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Dietary deficiencies in zinc (Zn) and vitamin A (VA) are among the leading micronutrient deficiencies globally and previous research has proposed a notable interaction between Zn and VA physiological status. This study aimed to assess the effects of zinc and vitamin A (isolated and combined) on intestinal functionality and morphology, and the gut microbiome (Gallus gallus). The study included nine treatment groups (n~11)—no-injection (NI); H2O; 0.5% oil; normal zinc (40 mg/kg ZnSO4) (ZN); low zinc (20 mg/kg) (ZL); normal retinoid (1500 IU/kg retinyl palmitate) (RN); low retinoid (100 IU/kg) (RL); normal zinc and retinoid (40 mg/kg; 1500 IU/kg) (ZNRN); low zinc and retinoid (ZLRL) (20 mg/kg; 100 IU/kg). Samples were injected into the amniotic fluid of the fertile broiler eggs. Tissue samples were collected upon hatch to target biomarkers. ZLRL reduced ZIP4 gene expression and upregulated ZnT1 gene expression (p < 0.05). Duodenal surface area increased the greatest in RL compared to RN (p < 0.01), and ZLRL compared to ZNRN (p < 0.05). All nutrient treatments yielded shorter crypt depths (p < 0.01). Compared to the oil control, ZLRL and ZNRN reduced (p < 0.05) the cecal abundance of Bifidobacterium and Clostridium genera (p < 0.05). These results suggest a potentially improved intestinal epithelium proceeding with Zn and VA intra-amniotic administration. Intestinal functionality and gut bacteria were modulated. Further research should characterize long-term responses and the microbiome profile.

Among food additive metal oxide nanoparticles (NP), titanium dioxide (TiO₂) and silicon dioxide (SiO₂) are commonly used as food coloring or anti-caking agents, while zinc oxide (ZnO) and iron oxide (Fe₂O₃) are added as antimicrobials and coloring agents, respectively, and can be used as micronutrient supplements. To elucidate potential perturbations associated with NP consumption on gastrointestinal health and development, this in vivo study utilized the Gallus gallus (broiler chicken) intraamniotic administration to assess the effects of physiologically relevant concentrations of food-grade metal oxide NP on brush border membrane (BBM) functionality, intestinal morphology and intestinal microbial populations in vivo. Six groups with 1 mL injection of the following treatments were utilized: non-injected, 18 MΩ DI H2O; 1.4 × 10−6 mg TiO2 NP/mL, 2.0 × 10−5 mg SiO2 NP/mL, 9.7 × 10−6 mg ZnO NP/mL, and 3.8 × 10−4 mg Fe2O3 NP/mL (n = 10 per group). Upon hatch, blood, cecum, and duodenum were collected to assess mineral (iron and zinc) metabolism, BBM functional, and pro-inflammatory-related protein gene expression, BBM morphometric analysis, and the relative abundance of intestinal microflora. Food additive NP altered mineral transporter, BBM functionality, and pro-inflammatory cytokine gene expression, affected intestinal BBM development and led to compositional shifts in intestinal bacterial populations. Our results suggest that food-grade TiO₂ and SiO₂ NP have the potential to negatively affect intestinal functionality; food-grade ZnO NP exposure effects were associated with supporting intestinal development or compensatory mechanisms due to intestinal damage, and food-grade Fe₂O₃ NP was found to be a possible option for iron fortification, though with potential alterations in intestinal functionality and health.

Catechin is a flavonoid naturally present in numerous dietary products and fruits (e.g., apples, berries, grape seeds, kiwis, green tea, red wine, etc.) and has previously been shown to be an antioxidant and beneficial for the gut microbiome. To further enhance the health benefits, bioavailability, and stability of catechin, we synthesized and characterized catechin pentaacetate and catechin pentabutanoate as two new ester derivatives of catechin. Catechin and its derivatives were assessed in vivo via intra-amniotic administration (Gallus gallus), with the following treatment groups: (1) non-injected (control); (2) deionized H2O (control); (3) Tween (0.004 mg/mL dose); (4) inulin (50 mg/mL dose); (5) Catechin (6.2 mg/mL dose); (6) Catechin pentaacetate (10 mg/mL dose); and (7) Catechin pentabutanoate (12.8 mg/mL dose). The effects on physiological markers associated with brush border membrane morphology, intestinal bacterial populations, and duodenal gene expression of key proteins were investigated. Compared to the controls, our results demonstrated a significant (p < 0.05) decrease in Clostridium genera and E. coli species density with catechin and its synthetic derivative exposure. Furthermore, catechin and its derivatives decreased iron and zinc transporter (Ferroportin and ZnT1, respectively) gene expression in the duodenum compared to the controls. In conclusion, catechin and its synthetic derivatives have the potential to improve intestinal morphology and functionality and positively modulate the microbiome.

Genistein is an isoflavone naturally present in numerous staple food crops, such as soybeans and chickpeas. This study utilized the Gallus gallus intraamniotic administration procedure to assess genistein administration effects on trace mineral status, brush border membrane (BBM) functionality, intestinal morphology, and intestinal microbiome in vivo. Eggs were divided into five groups with 1 mL injection of the following treatments: no-injection, DI H2O, 5% inulin, and 1.25% and 2.5% genistein (n = 8 per group). Upon hatch, blood, cecum, small intestine, and liver were collected for assessment of hemoglobin, intestinal microflora alterations, intestinal morphometric assessment, and mRNA gene expression of relevant iron and zinc transporter proteins, respectively. This study demonstrated that intraamniotic administration of 2.5% genistein increased villus surface area, number of acidic goblet cells, and hemoglobin. Additionally, genistein exposure downregulated duodenal cytochrome B (DcytB) and upregulated hepcidin expression. Further, genistein exposure positively altered the composition and function of the intestinal microbiota. Our results suggest a physiological role for genistein administration in improving mineral status, favorably altering BBM functionality and development, positively modulating the intestinal microbiome, as well as improving physiological status.

