Intestinal Brush Border Membrane Research Articles

194P - Prevalence and Prognostic Significance of Sodium-Dependent Phosphate Transporter 2B (Napi2B) Protein Expression in Non-Small Cell Lung Cancer (Nsclc)

ABSTRACT Aim: NaPi2b belongs to the type II family of sodium-dependent phosphate co-transporters, physiologically expressed in type II pneumocytes of lung and on the brush border membrane of small intestine. Increased expression of NaPi2b was recently described in ovary, thyroid and breast cancer. The aim of this study was to evaluate NaPi2b expression in lung cancer. Methods: Immunohistochemistry using the 10H1 primary antibody (Genentech) was performed on two cohorts of treatment naive resected NSCLC patients collected at MD Anderson, University of Texas, USA (training cohort, N = 415) and University of Oslo, Norway (testing cohort, N = 350). Moreover, 67 lung cancer cell lines were analyzed (51 non-small cell and 16 small cell lung cancer). EGFR and KRAS mutations were evaluated with the SnapShot assay. NaPi2b protein expression was scored using the H-score method (0-300). Expression was defined as high or low according to a H-score cut-off value of 200. Results: Patient characteristics did not differ significantly in the two cohorts. In the training and testing cohorts high levels of NaPi2b were detected in 48.4% and 64% of patients, respectively. Adenocarcinomas (AC) were found to express significantly higher levels of NaPi2b than squamous cell carcinoma (SqCC) (AC vs SqCC median H-score: 248 vs 11, p Conclusions: NaPi2b is a prognostic marker in NSCLC and is strongly associated with important clinicopathological and biological variables such as histology and driver mutation status. Further studies are warranted to clarify the role of NaPi2b in tumor development and progression, as well as its association with driver mutations. Disclosure: O.T. Brustugun received research funding from Roche, Astrazeneca and GlaxoSmithKline; Y. Xiao is a Genentech employee and owns stock in Roche Holdings; Y. Choi is en amployee of Genetech Inc and owns stock in Roche Holdings; Y. Wang is an employee of Genentech Inc and owns stock in Roche Holdings; R. Firestein is an employee of Genentech Inc and owns stock in Roche Holdings; A. Helland received research funding from Roche, AstraZeneca and GlaxoSmithKline; F.R. Hirsch received research funding from Celgene, Genentech, Ventana, Lilly, Imclone and Amgen, and received compensation from Genentech/Roche, Novartis, Pfizer, Brystol-Meyers Squibb, Amgen and Lilly for partecipating in their advisory boards; D. Shames is an employee of Genetech Inc and owns stock in Roche Holdings. All other authors have declared no conflicts of interest.

Effects of lactoferrin on intestinal epithelial cell growth and differentiation: an in vivo and in vitro study.

This study was designed to analyse the effects of human (h) and bovine lactoferrin (bLF) on the growth and differentiation of intestinal cells using the mice model supplemented with Lactoferrin (LF) and the enterocyte-like model of Caco-2 cells which spontaneously differentiate after confluency. In mice, bLF supplementation increased jejunal villus height and the expression of several intestinal brush border membrane enzymes activities. Addition of bLF or hLF to undifferentiated Caco-2 cells was able to increase cell proliferation with confluency being reached more rapidly. Moreover, when Caco-2 cells were grown in the presence of LF for 3weeks, brush-border membrane-associated enzyme activities i.e. sucrase, alkaline phosphatase and neutral aminopeptidase, as well as the L-glutamate transporter expression were all increased indicating an increased Caco-2 cell differentiation. Accordingly, cDNA Atlas array and Western blot analysis of cell cycle proteins shown a decreased expression of Cdck2 and an increased TAF1 expression; these proteins being implicated in the regulation of numerous genes related to cellular proliferation and differentiation. These modifications were associated with an inhibition of Caco-2 cell spontaneous apoptosis. Altogether, our results indicate that LF increase in vivo and in vitro enterocyte differentiation. In addition, LF was found to increase in vitro enterocyte proliferation resulting in higher cell density in cell flasks, an effect that was likely partly due to a reduction of the cellular apoptosis. The different stimulation patterns observed for the different parameters associated with cell differentiation in relationship with specific gene regulation is discussed.

