Intestinal Brush Border Research Articles

Galectin-3 Regulates Desmoglein-2 and Intestinal Epithelial Intercellular Adhesion

The desmosomal cadherins, desmogleins, and desmocollins mediate strong intercellular adhesion. Human intestinal epithelial cells express the desmoglein-2 isoform. A proteomic screen for Dsg2-associated proteins in intestinal epithelial cells identified a lectin referred to as galectin-3 (Gal3). Gal3 bound to N-linked β-galactosides in Dsg2 extracellular domain and co-sedimented with caveolin-1 in lipid rafts. Down-regulation of Gal3 protein or incubation with lactose, a galactose-containing disaccharide that competitively inhibits galectin binding to Dsg2, decreased intercellular adhesion in intestinal epithelial cells. In the absence of functional Gal3, Dsg2 protein was internalized from the plasma membrane and degraded in the proteasome. These results report a novel role of Gal3 in stabilizing a desmosomal cadherin and intercellular adhesion in intestinal epithelial cells.

Bilayer Tablet Formulation of Metformin HCl and Acarbose: A Novel Approach To Control Diabetes

The present investigation studied a novel bilayer tablet having an extended release system of metformin HCl with Eudragit RS 100 and RL 100 and an immediate release system of acarbose with polyvinylpyrrolidone K30 (PVP K30) and polyethylene glycol 6000 (PEG 6000) in different ratios using solvent evaporation and cogrinding techniques. Solid dispersions (SDs) were characterized by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), powder x-ray diffractometry (XRD), scanning electron microscopy (SEM), as well as by content uniformity, in vitro dissolution studies, and release kinetics. The selected SD system was subjected to bilayer tablet preparation by direct compression. Compressed tablets were evaluated for drug content, weight variation, friability, hardness, and thickness, and they underwent in vitro dissolution studies. The progressive disappearance of IR, x-ray, and thermotropic drug signals in SDs and physical mixtures were related to increasing amount of polymer. SEM studies suggested the homogenous dispersion of drug in polymers. FT-IR studies confirmed the formation of hydrogen bonding between drug and polymer. All tablet formulations showed compliance with pharmacopoeial standards. The formulations gave an initial burst effect to provide the loading dose of the drug followed by extended release for 12 h (Higuchi model via a non-Fickian diffusion controlled release mechanism). Stability studies conducted for the optimized formulation did not show any change in physical properties, drug content, or in vitro drug release. The goal of diabetes therapy today is to achieve and maintain as near normal glycemia as possible to prevent the long-term microvascular and macrovascular complications of elevated blood glucose levels. Oral therapeutic options for the treatment of type 2 diabetes mellitus, until recently, have been severely limited. Metformin, a biguanide, targets additional mechanisms of hyperglycemia by inhibiting hepatic glucose production and enhancing peripheral glucose uptake and thereby reducing insulin resistance; acarbose reversibly bind to pancreatic alpha-amylase and membrane-bound intestinal alpha-glucoside hydrolases. These enzymes inhibit hydrolysis of complex starches to oligosaccharides in the lumen of the small intestine and hydrolysis of oligosaccharides, trisaccharides, and disaccharides to glucose and other monosaccharides in the brush border of the small intestine. The two agents were found to have a remarkable effect on glycemic control. In the present investigation a bilayer tablet was prepared in which one layer gives instant action against diabetes and another layer maintain concentration of drug in plasma for longer periods.

The effect of intestinal alkaline phosphatase on intestinal epithelial cells, macrophages and chronic colitis in mice

The effect of intestinal alkaline phosphatase on intestinal epithelial cells, macrophages and chronic colitis in mice

Organogenesis of digestive system, visual system and other structures in Atlantic bluefin tuna (Thunnus thynnus) larvae reared with copepods in mesocosm system

Organogenesis of digestive system, visual system and other structures in Atlantic bluefin tuna (Thunnus thynnus) larvae reared with copepods in mesocosm system

Congenital glucose–galactose malabsorption: A descriptive study of clinical characteristics and outcome from Western Saudi Arabia

Congenital glucose–galactose malabsorption: A descriptive study of clinical characteristics and outcome from Western Saudi Arabia

Exacerbation of intestinal brush border enzyme activities and oxidative stress in streptozotocin-induced diabetic rats by monocrotophos

Exacerbation of intestinal brush border enzyme activities and oxidative stress in streptozotocin-induced diabetic rats by monocrotophos

Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells.

The intestinal brush border Na(+)/H(+) exchanger NHE3 is tightly regulated through changes in its endocytosis and exocytosis. Myosin VI, a minus-end-directed actin motor, has been implicated in endocytosis at the inter-microvillar cleft and during vesicle remodeling in the terminal web. Here, we asked whether myosin VI also regulates NHE3 movement down the microvillus. The basal NHE3 activity and its surface amount, determined by fluorometry of the ratiometric pH indicator BCECF and biotinylation assays, respectively, were increased in myosin-VI-knockdown (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immunoelectron microscopy results showed that NHE3 was preferentially localized in the basal half of control microvilli but in the distal half in myosin VI KD cells. Treatment with dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis. However, NHE3 had an intermediate distribution along the microvillus (between that in myosin VI KD and untreated cells) in dynasore-treated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells, and suggest that myosin VI also moves NHE3 down the microvillus.