The use of enzymes from the brush border membrane (BBM) in simulated gastrointestinal digestion of milk proteins has been evaluated. With this purpose, the resistant sequences from casein and milk whey proteins after INFOGEST in vitro digestion with and without BBM have been analyzed by tandem mass spectrometry. The use of BBM revealed additional cleavages to those found with pancreatic enzymes, although the number of total identified peptides decreased due to the reduction of the peptide size. These new cleavages were mainly attributed to the activity of amino- and carboxy-peptidases, which was also reflected in the higher concentration of free amino acids found in the gastrointestinal digests with BBM. The peptidome of the simulated gastrointestinal digests was compared with that previously obtained in digests aspirated from human jejunum after oral administration of the same substrates. The addition of BBM did not change significantly the peptide profile, although it allowed the identification of peptides found in human digests. However, none of the models was able to reproduce the large variety of peptides found in vivo. In addition, in vitro transepithelial transport of six β-casein derived peptides resistant to gastrointestinal digestion, including the opioid β-casomorphin-7, was also evaluated. The results point to the importance of the nature of the N- and C-terminal end for the transport rate through the Caco-2 cell monolayer. Therefore, the use of BBM as a supplementary step after simulated pancreatic digestion can be considered in bioavailability studies since the final sequence can determine the absorption of peptides., This work has received financial support from grants PID2019-107663RB-I00 funded by MCIN/AEI/10.13039/501100011033. J. S., M.S. and S.V. were supported by grants BES-2016-076348 (MCIN/AEI/10.13039/501100011033), FPU15-03616 (Spanish Ministry of Education), and project 20197C061 (CSIC), respectively.

This is a preliminary study evaluating the effect of different fractions of Concord grapes (Vitis labrusca L.) on the brush border membrane (BBM) morphology, duodenal gene expression, and specific gut bacterial populations. For this study, we utilized a unique intraamniotic approach, wherein, the test substances are administered into the amnion of the Gallus gallus egg (on day 17). The embryo orally consumes the amniotic fluid along with the injected test substance before the hatch. We randomly divided ~50 fertilized eggs into 5 groups including 6% grape (juice, puree, and pomace) along with controls (no injection and diluent—H2O). The grape juice was prepared by crushing the grapes; the grape residues were used as pomace. The grape puree included the grape skin, endocarp, mesocarp, and juice but not the seeds. On day 21, the hatch day, the blood, pectoral muscle, liver, duodenum, and large intestine were harvested. Our results showed no significant differences in blood glucose, pectoral glycogen level, or body weight. However, significant (p < 0.05) differences in duodenal and liver gene expression were observed between the treatment groups. The grape puree treatment resulted in higher Clostridium numbers and lower Bifidobacterium numbers when compared to all other groups. In summary, the dietary consumption of grape polyphenols has the potential to beneficially modulate aspects of intestinal health provided their concentration is limited.

Nicotinamide riboside (NR) acts as a nicotinamide adenine dinucleotide (NAD+) precursor where NR supplementation has previously been shown to be beneficial. Thus, we synthesized and characterized nicotinamide riboside tributyrate chloride (NRTBCl, water-soluble) and nicotinamide riboside trioleate chloride (NRTOCl, oil-soluble) as two new ester derivatives of nicotinamide riboside chloride (NRCl). NRCl and its derivatives were assessed in vivo, via intra-amniotic administration (Gallus gallus), with the following treatment groups: (1) non-injected (control); and injection of (2) deionized H2O (control); (3) NRCl (30 mg/mL dose); (4) NRTBCl (30 mg/mL dose); and (5) NRTOCl (30 mg/mL dose). Post-intervention, the effects on physiological markers associated with brush border membrane morphology, intestinal bacterial populations, and duodenal gene expression of key proteins were investigated. Although no significant changes were observed in average body weights, NRTBCl exposure increased average cecum weight. NR treatment significantly increased Clostridium and NRCl treatment resulted in increased populations of Bifidobacterium, Lactobacillus, and E. coli. Duodenal gene expression analysis revealed that NRCl, NRTBCl, and NRTOCl treatments upregulated the expression of ZnT1, MUC2, and IL6 compared to the controls, suggesting alterations in brush border membrane functionality. The administration of NRCl and its derivatives appears to trigger increased expression of brush border membrane digestive proteins, with added effects on the composition and function of cecal microbial populations. Additional research is now warranted to further elucidate the effects on inflammatory biomarkers and observe changes in the specific intestinal bacterial populations post introduction of NR and its derivatives.

Rosa canina L. is a natural polyphenol-rich medicinal plant that exhibits antioxidant and anti-inflammatory activities. Recent in vivo studies have demonstrated that a methanol extract of Rosa canina L. (RCME) has reversed an inflammatory bowel disease (IBD)-like phenotype that has been triggered by dextran sulfate sodium (DSS) in mice. In the current study, we investigated the effects of RCME on perturbations of cellular mechanisms induced by DSS-treatment of intestinal Caco-2 cells, including stress response in the endoplasmic reticulum (ER), protein trafficking and sorting as well as lipid rafts integrity and functional capacities of an intestinal enzyme. 6 days post-confluent cells were treated for 24 h with DSS (3%) or simultaneously with DSS (3%) and RCME (100 &micro, g/mL) or exclusively with RCME (100 &micro, g/mL) or not treated. The results obtained demonstrate the ability of RCME to counteract the substantial increase in the expression levels of several ER stress markers in DSS-treated cells. Concomitantly, the delayed trafficking of intestinal membrane glycoproteins sucrase-isomaltase (SI) and dipeptidyl peptidase 4 (DPP4) induced by DSS between the ER and the Golgi has been compromised by RCME. Furthermore, RCME restored the partially impaired polarized sorting of SI and DPP4 to the brush border membrane. An efficient sorting mechanism of SI and DPP4 is tightly associated with intact lipid rafts structures in the trans-Golgi network (TGN), which have been distorted by DSS and normalized by RCME. Finally, the enzymatic activities of SI are enhanced in the presence of RCME. Altogether, DSS treatment has triggered ER stress, impaired trafficking and function of membrane glycoproteins and distorted lipid rafts, all of which can be compromised by RCME. These findings indicate that the antioxidants in RCME act at two major sites in Caco-2 cells, the ER and the TGN and are thus capable of maintaining the membrane integrity by correcting the sorting of membrane-associated proteins.