Leukotriene D4 dephosphorylates ASCT1 through Ca2+ dependent PKCα‐Akt pathway in intestinal epithelial cells (1095.11)

Arachidonic acid metabolite derived via Lipoxygenase pathway, leukotriene D4 (LT) inhibits Na+‐dependent alanine cotransport (ASCT1, solute carrier, SLC1A4) in intestinal epithelial cell brush border membrane (BBM) by decreasing the affinity of cotransporter. However, the intracellular mechanism of LT mediated inhibition of ASCT1 is unknown. This study was designed to investigate the intracellular mechanism of ASCT1 inhibition by LT in enterocytes. [3H]‐Ala uptake was measured in 10 days postconfluent rat intestinal epithelial cells (IEC‐6) grown on transwell plates. IEC‐6 cells were treated with different inhibitors intercepting different checkpoints of pathways for LT mediated inhibition of ASCT1 at 8 days postconfluence. LT treatment decreased ASCT1 activity and increased more than 2.5‐fold intracellular cAMP. However, protein kinase A (PKA)inhibitor did not reverse the LT mediated inhibition of ASCT1. LT increased 2‐fold cytosolic Ca2+ and PKC‐α antagonized LT effect on ASCT1 activity. In contrast, PKC‐δ and ‐θ inhibitor did not reverse LT mediated inhibition of ASCT1. Further downstream of PKC‐α pathway, tyrosine kinase (Akt) inhibitor also reversed LT mediated inhibition of ASCT1 activity. Kinetics, western blotting, and qRT‐PCR studies demonstrated that the mechanism of reversal of LT mediated inhibition of ASCT1 by Akt inhibitor was due to the restoration of affinity of the cotransporter. Immunocytochemical and dephosphorylation studies revealed that ASCT1 was dephosphorylated by LT treatment. Therefore, we conclude that LT inhibits ASCT1 activity by dephosphorylation through Ca2+‐dependent PKCα‐Akt pathway in enterocytes.Grant Funding Source: NSF

Improvement of Intestinal Absorption of Poorly Absorbable Drugs by Various Sugar Esters

Effects of sucrose fatty acid esters (sugar esters) on the intestinal absorption of poorly absorbable drugs were examined by an in situ closed loop method in rats. 5(6)-Carboxyfluorescein (CF) and fluorescein isothiocyanate-dextrans (FDs) with various molecular weights were used as model drugs of poorly absorbable drugs. The absorption of CF from the rat small intestine was significantly enhanced in the presence of various sugar esters and a maximal absorption enhancing effect was observed in the presence of 0.5%(w/v) S-1670. The absorption enhancing effect of S-1670 in the small intestine decreased as the molecular weights of drugs increased. Moreover, we evaluated the intestinal membrane damage with or without various sugar esters. These sugar esters (0.5%(w/v)) did not increase the activities of lactate dehydrogenase (LDH), suggesting that these sugar esters did not cause serious membrane damage to the intestinal epithelium. Furthermore, these sugar esters increased membrane fluidity of lipid layers of the intestinal brush border membranes and decreased the transepithelial electrical resistance (TEER) of Caco-2 cells. Therefore, these findings suggested that these sugar esters might improve the intestinal absorption of poorly absorbable drugs via a transcellular and a paracellular pathways.

Hepatectomy-Related Hypophosphatemia

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na(+)-dependent phosphate (Pi) uptake decreased by 50%-60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na(+)-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.