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.

Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea)

The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine-alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine-alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period.

Generation of Stable Lipid Raft Microdomains in the Enterocyte Brush Border by Selective Endocytic Removal of Non-Raft Membrane

The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB) was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.

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

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

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.

Calcium homeostasis, and clinical or subclinical vitamin D deficiency – Can a hypothesis of “intestinal calcistat” explain it all?

Calcium homeostasis, and clinical or subclinical vitamin D deficiency – Can a hypothesis of “intestinal calcistat” explain it all?

Abnormally short serum half-lives of chimeric and human IgGs in NOD-SCID mice (P4184)

Abstract The immunodeficient mice are the standard animal model in antibody research, which include the inbred NOD-SCID strain. We report here the aberrant pharmacokinetics of chimeric and human IgGs with a human IgG1 Fc domain in NOD-SCID mice. These IgGs displayed very short in vivo half-lives (25.0±0.4 hrs and 27.6±1.3 hrs, respectively) when injected at a dose of 10 μg. At a dose of 500 μg the half-lives were extended (131.1±3.1 hrs and 220.3±14.3 hrs, respectively), but still shorter than those observed in other strains of mice. The half-lives of these IgGs in NOD MrkTac, BALB/c, Swiss Webster and another immunodeficient strain of mice, C.B-17 SC-R, were similar to previous reports (300-400 hrs). In contrast, no differences in half-lives were observed for murine, rat, bovine and goat IgGs in any mouse strain studied here. RT-PCR for FcRn cDNA showed no difference in FcRn gene sequence and the binding affinity of chimeric and human IgGs to the FcRn-positive intestinal brush border of suckling mice was similar for all mice. However, chimeric and human IgGs showed higher in vitro binding affinities for Fc gamma receptors on macrophages from NOD-SCID mice vs. other strains of mice studied, suggesting that at low doses, these antibodies might bind preferentially to Fc gamma receptors-expressing macrophages, impairing their pharmacokinetics. Taken together, the NOD-SCID mice and their derivatives may not be suitable models for studying the in vivo activity of chimeric and human IgGs.

Tu1620 Intestinal Alkaline Phosphatase Is an Endogenous Anti-Inflammatory Factor

Tu1620 Intestinal Alkaline Phosphatase Is an Endogenous Anti-Inflammatory Factor

Maturation and trafficking of a HMW sucrase‐isomaltase species expressed via maltose sensing

As the primary product of starch digestion by pancreatic α‐amylase, maltose and maltooligosaccharides, are the dominant substrates for α‐glucosidases at the intestinal brush border. Here, immunoblotting of SI from Caco‐2 cells showed maltose induce the formation of a higher molecular weight (HMW) SI species. As this finding is potentially indicative of a maltose sensing ability by enterocytes, our goal is to investigate the molecular mechanisms associated with the maturation and trafficking of this HMW SI. Biosynthetic labeling using [35S]‐methionine revealed a higher rate for early trafficking and maturation of the HMW SI species. Endoglycosidase treatment of the immunoprecipitated SI showed the increased molecular weight of SI is a consequence of altered N‐ and O‐glycosylation of this enzyme. In comparison to the control, the HMW SI was found to be more associated with Triton X‐100 lipid rafts which results in higher apical sorting of these species, as defined by isolation and characterization of the brush border membrane of Caco‐2 cells. Thus, maltose sensing ability of enterocytes influences the intracellular processing of the glycoproteins, speculatively to enhance their digestive property.

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.

The intestinal calcistat

The main physiological function of vitamin D is maintenance of calcium homeostasis by its effect on calcium absorption, and bone health in association with parathyroid gland. Vitamin D deficiency (VDD) is defined as serum 25-hydroxy vitamin D (25OHD) levels <20 ng/ml. Do all subjects with VDD have clinical disease according to this definition? We hypothesize that there exist an intestinal calcistat, which controls the calcium absorption independent of PTH levels. It consists of calcium sensing receptor (CaSR) on intestinal brush border, which senses calcium in intestinal cells and vitamin D system in intestinal cells. CaSR dampens the generation of active vitamin D metabolite in intestinal cells and decrease active transcellular calcium transport. It also facilitates passive paracellular diffusion of calcium in intestine. This local adaptation adjusts the fractional calcium absorption according the body requirement. Failure of local adaptation due to decreased calcium intake, decreased supply of 25OHD, mutation in CaSR or vitamin D system decreases systemic calcium levels and systemic adaptations comes into the play. Systemic adaptations consist of rise in PTH and increase in active vitamin D metabolites. These adaptations lead to bone resorption and maintenance of calcium homeostasis. Not all subjects with varying levels of VDD manifest with secondary hyperparathyroidism and decreased in bone mineral density. We suggest that rise in PTH is first indicator of VDD along with decrease in BMD depending on duration of VDD. Hence, subjects with any degree of VDD with normal PTH and BMD should not be labeled as vitamin D deficient. These subjects can be called subclinical VDD, and further studies are required to assess beneficial effect of vitamin D supplementation in this subset of population.