The inclusion of pulses in traditional wheat-based food products is increasing as the food industry and consumers are recognizing the nutritional benefits due to the high protein, antioxidant activity, and good source of dietary fiber of pulses. Iron deficiency is a significant global health challenge, affecting approximately 30% of the world&rsquo, s population. Dietary iron deficiency is the foremost cause of anemia, a condition that harms cognitive development and increases maternal and infant mortality. This study intended to demonstrate the potential efficacy of low-phytate biofortified pea varieties on dietary iron (Fe) bioavailability, as well as on intestinal microbiome, energetic status, and brush border membrane (BBM) functionality in vivo (Gallus gallus). We hypothesized that the low-phytate biofortified peas would significantly improve Fe bioavailability, BBM functionality, and the prevalence of beneficial bacterial populations. A six-week efficacy feeding (n = 12) was conducted to compare four low-phytate biofortified pea diets with control pea diet (CDC Bronco), as well as a no-pea diet. During the feeding trial, hemoglobin (Hb), body-Hb Fe, feed intake, and body weight were monitored. Upon the completion of the study, hepatic Fe and ferritin, pectoral glycogen, duodenal gene expression, and cecum bacterial population analyses were conducted. The results indicated that certain low-phytate pea varieties provided greater Fe bioavailability and moderately improved Fe status, while they also had significant effects on gut microbiota and duodenal brush border membrane functionality. Our findings provide further evidence that the low-phytate pea varieties appear to improve Fe physiological status and gut microbiota in vivo, and they highlight the likelihood that this strategy can further improve the efficacy and safety of the crop biofortification and mineral bioavailability approach.

Recent studies suggested that gliadin proteins from the ancient diploid einkorn wheat Triticum monococcum retained a reduced number of immunogenic peptides for celiac disease patients because of a high in vitro digestibility with respect to hexaploid common wheat. In this study, we compared the immunological properties of gliadins from two Triticum monococcum cultivars (Hammurabi and Norberto-ID331) with those of a Triticum durum cultivar (Adamello). Gliadins were digested by mimicking the in vitro gastrointestinal digestion process that includes the brush border membrane peptidases. Competitive ELISA, based on R5 monoclonal antibody, showed that gastrointestinal digestion reduced the immunogenicity of Triticum monococcum gliadins; conversely, the immunogenic potential of Triticum durum gliadins remained almost unchanged by the in vitro digestion. The immune stimulatory activity was also evaluated by detecting the IFN-γ production in gliadin-reactive T-cell lines obtained from the small intestinal mucosa of HLA-DQ2+ celiac disease patients. Interestingly, gastrointestinal digestion markedly reduced the capability of Triticum monococcum gliadins (p

Multidrug resistance-associated protein 2 (MRP2) is an ATP-dependent transporter expressed at the brush border membrane of the enterocyte that confers protection against absorption of toxicants from foods or bile. Acute, short-term regulation of intestinal MRP2 activity involving changes in its apical membrane localization was poorly explored. We evaluated the effects of dibutyryl-cAMP (db-cAMP), a permeable analog of cAMP, and estradiol-17β-d-glucuronide (E217G), an endogenous derivative of estradiol, on MRP2 localization and activity using isolated rat intestinal sacs and Caco-2 cells, a model of human intestinal epithelium. Changes in MRP2 localization were studied by Western blotting of plasma membrane (PM) vs. intracellular membrane (IM) fractions in both experimental models, and additionally, by confocal microscopy in Caco-2 cells. After 30 min of exposure, db-cAMP-stimulated sorting of MRP2 from IM to PM both in rat jejunum and Caco-2 cells at 10 and 100 µM concentrations, respectively, with increased excretion of the model substrate 2,4-dinitrophenyl-S-glutathione. In contrast, E217G (400 µM) induced internalization of MRP2 together with impairment of transport activity. Confocal microscopy analysis performed in Caco-2 cells confirmed Western blot results. In the particular case of E217G, MRP2 exhibited an unusual pattern of staining compatible with endocytic vesiculation. Use of selective inhibitors demonstrated the participation of cAMP-dependent protein kinase and classic calcium-dependent protein kinase C in db-cAMP and E217G effects, respectively. We conclude that localization of MRP2 in intestine may be subjected to a dynamic equilibrium between plasma membrane and intracellular domains, thus allowing for rapid regulation of MRP2 function. Fil: Tocchetti, Guillermo Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. University of Heidelberg; Alemania Fil: Arias, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Arana, Maite Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Rigalli, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina. University of Heidelberg; Alemania Fil: Dominguez, Camila Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Zecchinati, Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Ruiz, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Villanueva, Silvina Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Mottino, Aldo Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina

The intestine presides over a series of vital functions in the human body, among which the digestion/absorption of nutrients. Despite their major digestion role, the impact of the enzymes of the luminal intestinal surface on food components has been considered in relatively few experiments of simulated gastrointestinal digestion. In contrast, the identification of proteolitically stable peptides which survived digestion in multiphasal models that also included a step with small intestinal brush border membrane (BBM) peptidases has provided physiologically consistent results. Herein, we critically review the use of BBM enzymes to simulate the intestinal digestion of dietary polypeptides. Addressing the controversial issue of the in vitro-in vivo correspondence of the digestion models, the review emphasizes the need to establish consensus protocols to simulate the intestinal step, for instance using the BBM hydrolases at least in a selected number of cases. The factors that have limited the development of relevant models of intestinal degradationare discussed together with hints to possible alternatives, forthcoming approaches and future perspectives to reproduce the physiopathology of the human small intestine. (C) 2015 Elsevier Ltd. All rights reserved.

Cholesterol homeostasis is maintained through a balance of de novo synthesis, intestinal absorption, and excretion from the gut. The small intestine contributes to cholesterol homeostasis by absorbing and excreting it, the latter of which is referred to as trans-intestinal cholesterol efflux (TICE). Because the excretion efficiency of endogenous cholesterol is inversely associated with the development of atherosclerosis, TICE provides an attractive therapeutic target. Thus, elucidation of the mechanism is warranted. We have shown that intestinal cholesterol absorption and TICE are inversely correlated in intestinal perfusion experiments in mice. In this review, we summarized 28 paired data sets for absorption efficiency and fecal neutral sterol excretion, a surrogate marker of TICE, obtained from 13 available publications in a figure, demonstrating the inverse correlation were nearly consistent with the assumption. We then offer a bidirectional flux model that accommodates absorption and TICE occurring in the same segment. In this model, the brush border membrane (BBM) of intestinal epithelial cells stands as the dividing ridge for cholesterol fluxes, making the opposite fluxes competitive and being coordinated by shared BBM-localized transporters, ATP-binding cassette G5/G8 and Niemann-Pick C1-like 1. Furthermore, the idea is applied to address how excess plant sterol/stanol (PS) intake reduces circulating cholesterol level, because the mechanism is still unclear. We propose that unabsorbable PS repeatedly shuttles between the BBM and lumen and promotes concomitant cholesterol efflux. Additionally, PSs, which are chemically analogous to cholesterol, may disturb the trafficking machineries that transport cholesterol to the cell interior.