Ontogeny of the digestive enzyme activities in hatchery produced Beluga (Huso huso)

The development of digestive enzymes of the stomach (pepsin), pancreas (trypsin, chymotrypsin, amylase, and lipase) and intestine (alkaline phosphatase) were studied in Great sturgeon or Beluga (Huso huso) from hatching time to 50th day post hatching. Sampling was carried out immediately after hatching, 111.3, 235.2, 306, 400.2, 490, 612.5, 756 and 906.5 degree days post hatching, ddph (0, 7, 14, 18, 23, 28, 35, 42 and 50days post hatching, dph, respectively). Beluga larvae had near 70% mortality during rearing procedure. The highest mortality rate was accrued in two stages; firstly, during endogenous feeding (2–9dph; 30.4–144.9ddph) and then from a mixed feeding stage (12dph; 253.5ddph) until the starting time of weaning (28dph; 490ddph), with values ranging from 50 to 40% of total mortality, respectively. Furthermore, the results showed that at the onset of exogenous feeding, gastric glands were already functional as indicated by the steady increase in pepsin specific activity. In contrast, trypsin and chymotrypsin significantly decreased after the onset of exogenous feeding, showing the importance of these types of enzymes in the cleavage of yolk proteins during the endogenous feeding phase. Moreover, amylolytic activity fluctuated considerably over the study period; after onset of exogenous feeding alpha-amylase activity increased (P<0.05), whereas it reached a maximum value at the end of the study (P<0.05). Lipolytic activity was low in the first week (P<0.05), whereas it increased until the onset of exogenous feeding and then, until 490ddph (28dph) went down again and reached the same amount in the first week. At the end of the study, lipolytic activity reached 4 times more relative to the activity levels recorded during the first 4weeks of exogenous feeding. Furthermore, changes in enzyme activities from the stomach and pancreas were coupled with those in the intestine (brush border membrane), whereas the increase in alkaline phosphatase from hatching until 111.3ddph (7dph) and reaching a maximum value between 111.3ddph and onset of exogenous feeding at 235.2ddph (14dph), suggested that the Beluga larvae had a developed intestine at the onset of exogenous feeding. The results obtained from this study proposed that, more Beluga weaned earlier, more larvae will live and hence weaning should begin immediately after commencement of exogenous feeding.

Characterization of Monocarboxylate Transporter 6: Expression in Human Intestine and Transport of the Antidiabetic Drug Nateglinide

Monocarboxylate transporter (MCT) 6, encoded by SLC16A5, is a member of the monocarboxylate transporter family. Nateglinide, an oral hypoglycemic agent, quickly reaches the maximal serum concentration after its premeal administration. Although the functional existence of uptake systems for nateglinide in the intestine has been demonstrated, these transport systems have not yet been identified at the molecular level. The aim of this study was to demonstrate the localization of MCT6 in the human small intestine and characterize the transport properties of nateglinide via MCT6. Immunohistochemical analysis of the human small intestine revealed that anti-MCT6 antiserum stained the luminal side of the epithelial cells. When expressed in Xenopus laevis oocytes, MCT6-mediated uptake of [(14)C]nateglinide was sensitive to extracellular pH and membrane potential. Furthermore, the K(t) value of nateglinide (45.9 μM) for MCT6 was lower than those previously reported in Caco-2 cells and rat intestinal brush-border membrane vesicles. In addition, probenecid, fluorescein, valproic acid, and salicylic acid, which are inhibitors of nateglinide uptake in Caco-2 cells and rat intestine, did not inhibit the uptake of nateglinide via MCT6. These results suggest that MCT6 may play a role in the intestinal absorption of nateglinide, although other transporters are also likely involved.

