporter SN2/SNAT5 in rabbit intestinal crypt cells during chronic enteritis

The only Na‐nutrient cotransporter described in mammalian small intestinal crypt cells is SN2/SNAT5, which facilitates glutamine uptake. In a rabbit model of chronic intestinal inflammation, SN2 stimulation is secondary to an increase in affinity of the cotransporter for glutamine. However, the immune regulation of SN2 in the crypt cells during chronic intestinal inflammation is unknown. We sought to determine the mechanism of regulation of Na‐nutrient cotransporter SN2 by arachidonic acid metabolites in crypt cells. The small intestines of New Zealand white male rabbits were inflamed via inoculation with Eimeria magna oocytes. After 2‐week incubation, control and inflamed rabbits were subjected to intramuscular injections of arachidonyl trifluoromethyl ketone (ATK), piroxicam and MK886 for 48 hrs. After injections, the rabbits were euthanized and crypt cells from small intestines were harvested and used. Results: Treatment of rabbits with ATK prevented the release of AA and reversed stimulation of SN2. Inhibition of cyclooxygenase (COX) with piroxicam did not affect stimulation of SN2. However, inhibition of lipoxygenase (LOX) with MK886, thus reducing leukotriene formation during chronic enteritis, reversed the stimulation of SN2. Kinetic studies showed that the mechanism of restoration of SN2 by ATK or MK886 was secondary to the restoration of the affinity of the cotransporter for glutamine. For all treatment conditions, Western blot analysis revealed no change in SN2 protein levels. COX inhibition proved ineffective at reversing the stimulation of SN2. Thus, this study provides evidence that SN2 stimulation in crypt cells is mediated by the leukotriene pathway during chronic intestinal inflammation.

Cyclooxygenase pathway mediates the inhibition of Na-glutamine co-transporter B0AT1 in rabbit villus cells during chronic intestinal inflammation

In the mammalian intestine, glutamine assimilation by the absorptive villus cells is mediated by Na-glutamine co-transport, specifically by B0AT1. In a rabbit model of chronic intestinal inflammation, B0AT1 is inhibited secondary to a decrease in the number of co-transporters in the brush border membrane (BBM). This inhibition can be reversed by treatment with a broad-spectrum immune modulator such as glucocorticoid suggesting that immune inflammatory mediators may regulate B0AT1 during chronic intestinal inflammation. Arachidonic acid (AA) metabolites (AAM) are increased during chronic intestinal inflammation. However, whether AAM may regulate B0AT1 during chronic intestinal inflammation is unknown. Treatment of rabbits with ATK, to prevent the release of AAM reversed the inhibition of B0AT1. AAM are products of cyclooxygenase (COX) and/or lipoxygenase (LOX) pathways. Inhibition of COX with piroxicam, therefore reduction of prostaglandin formation in the chronically inflamed intestine, reversed the inhibition of B0AT1 to its normal levels. In contrast, inhibition of LOX with MK886, thus reduction of leukotriene formation during chronic enteritis, did not affect the inhibition of B0AT1. Kinetic studies showed that the mechanism of restoration of B0AT1 by ATK or piroxicam was secondary to the restoration of BBM co-transporter numbers. Western Blot analysis also demonstrated restoration of BBM B0AT1 co-transporter numbers. In conclusion, this study demonstrates that Na-glutamine co-transport mediated by B0AT1 in villus cells is regulated by prostaglandins rather than leukotrienes in the chronically inflamed intestine.

Tu1398 Direct and Specific Inhibition of Constitutive Nitric Oxide Mediated Regulation of Brush Border Membrane NHE3 and SGLT1 in Intestinal Epithelial Cells