This work aimed to study the opioid peptide β-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

The aim of the present study was to identify and characterize the celiacogenic/immunogenic proteins and peptides released during digestion of pasta (Triticum durum semolina). Cooked pasta was digested using a harmonized in vitro static model of oral-gastro-duodenal digestion. The course of pasta protein digestion was monitored by SDS-PAGE, and gluten proteins were specifically analyzed by Western blot using sera of celiac patients. Among the allergens, nonspecific lipid-transfer protein was highly resistant to gastroduodenal hydrolysis, while other digestion-stable allergens such as alpha-amylase/trypsin inhibitors were not detected being totally released in the pasta cooking water. To simulate the final stage of intestinal degradation, the gastroduodenal digesta were incubated with porcine jejunal brush-border membrane hydrolases. Sixty-one peptides surviving the brush-border membrane peptidases were identified by liquid chromatography-mass spectrometry, including several gluten-derived sequences encrypting different motifs responsible for the induction of celiac disease. These results provide new insights into the persistence of wheat-derived peptides during digestion of cooked pasta samples.

This research investigated the ileum morphometry and enzymatic activity, the caecal volatile fatty acid production and the apparent nutrient digestibility in laying hens fed a Hermetia illucens larvae meal (HILM) as a complete replacement of diet soybean meal (SBM). The hens fed HILM exhibited a lower live weight (P < 0.05) and a higher incidence of the full digestive tract (P < 0.05) than the SBM group. In the duodenum, the maltase exhibited a higher (P < 0.05) activity in the HILM group while the intestinal alkaline phosphatase (IAP) had a higher (P < 0.05) activity in the SBM group. In the ileum, the maltase and saccarase had a higher activity in the HILM hens (P ≤ 0.01) while the IAP and ɤ glutamil transferase had a higher activity in the SBM group (P < 0.05 and P < 0.01, respectively). The HILM group showed a higher (P < 0.05) villi height in the duodenum, while the opposite happened in the jejunum and the ileum. Only in the ileum the crypt depth resulted higher (P < 0.05) in the HIML group than in the SBM. The higher production of acetate (P < 0.05) and butyrate (P < 0.01) affected the total production of volatile fatty acids of the HILM group. The coefficient of apparent digestibility of dry and organic matter as well as of crude protein was higher (P < 0.05) in SBM group. The total replacement of SBM with HILM in laying hens diet from 24 to 45 weeks of age resulted in a higher caecal production of butyric acid while the enzymatic activities of brush border membrane were partially reduced.

Calcium (Ca) is one of the most abundant inorganic elements in the human body and has many important physiological roles. Prebiotics and bioactive peptides are two important substances used to promote calcium uptake. However, the difference in mechanisms of the calcium uptake from these two supplements is not clear. By using the Gallus gallus model and the intra-amniotic administration procedure, the aim of this study was to investigate whether Ca status, intestinal functionality, and health-promoting bacterial populations were affected by prebiotics extracted from chickpea and lentil, and duck egg white peptides (DPs). Eleven groups (non-injected; 18 MΩ H2O; 4 mmol/L CaCl2; 50 mg/mL chickpea + 4 mmol/L CaCl2; 50 mg/mL lentil + 4 mmol/L CaCl2; 40 mg/mL DPs + 4 mmol/L CaCl2; 5 mg/mL Val-Ser-Glu-Glu (VSEE) + 4 mmol/L CaCl2; 50 mg/mL chickpea; 50 mg/mL lentil; 40 mg/mL DPs; 5 mg/mL VSEE) were utilized. Upon hatch, blood, cecum, small intestine, liver and bone were collected for assessment of serum bone alkaline phosphate level (BALP), the relative abundance of intestinal microflora, expression of Ca-related genes, brush border membrane (BBM) functional genes, and liver and bone mineral levels, respectively. The BALP level increased in the presence of lentil, DPs and VSEE (p < 0.05). The relative abundance of probiotics increased significantly (p < 0.05) by VSEE + Ca and chickpea. The expression of CalbindinD9k (Ca transporter) increased (p < 0.05) in Ca, chickpea + Ca and lentil + Ca groups. In addition, the brush border membrane functionality genes expressions increased (p < 0.05) by the chickpea or lentil extracts. Prebiotics and DPs beneficially affected the intestinal microflora and duodenal villus surface area. This research expands the understanding of the prebiotics’ properties of chickpea and lentil extracts, and peptides’ effects on calcium metabolism and gut health.

Sodium nitrite (SNT) widely used as a curative agent in meat processing industry possesses cell-transforming mutagenic and cytogenic properties. Dietary omega-3 polyunsaturated fatty acids (ω-3 PUFA) has been shown to reduce the severity of certain types of cancers, cardiovascular and renal diseases. The present study examined whether feeding of fish oil (FO)/flaxseed oil (FXO) has protective effect against SNT-induced toxicity. SNT significantly altered the activities of serum creatinine (Crt), blood urea nitrogen (BUN), metabolic and brush border membrane (BBM) enzymes. SNT caused significant imbalances in the antioxidant system associated with increased lipid peroxidation (LPO). Feeding of FO and FXO with SNT ameliorated the changes in various parameters caused by SNT. Nephrotoxicity parameters lowered and enzyme activities of carbohydrate metabolism, BBM and radioactively labeled inorganic phosphate (32Pi) transport were improved to near control values. The results of the present study suggest that ω-3 PUFA-enriched FO and FXO from sea-foods and plant sources respectively are similarly effective in reducing SNT-induced nephrotoxicity and oxidative damage.