CGMP-dependent kinase 2, Na+/H+ regulatory factor 2, and Na+/H+ exchanger isoform 3 assemble within lipid rafts in murine small intestinal brush border membrane

Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of the very homologous NHERFs. We studied the association of NHE3, as well as NHERF1 (EBP50), NHERF2 (E3KARP) and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM and their possible association with signaling molecules. NHE3 was found to partially associate with glycosphingolipid-enriched microdomains in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity as well as on second-messenger-dependent regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. A search for other signalling molecules that are implicated in regulating NHE3 through a NHERF interaction, cGMP-dependent kinase II, which together with NHERF2 is essential for guanylin/heat stable enterotoxin of E.coli (STa)-mediated NHE3 inhibition in the intestine, was found exclusively lipid-raft associated. In conclusion, the differential association of the NHERFs, as well as kinases, with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is likely one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

Chronic alcohol feeding inhibits physiological and molecular parameters of intestinal and renal riboflavin transport

Vitamin B2 (riboflavin, RF) is essential for normal human health. Mammals obtain RF from exogenous sources via intestinal absorption and prevent its urinary loss by reabsorption in the kidneys. Both of these absorptive events are carrier-mediated and involve specific RF transporters (RFVTs). Chronic alcohol consumption in humans is associated with a high prevalence of RF deficiency and suboptimal levels, but little is known about the effect of chronic alcohol exposure on physiological and molecular parameters of the intestinal and renal RF transport events. We addressed these issues using rats chronically fed an alcohol liquid diet and pair-fed controls as a model. The results showed that chronic alcohol feeding significantly inhibits carrier-mediated RF transport across the intestinal brush-border and basolateral membrane domains of the polarized enterocytes. This inhibition was associated with a parallel reduction in the expression of the rat RFVT-1 and -3 at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Chronic alcohol feeding also caused a significant inhibition in RF uptake in the colon. Similarly, a significant inhibition in carrier-mediated RF transport across the renal brush-border and basolateral membrane domains was observed, which again was associated with a significant reduction in the level of expression of RFVT-1 and -3 at the protein, mRNA, and hnRNA levels. These findings demonstrate that chronic alcohol exposure impairs both intestinal absorption and renal reabsorption processes of RF and that these effects are, at least in part, mediated via transcriptional mechanism(s) involving the slc52a1 and slc52a3 genes.

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT.

Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.

CFTR and V‐ATPase trafficking and function in the intestinal brush border membrane

The multi‐subunit proton pump, vacuolar ATPase (V‐ATPase) and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are both regulated by cAMP‐dependent traffic from endosomes to the plasma membrane in ion transporting epithelia. We hypothesized that V‐ATPase traffic and function are linked to CFTR in the intestine. The distribution of V‐ATPase Voa1, Voa2 and V1E subunits and CFTR were examined in wild‐type (WT), CFTR−/− mouse intestine and polarized intestinal CaCo‐2 BBe cells following cAMP stimulation. Functional dependence of V‐ATPase on CFTR was determined in CaCo‐2 BBe cells using the pH sensitive dye BCECF. Co‐immunoprecipitation confirmed Voa1, Voa2 and V1E interaction with CFTR. V‐ATPase Voa1 and Voa2 subunits were localized to the apical and basolateral domains in CaCo‐2 BBe cells. V‐ATPase Voa1 and Voa2 subunits were localized to distinct domains in untreated and treated enterocytes from WT and CFTR−/−intestine. Following cAMP activation, both transporters co‐localized in the apical membrane. Inhibition of V‐ATPase or CFTR significantly decreased proton efflux in CaCo2 BBe cells. These studies suggest functional synergy between both transporters in maintaining intracellular pH; and localization in the intestinal brush border membrane. NIH Grants: R01‐DK‐077065 to N. Ameen and P30‐DK‐34989 to the Yale Liver Center.