Background and aim:diarrhea is a common disease in gastroenterology which is caused by various affects such as intestinal infection, non-infectious inflammation, tumors and so on. The imbalance of fluid/sodium absorption and secretion is considered playing a crucial role in diarrhea. Sodium, as an important electrolyte, involves three mechanisms to transport. Of note, sodium/hydrogen exchanger (NHE) may occupy a crucial position in modulating intestinal water and sodium absorption. Previous study had shown that somatostatin (SST) could stimulate NHE8 expression in physiological intestine. To determine the benefical effect of SST on NHE8 protein expression in colitis mice and its mechanisms, experimental colitis was induced in mice utilizing dextran sulfate sodium (DSS), models of Caco-2 cells intervened by TNF-α were also established to further explore the mechanism of somatostatin modulating NHE8 expression. Methods: To induce diarrhea via intestinal inflammation, mice were fed with 3% Dextran sulfate sodium (DSS) water for seven days. On the eighth day, treatment groups were administrated with octreotide at dose of 50 μg/ kg body weight three times a day for three days. On the eleventh day, mice were euthanized and colonic tissues were collected. Diarrheal symptoms were assessed every other day. Diarrheal score was recorded based on fecal shape, color and hardness. For the TNF-α study, Caco-2 cells were incubated with TNF-α for 18 hours before adding somatostatin. Cells were exposed to somatostatin for 1 hour before harvest. To further explore the mechanism of somatostatin modulating NHE8 expression, Caco-2 cells were incubated with TNF-α for 18 hours. Subsequently, Caco-2 cells pretreated with MAPKK inhibitor (PD98059) were administratedwith SST for 1 hour before harvest.Results: For DSS colitis mice, the expression of somatostatin in colon were decreased in DSS colitis mice compared with the control mice. Moreover, SST could not only ameliorate diarrhea in inflammatory colitis (diarrheal score: 1.7 ± 0.78 vs. 3.6 ± 0.16, n = 3, P<0.05) but also stimulate NHE8 expression in proximal and distal colon compared with DSS colitis mice (proximal colon: 0.61±0.08 vs. 0.31±0.04, n=3, P<0.05; distal colon: 0.74±0.1 vs. 0.36±0.06, n=3, P<0.05). For TNF-α intervened Caco-2 cells, SST could stimulate NHE8 expression compared with TNF-α intervened cells (0.5 ± 0.04 vs. 0.25 ± 0.01; n=3, P<0.05). Furthermore, compared with TNF-α intervened cells, SST could decline the phosphorylation of ERK1/2 (P<0.05). conclusions :In conclusion, the present study suggested that somatostatin could up-regulate the expression of NHE8 protein in both DSS colitis mice and TNF-α intervened Caco-2 cells by blocking the activation of the MAPK-ERK1/2 pathway.

Su1887 Mechanism of Inhibition of Na-Glutamine Co-Transport by Prostaglandin E2 in Intestinal Epithelial Cells

generate PCTV to transport the chylomicron from ER to Golgi. The chylomicron output into lymph is correlated to intestinal luminal phosphatidylcholine (PC). Luminal PC is absorbed as lyso-phosphatidylcholine (lyso-PC). We previously showed that the dietary lipids are absorbed from the apical membrane by Caveolin-1 containing Cytosolic Endocytic Vesicles (CEV). We tested the hypothesis that lyso-PC activates the PKCζ; detach it from CEV, enabling PKCζ to phosphorylate Sar1b. Methods: Cytosol was isolated from rats whose intestinal PC was altered by (A) bile diversion, no PC (B) saline infusion, low PC (C) chow fed, normal PC (D) fat fed, high PC and (E) PC infusion, very high PC. PKCζ activity was measured by phosphorylation of PKCζ pseudo substrate. Lyso-PC was measured by HPLC. Results: PKCζ was activated by lyso-PC, non-linear regression curve for PKCζ vs. lyso-PC, calculated as Km=1.49 ± 0.244 nM and Vmax = 1.12 ± 1.058 nM. The amount of cytosolic lyso-PC in A to E ranges from 0 to 0.45 nM, suggesting that the amount of cytosolic lysoPC is always within a range to control PKCζ activation. PKCζ activity ranges from 0 to 0.7 Arbitrary Unit in A to E. Post absorptive CEV contain PKCζ by western blot but PKCζ detachment from the CEV is proportional to cytosolic lyso-PC as estimated by western blot of CEV. Biotinylation of r-PKCζ showed a conformational change on activation by lyso-PC. We conclude that PKCζ on CEV was activated by lyso-PC, changes its conformation and eluted from vesicles, enter into the cytosol and phosphorylate Sar1b, split the heteroquatramer protein complex to release FABP1. Now free FABP1 can bind to ER membrane for PCTV formation, which transports chylomicrons from ER to Golgi. Fig. 1 illustrated the scheme for control of dietary lipid transport.