Two strains of Saccharomyces cerevisiae were tested as probiotics for rainbow trout fry, during the first month of feeding. Each strain was introduced into separate diets, at the rate of 106 CFU g− 1 and their effects were compared with those of a control diet. Two rearing conditions were simultaneously compared, to test the adaptability of the probiotic treatment. From start feeding onwards, the water supply came from either spring or river, resulting in two different temperature ranges, 11¿11.5 and 7¿8 °C respectively. Growth and development were optimal in spring water, while some delay was observed with colder river water. A slight but significant increase in mortality was also observed in the river group. In all groups, the counts of bacteria associated with trout intestine were maximum 10 days post start feeding (dpsf; 107 CFU g− 1). The counts of probiotic yeast were also maximum at 10 dpsf (104¿105 CFU g− 1), but the decrease was slower in river than in spring water. An autochthonous yeast, Debaryomyces hansenii, was also retrieved associated to the intestine of the control group in high numbers after 240 degree days of experiment (104¿105 CFU g− 1), while the colonization level was significantly less in trout fed the probiotic diets. The effect of the dietary yeast was observed by assaying the activity of three enzymes in the brush border membrane of the enterocytes: alkaline phosphatase (AP), γ-glutamyl-transpeptidase (GGT), and leucine-amino-peptidase N (LAP). At 10 and 20 dpsf, the trout reared in spring water had higher activities of the three enzymes when they were fed the strain S. cerevisiae var. boulardii, suggesting an earlier maturation of the digestive system in this group, compared with trout fed either the other strain of S. cerevisiae or the control diet. The effect was not observed in trout reared in river water with slower growth. Both S. boulardii and D. hansenii seemed to stimulate digestive maturation in fish, but the natural colonization by D. hansenii was likely too late for trout reared at optimal temperature. The supplementation of trout starter diet with S. boulardii may be particularly useful in fast growing conditions.

Transport characteristics of D-glucose were studied in the microvillous vesicles isolated from the human term placenta. Transport occurred by selective and rapid facilitated diffusion system which was inhibitable by phloretin and HgCl2. The transport was dependent on a transmembrane. Na+-gradient indicating a "secondary active transport" system operating. The transport influx was saturable and the kinetic analysis based on Hanes-Woolf plot produced a kt and Jmax value of 1.2 mM and 34 nmoles. mgprotein-1.min-1, respectively. The efflux of D-glucose from the membrane vesicles in a pre-equilibrated assay conditions showed a distinct biphasic pattern differing significantly in the half time efflux. The t1/2 of the fast and slow components was found to be 15 sec and 660 sec, respectively. The transport showed distinct sensitivity to temperature and the Ea values both below and above the transition temperature of 37 ºC, as calculated from the Arrhenius plot were found to be 7600 and 5472 kCa1.mol-1, respectively. Inhibition studies with a number of sugars for hexose transport pathway showed that the glucose epimers, phosphorylated sugars, and even the disaccharides and the pentose sugars competed effectively with D-glucose. The influx was also inhibited by a number of steroids such as progesterone, 17α-hydroxyprogesterone, testosterone and estrogen. Insulin was found to increase glucose transport in a dose- dependent fashion at a concentration of 0.2-1 unit.ml-1. Ouabain, dinitrophenoi and nicotine strongly inhibited D-glucose uptake in the membrane vesicles.Se estudiaron las características del transporte de la D-glucosa en las vesículas con microvellosidades aisladas de la placenta humana a término. El transporte ocurría por un sistema de difusión selectiva y facilitada rápida que podía inhibirse por floretina y por HgCl2. El transporte dependía de un gradiente de Na+ transmembrana,indicativo de un sistema operativo de "transporte activo secundario". El flujo de entrada del transporte era saturable y el análisis cinético basado en el gráfico de Hanes- Woolf produjo una k t y una Jmax de 1,2 mM y 34 nmoles• mg de proteína4•min-1, respectivamente. El flujo de salida de la D-glucosa desde las vesículas de membrana en condiciones de ensayo de pre-equilibrio mostró un patrón bifásico distintivo que difería significativamente en la mitad del flujo de salida. Se halló que la t1/2 de los componentes rápido y lento 15 seg. y 660 seg., respectivamente. El transporte mostró una sensibilidad distintiva a la temperatura, y se encontró que los valores de Ea, tanto por encima como por debajo de la temperatura de transición de 37º C, calculada por el gráfico de Arrhenius, fueron de 7.600 y 5.472 kCa1.mol-1, respectivamente. Los estudios de inhibición con una serie de azúcares para la ruta del transporte de la hexosa mostraron que los epímeros de la glucosa, los azúcares fosforilados, e incluso los disacáridos y los azúcares pentosa, competían de forma eficaz con la D-glucosa. El flujo de entrada también se inhibió por una serie de esteroides como la progesterona, la 17α-hidroxiprogesterona, la testosterona y los estrógenos. Se encontró que la insulina aumentaba el transporte de glucosa de manera dependiente de la dosis a una concentración de 0,2-1 unidades•ml-1. La ouabaína, el dinitrofenol y la nicotina inhibieron fuertemente la captación de D-glucosa por las vesículas de membrana.

The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (Vmax) and increase in affinity constant (Km) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM.

The Na+/phosphate cotransporter NaPi-IIa (SLC34A1) is the major transporter mediating the reabsorption of Piin the proximal tubule. Expression and activity of NaPi-IIa is regulated by several factors, including parathyroid hormone, dopamine, metabolic acidosis, and dietary Piintake. Dopamine induces natriuresis and phosphaturia in vivo, and its actions on several Na+-transporting systems such as NHE3 and Na+-K+-ATPase have been investigated in detail. Using freshly isolated mouse kidney slices, perfused proximal tubules, and cultured renal epithelial cells, we examined the acute effects of dopamine on NaPi-IIa expression and localization. Incubation of isolated kidney slices with the selective D1-like receptor agonists fenoldopam (10 μM) and SKF-38393 (10 μM) for 1 h induced NaPi-IIa internalization and reduced expression of NaPi-IIa in the brush border membrane (BBM). The D2-like selective agonist quinpirole (1 μM) had no effect. The D1and D2agonists did not affect the renal Na+/sulfate cotransporter NaSi in the BBM of the proximal tubule. Studies with isolated perfused proximal tubules demonstrated that activation of luminal, but not basolateral, D1-like receptors caused NaPi-IIa internalization. In kidney slices, inhibition of PKC (1 μM chelerythrine) or ERK1/2 (20 μM PD-098089) pathways did not prevent the fenoldopam-induced internalization. Inhibition with the PKA blocker H-89 (10 μM) abolished the effect of fenoldopam. Immunoblot demonstrated a reduction of NaPi-IIa protein in BBMs from kidney slices treated with fenoldopam. Incubation of opossum kidney cells transfected with NaPi-IIa-green fluorescent protein chimera shifted fluorescence from the apical membrane to an intracellular pool. In summary, dopamine induces internalization of NaPi-IIa by activation of luminal D1-like receptors, an effect that is mediated by PKA.