Role of Myosin1a in regulated exocytosis of CFTR in villus enterocytes

Insertion of CFTR channels into the intestinal brush border membrane (BBM) is necessary for anion secretion, but the mechanisms of CFTR delivery into the BBM are poorly understood. BB myosins move cargo and regulate membrane trafficking. Myosin1a (Myo1a) regulates CFTR BBM localization and function in villus enterocytes. cAMP and cGMP regulate CFTR traffic into the enterocyte BBM from subapical endosomes. We hypothesized that Myo1a regulates cAMP/cGMP traffic of CFTR into the BBM. Myo1a KO and control mouse intestine were treated with cAMP or cGMP agonists. CFTR distribution was examined by confocal microscopy and surface biotinylation. Immunolocalization revealed absence of BBM CFTR and its accumulation in subapical endosomes in Myo1aKO villus enterocytes. Surface biotinylation confirmed no change in surface CFTR in stimulated Myo1aKO tissues. Specificity of Myo1a was confirmed by lack of changes in BBM localization of ALP, SLC26A6 (PAT‐1), and NHE3. Surface biotinylation of cAMP‐stimulated polarized Caco2BB‐e cells lacking Myo1a expression (shRNA silencing) confirmed CFTR specificity. Surface CFTR was reduced by 98% in Myo1a knockdown (KD) cells compared to control, while surface PAT1 was unchanged in control and Myo1a KD cells. These data support Myo1a as the motor for regulated exocytosis of CFTR. Future studies will seek to dissect the molecular mechanisms regulating Myo1a‐dependent CFTR traffic.

Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na⁺/H⁺ exchanger NHE3 is regulated by the Na⁺/H⁺ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO₂/HCO₃^- mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.

Seasonal plasticity of gut morphology and small intestinal enzymes in free-living Mongolian gerbils

The phenotypic plasticity of the digestive system may determine the diversity of animal diets and, thus, their niche width. This study examines the effects of seasonal fluctuations in food quality and temperature on the gut morphology and the activity of sucrase, maltase, and aminopeptidase-N in the small intestinal brush-border membrane of male Mongolian gerbils (Meriones unguiculatus). Based on the adaptive modulation hypothesis and the principle of optimal gut function design, we hypothesize that the gut size, tissue-specific activity, and total hydrolytic capacity of intestinal digestive enzyme are upregulated in winter and downregulated in summer in response to diet shifts and energy demand in free-living Mongolian gerbils. Various seasonal modulation patterns in digestive enzyme activity in different regions of the small intestines were observed. The results show that male gerbils have the longest and heaviest small intestines in winter. This mechanism may be adapted to increase their food intake during winter. Male gerbils also exhibit the highest tissue-specific and total sucrase, maltase, and aminopeptidase-N activity in winter and in spring. Seasonal modulations are more distinct in the jejunum than in the duodenum and the ileum of the small intestines. The digestive phenotypic flexibility of male gerbils effectively corresponded with seasonal diet shifts and temperature fluctuations.

Inhibition of Maltase‐Glucoamylase Activity to Hydrolyze α‐1,4 Linkages by the Presence of Undigested Sucrose

Inhibition of Maltase‐Glucoamylase Activity to Hydrolyze α‐1,4 Linkages by the Presence of Undigested Sucrose

Congenital Sucrase‐Isomaltase Deficiency

Congenital Sucrase‐Isomaltase Deficiency

Comparison of high-protein soybean meal and commercial soybean meal partly replacing fish meal on the activities of digestive enzymes and aminotransferases in juvenile Japanese seabass,Lateolabrax japonicus(Cuvier, 1828)

This study was conducted to compare the effects of fish meal (FM) replacement by two types of soybean meals (SBMs) on activities of digestive enzyme, intestinal brush border enzyme and aminotransferases of juvenile Japanese seabass (initial weight 8.3 ± 0.2 g). Nine isonitrogenous (crude protein 44%) and isoenergetic (20 kJ g−1) experimental diets replacing 0 (the control), 15%, 30%, 45% and 60% FM protein by high-protein SBM (HSB) or commercial SBM (CSB) were formulated. The concentrations of anti-nutritional factors (ANFs) in HSB were relatively lower compared with CSB (P < 0.05). After 10-week feeding experiment, activities of digestive enzymes, intestinal brush border enzymes and aminotransferases in liver decreased with increasing dietary SBM. When the substitution level was above 30% with CSB and 60% with HSB, activities of pepsin, disaccharidase in the intestinal brush border membrane and aspartate aminotransferase (AST) in liver were significantly lower than the control. However, activities of alanine aminotransferase (ALT) and AST in serum were significantly higher than the control when the substitution level was above 30% with CSB and 60% with HSB. Regression analysis showed that effects of FM substitution by HSB on digestion and metabolism of juvenile seabass were relatively smaller compared with CSB.

Intestinal peptidases form functional complexes with the neutral amino acid transporter B(0)AT1.

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

Modulation of intestinal glucose transport in response to reduced nitrogen supply in young goats1

The reduction of dietary protein is a common approach in ruminants to decrease the excretion of N because ruminants are able to recycle N efficiently by the rumino-hepatic circulation. In nonruminant species an impact on other metabolic pathways such as glucose metabolism was observed when dietary protein intake was reduced. However, an impact of dietary N reduction in goats on glucose metabolism especially on intestinal glucose absorption is questionable because ruminants have very efficient endogenous recycling mechanisms. Therefore, the aim of the present study was to characterize the intestinal absorption of glucose in growing goats kept on different N supply under isoenergetic conditions. The different CP concentrations (20, 16, 10, 9, and 7% CP) of the experimental diets were adjusted by adding urea to the rations. Intestinal flux rates of glucose were determined by Ussing chamber experiments. For a more mechanistic approach, the Na(+)-dependent uptake of glucose into intestinal brush-border membrane vesicles (BBMV) and the expression patterns of the Na(+)-dependent glucose transporter SGLT1 and the glucose transporter 2 (GLUT2) were determined. Reduced N intake resulted in a decrease of plasma glucose (P < 0.001) and insulin (P = 0.004) concentrations whereas the intestinal flux rates of glucose were elevated (P < 0.001), which were inhibited by phlorizin. However, the uptake of glucose into intestinal BBMV was not changed whereas the expression of SGLT1 on mRNA (P < 0.05) and protein abundance (P = 0.03) was decreased in response to a reduced N intake. The mRNA expression of GLUT2 was not affected. From these data, it can be concluded that the intestinal absorption of glucose was modulated by changes in dietary N intake. It is suggested that intracellular metabolism or basolateral transport systems or both might be activated during this feeding regimen because the apical located SGLT1 might not be involved. Therefore, an impact of dietary N reduction on glucose metabolism in growing goats occurred as in monogastric animals.

A Na+-phosphate cotransporter homologue (SLC17A4 protein) is an intestinal organic anion exporter

The SLC17 anion transporter family comprises nine members that transport various organic anions in membrane potential (Δψ)- and Cl(-)-dependent manners. Although the transport substrates and physiological relevance of the majority of the members have already been determined, little is known about SLC17A4 proteins known to be Na(+)-phosphate cotransporter homologue (NPT homologue). In the present study, we investigated the expression and transport properties of human SLC17A4 protein. Using specific antibodies, we found that a human NPT homologue is specifically expressed and present in the intestinal brush border membrane. Proteoliposomes containing the purified protein took up radiolabeled p-aminohippuric acid (PAH) in a Cl(-)-dependent manner at the expense of an electrochemical gradient of protons, especially Δψ, across the membrane. The Δψ- and Cl(-)-dependent PAH uptake was inhibited by diisothiocyanostilbene-2,2'-disulfonic acid and Evans blue, common inhibitors of SLC17 family members. cis-Inhibition studies revealed that various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs, pravastatin, and urate inhibited the PAH uptake. Proteoliposomes took up radiolabeled urate, with the uptake having properties similar to those of PAH uptake. These results strongly suggested that the human NPT homologue acts as a polyspecific organic anion exporter in the intestines. Since SLC17A1 protein (NPT1) and SLC17A3 protein (NPT4) are responsible for renal urate extrusion, our results reveal the possible involvement of a NPT homologue in urate extrusion from the intestinal duct.