cope The ancient diploid Triticum monococcum is of special interest as a candidate low-toxic wheat species for celiac disease patients. Here, we investigated how an in vitro gastro-intestinal digestion, affected the immune toxic properties of gliadin from diploid compared to hexaploid wheat. Methods and results Gliadins from Triticum monococcum, and Triticum aestivum cultivars were digested using either a partial proteolysis with pepsin-chymotrypsin, or an extensive degradation that used gastrointestinal enzymes including the brush border membrane enzymes. The immune stimulatory properties of the digested samples were investigated on T-cell lines and jejunal biopsies from celiac disease patients. The T-cell response profile to the Triticum monococcum gliadin was comparable to that obtained with Triticum aestivum gliadin after the partial pepsin-chymotrypsin digestion. In contrast, the extensive gastrointestinal hydrolysis drastically reduced the immune stimulatory properties of Triticum monococcum gliadin. MS-based analysis showed that several Triticum monococcum peptides, including known T-cell epitopes, were degraded during the gastrointestinal treatment, whereas many of Triticum aestivum gliadin survived the gastrointestinal digestion. Conclusion The pattern of Triticum monococcum gliadin proteins is sufficiently different from those of common hexaploid wheat to determine a lower toxicity in celiac disease patients following in vitro simulation of human digestion.

We investigated the effect of thyroid hormone status on renal handing of Ca2+. Further, like kinetics of Ca2+ transport across brush-border membrane (BBM) and basolateral membrane (BLM) of renal epithelial cells was carried out. FE(Ca) was decreased in hyperthyroid (Hyper-T) rats and increased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Ca2+ uptake into renal brush-border membrane vesicles (BBMV) was increased in Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats. K(m) was lower in Hyper-T rats and higher in Hypo-T rats as compared to Eu-T rats whereas, V(max) remained unaltered. The transition temperature for calcium uptake varied inversely with the thyroid hormone status. Renal BBM of Hyper-T rats showed decreased anisotropy and polarisation of DPH as compared to EU-T rats whereas these values were increased in Hypo-T rats. Thus, the altered BBM fluidity appears to modulate Ca2+ transport across BBM. Na+/Ca2+ exchange activity of renal cells was increased in Hyper-T and decreased in Hypo-T rats as compared to Eu-T rats. V(max) for Na+/Ca2+ exchange was increased in Hyper-T rats and deceased in Hypo-T rats as compared to Eu-T rats, whereas, [Na+](0.5) was similar in all three groups. The c-AMP levels of renal cortex of Hyper-T rats was increased and that of Hypo-T rats decreased as compared to Eu-T rats. Thus, thyroid hormones increased Ca2+ reabsorption in the kidney of rat. Thyroid hormone-mediated modulation of BBM fluidity appears to stimulate Ca2+ uptake into renal BBMV. Thyroid hormones possibly activated the Na+/Ca2+ exchanger through cAMP-dependent pathway.

This study investigates the effectiveness of two types of prebiotics—stachyose and raffinose—which are present in staple food crops that are widely consumed in regions where dietary Fe deficiency is a health concern. The hypothesis is that these prebiotics will improve Fe status, intestinal functionality, and increase health-promoting bacterial populations in vivo (Gallus gallus). By using the intra-amniotic administration procedure, prebiotic treatment solutions were injected in ovo (day 17 of embryonic incubation) with varying concentrations of a 1.0 mL pure raffinose or stachyose in 18 MΩ H2O. Four treatment groups (50, 100 mg·mL−1 raffinose or stachyose) and two controls (18 MΩ H2O and non-injected) were utilized. At hatch the cecum, small intestine, liver, and blood were collected for assessment of the relative abundance of the gut microflora, relative expression of Fe-related genes and brush border membrane functional genes, hepatic ferritin levels, and hemoglobin levels, respectively. The prebiotic treatments increased the relative expression of brush border membrane functionality proteins (p < 0.05), decreased the relative expression of Fe-related proteins (p < 0.05), and increased villus surface area. Raffinose and stachyose increased the relative abundance of probiotics (p < 0.05), and decreased that of pathogenic bacteria. Raffinose and stachyose beneficially affected the gut microflora, Fe bioavailability, and brush border membrane functionality. Our investigations have led to a greater understanding of these prebiotics’ effects on intestinal health and mineral metabolism.

To investigate maturational changes of membrane food protein binding capacity, we studied binding characteristics of brush border membranes isolated from small intestines of newborn and adult rats. Binding of biotinylated gliadin peptides, cow's milk proteins (alpha-casein, beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin) and lectins was assessed by a sensitive chemiluminescence blot assay. We found specific food protein binding with regard to saturation and inhibition. Maximal binding of most food proteins and several lectins to brush border membranes of newborn and adult rats was comparable, whereas binding of beta-lactoglobulin was substantially less. Common or adjoining binding sites for the different food proteins tested were indicated by corresponding membrane protein binding patterns and by inhibition properties of unrelated proteins. Compared to newborns, adult membrane vesicles as well as isolated membrane proteins showed higher binding capacities. Thus postnatal maturation of small intestinal brush border membranes correlated with increased food protein binding capacity.

Subtotal nephrectomy alters tubular function: Effect of phosphorus restriction. Few studies have examined tubular function after subtotal nephrectomy (Nx) and conservative treatments. The effects of 70% and 80% Nx (associated with dietary phosphate restriction in the latter case) on the apical brush border membrane (BBM) enzymes 5′-nucleotidase, γ glutamyl-transferase and alkaline-phosphatase, and one BBM Na-phosphate cotransporter (NaPi-2) were studied in rats after a six week period. Changes in activity and mRNA abundance of the BBM enzymes and in NaPi-2 protein and mRNA abundance were compared with changes in the distal markers of Na,K-ATPase activity and epidermal growth factor (EGF) production. The activity, but not the mRNA of BBM enzymes, was moderately reduced by the 70% Nx. Both the mRNA and activity of γ glutamyl-transferase and alkaline-phosphatase were decreased in the 80% Nx, and the NaPi-2 mRNA, protein and Na,K-ATPase activities were also reduced. These effects (except for 5′ nucleotidase and Na,K-ATPase) were partly reversed by phosphate restriction. Overproduction of EGF occurred after the 70% Nx, was blunted in the 80% Nx, and then partially restored by phosphate restriction. Aggravation of tubular alteration was associated with enhanced renal hyperplasia (increased DNA mass), reduced GFR and hyperphosphatemia, and high PTH levels, but reduced cAMP excretion. Improvement following phosphate restriction was associated with reduced hyperplasia and lowering of phosphatemia and PTH levels. These data demonstrate that Nx selectively affected BBM function through transcriptional changes that were partially reversed by phosphate restriction. Regulatory factors involved in these changes may include intracellular phosphate content and growth factors, but not the PTH effects that are impaired in chronic renal failure.

Background and aim: Feijoa sellowiana fruits grow throughout the Mediterranean area and are used for human food. These fruits and especially the acetonic extract have been shown to possess biological activities, such as antioxidant and anti-cancer properties. We aim to investigate the effects of the acetonic extract of Feijoa sellowiana fruits on the viability, proliferation and brush border membrane enzyme activities of the epithelial intestinal cells. Material and methods: Caco-2 cells were seeded into a 6-wells multiwells plate and cultured with appropriate medium. At confluence, cells were star- vated for 24h and then exposed to the acetonic extract (500 ug/mL), for 24h and 48h. Brush border membrane enzymes lactase and sucrase activity, cell proliferation and viability were measured with the modified Dahlqvist method, bromodeoxyuridine incorporation assay and MTT vitality test, respectively. Cells with medium alone were used as control. Data are expressed as mean ± SD. Results: Compared to control conditions, the acetonic extract significantly increased lactase activity, both after 24h (+7.1 ± 1.0 fold increase vs control, p < 0.05) and 48h (+2.9 ± 0.8 fold increase vs control, p < 0.05) and sucrase acivity, both after 24h (+3.2 ± 0.7 fold increase vs control, p < 0.05) and 48h (+3.6 ± 0.8 fold increase vs control, p < 0.05). The acetonic extract signifi- cantly decreased proliferation respect to control, both after 24h (0.17 ± 0.01 vs 0.23 ± 0.01 450–540nm; p < 0.01) and 48h (0.22 ± 0.02 vs 0.39 ± 0.03 450– 540nm; p < 0.01). After 24h, the acetonic extract had no effects on cells viability, while after 48h, the acetonic extract significantly increased viability respect to control (+50 ± 7% increase vs control, p < 0.05). Conclusions: We demonstrate that the treatment with the acetonic extract from Feijoa sellowiana fruits improves lactase and sucrase activity in intestinal epithelial cells. We also show that the acetonic extract inhibits cell proliferation and have no cytotoxic effects. These changes induced by the acetonic extract in the brush border enzyme activity may be exploited in case of enzymatic deficit of lactase and sucrase

OBJECTIVES Angiotensin converting enzyme (ACE) has been shown to be an important peptidasse that play a role in digestion and assimilation of protein rich in proline such as casein, gliadin and collagen. Despite that ACE inhibitors have been popular for various types of hypertension and congestive heart failure, the effects of their long-term treatment on intestinal ACE activities are not known. Therefore, we measured intestinal specific activities in rats after four weeks' treatment of ACE inhibitors. METHODS Thirty Wistar rats weighing about 200g in average were divided into three groups, and supplied with tap water, captopril solution and enalapril solution respectively for four weeks. After sacrificing, intestinal ACE specific activities were measured in homogenate and brush border membrane fraction respectively, which was prepared from three equally divided segments of removed small intestine. RESULTS ACE specific activities of proximal, middle and distal segments of control group were 178.6 +/- 64.2, 180.3 +/- 60.2 and 48.6 +/- 13.1 in brush border membrane (mean +/- SD, nmol/min/mg protein) respectively. Those of captopril group were 314.2 +/- 72.5, 281.0 +/- 69.8 and 67.7 +/- 21.8 respectively, showing tendency of increase in proximal and middle segments (p < 0.01 and 0.05 respectively). By contrast, those of enalapril group were 48.5 +/- 27.6, 70.7 +/- 15.6 and 11.6 +/- 4.4 respectively, which were significantly lower (p < 0.01) than those of control group. CONCLUSION Rat intestinal ACE specific activities were not inhibited by captopril treatment, but inhibited by enalapril treatment. This finding may explain why there has not been any case report of malabsorption in patients taking captopril. But the malabsorption of prolyl peptide could be possible in cases with long-term administration of enalapril.

International audience; Our understanding of the molecular mechanisms responsible for fat-soluble vitamin uptake and transport at the intestinal level has advanced considerably over the past decade. On one hand, it has long been considered that vitamin D and E as well as beta-carotene (the main provitamin A carotenoid in human diet) were absorbed by a passive diffusion process, although this could not explain the broad inter-individual variability in the absorption efficiency of these molecules. On the other hand, it was assumed that preformed vitamin A (retinol) and vitamin K1 (phylloquinone) absorption occurred via energy-dependent processes, but the transporters involved have not yet been identified. The recent discovery of intestinal proteins able to facilitate vitamin E and carotenoid uptake and secretion by the enterocyte has spurred renewed interest in studying the fundamental mechanisms involved in the absorption of these micronutrients. The proteins identified so far are cholesterol transporters such as SR-BI (scavenger receptor class B type I), CD36 (cluster determinant 36), NPC1L1 (Niemann-Pick Cl-like 1) or ABCA1 (ATP-Binding Cassette A1) displaying a broad substrate specificity, but it is likely that other membrane proteins are also involved. After overviewing the metabolism of fat-soluble vitamins and carotenoids in the human upper gastrointestinal lumen, we will focus on the putative or identified proteins participating in the intestinal uptake, intracellular transport and basolateral secretion of these fat-soluble vitamins and carotenoids, and outline the uncertainties that need to be explored in the future. Identifying the proteins involved in intestinal uptake and transport of fat-soluble vitamins and carotenoids across the enterocyte is of great importance, especially as some of them are already targets for the development of drugs able to slow cholesterol absorption. Indeed, these drugs may also interfere with lipid vitamin uptake. A better understanding of the molecular mechanisms involved in fat-soluble vitamin and carotenoid absorption is a priority to better optimize their bioavailability.

International audience; The sustainable composition of diets of high nutritional value is of the utmost importance for intensive aquaculture. Digestion and absorption of nutrients depend on the activity of the digestive enzymes, in particular those located in the brush border membrane of enterocytes, which are responsible for the final stages of breaking down and absorption of nutrients. In the present study, the substitution of fish meal by lupin or rapeseed meal in the diet was evaluated on gilthead sea bream (Sparus aurata) and goldfish (Carassius auratus). The objectives were to compare the activities of intestinal brush border enzymes in both species fed the control and experimental diets. When gilthead sea bream were fed the vegetable diets, significantly lower activities compared with the control group were observed for alkaline phosphatase and gamma-glutamyl transpeptidase, but these differences were not significant in goldfish. Maltase activity was found decreased in the group fed lupin meal, both in sea bream and in goldfish. However, in spite of these differences in enzyme activities, growth characteristics of the fishes were similar with the three diets. It seemed that both fish were able to adapt to partial substitution of fish meal, but it remains to investigate the mechanism for compensating the decrease in specific enzymatic activity in the enterocytes of carnivorous gilthead sea bream. (C) 2010 Elsevier B.V. All rights reserved.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

International audience

Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Tabanidae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H. pellucidula and E. danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I. grammatica and H. bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E. danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H. pellucidula > I. grammatica > H. bimaculata > E. danica. Moreover, in H. pellucidula and I. grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E. danica, leucine uptake was sodium-independent at all pH values. The highest value of Vmax (45.3 pmol·s–1·mg proteins–1) was in E. danica, which, however, displayed the lowest affinity (Km 137 μM) when compared to the kinetic parameters of other taxa. The Vmax and Km values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H. bimaculata, H. pellucidula, and I. grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects. Arch. Insect Biochem. Physiol. 63:110–122, 2006. © 2006 Wiley-Liss, Inc.

Transport characteristics of D-glucose were studied in the microvillous vesicles isolated from the human term placenta. Transport occurred by selective and rapid facilitated diffusion system which was inhibitable by phloretin and HgCl2. The transport was dependent on a transmembrane. Na+-gradient indicating a "secondary active transport" system operating. The transport influx was saturable and the kinetic analysis based on Hanes-Woolf plot produced a kt and Jmax value of 1.2 mM and 34 nmoles. mgprotein-1.min-1, respectively. The efflux of D-glucose from the membrane vesicles in a pre-equilibrated assay conditions showed a distinct biphasic pattern differing significantly in the half time efflux. The t1/2 of the fast and slow components was found to be 15 sec and 660 sec, respectively. The transport showed distinct sensitivity to temperature and the Ea values both below and above the transition temperature of 37 ºC, as calculated from the Arrhenius plot were found to be 7600 and 5472 kCa1.mol-1, respectively. Inhibition studies with a number of sugars for hexose transport pathway showed that the glucose epimers, phosphorylated sugars, and even the disaccharides and the pentose sugars competed effectively with D-glucose. The influx was also inhibited by a number of steroids such as progesterone, 17α-hydroxyprogesterone, testosterone and estrogen. Insulin was found to increase glucose transport in a dose- dependent fashion at a concentration of 0.2-1 unit.ml-1. Ouabain, dinitrophenoi and nicotine strongly inhibited D-glucose uptake in the membrane vesicles. Se estudiaron las características del transporte de la D-glucosa en las vesículas con microvellosidades aisladas de la placenta humana a término. El transporte ocurría por un sistema de difusión selectiva y facilitada rápida que podía inhibirse por floretina y por HgCl2. El transporte dependía de un gradiente de Na+ transmembrana,indicativo de un sistema operativo de "transporte activo secundario". El flujo de entrada del transporte era saturable y el análisis cinético basado en el gráfico de Hanes- Woolf produjo una k t y una Jmax de 1,2 mM y 34 nmoles• mg de proteína4•min-1, respectivamente. El flujo de salida de la D-glucosa desde las vesículas de membrana en condiciones de ensayo de pre-equilibrio mostró un patrón bifásico distintivo que difería significativamente en la mitad del flujo de salida. Se halló que la t1/2 de los componentes rápido y lento 15 seg. y 660 seg., respectivamente. El transporte mostró una sensibilidad distintiva a la temperatura, y se encontró que los valores de Ea, tanto por encima como por debajo de la temperatura de transición de 37º C, calculada por el gráfico de Arrhenius, fueron de 7.600 y 5.472 kCa1.mol-1, respectivamente. Los estudios de inhibición con una serie de azúcares para la ruta del transporte de la hexosa mostraron que los epímeros de la glucosa, los azúcares fosforilados, e incluso los disacáridos y los azúcares pentosa, competían de forma eficaz con la D-glucosa. El flujo de entrada también se inhibió por una serie de esteroides como la progesterona, la 17α-hidroxiprogesterona, la testosterona y los estrógenos. Se encontró que la insulina aumentaba el transporte de glucosa de manera dependiente de la dosis a una concentración de 0,2-1 unidades•ml-1. La ouabaína, el dinitrofenol y la nicotina inhibieron fuertemente la captación de D-glucosa por las vesículas de membrana.

Inorganic phosphate (P(i)) is reabsorbed in the renal proximal tubule mainly via the type-IIa sodium-phosphate cotransporter (NaPi-IIa). This protein is regulated tightly by different factors, among them dietary P(i) intake and parathyroid hormone (PTH). A number of PDZ-domain-containing proteins have been shown to interact with NaPi-IIa in vitro, such as Na(+)/H(+) exchanger-3 regulatory factor-1 (NHERF1) and PDZK1. PDZK1 is highly abundant in kidney and co-localizes with NaPi-IIa in the brush border membrane of proximal tubules. Recently, a knock- out mouse model for PDZK1 (Pdzk1(-/-)) has been generated, allowing the role of PDZK1 in the expression and regulation of the NaPi-IIa cotransporter to be examined in in vivo and in ex vivo preparations. The localization of NaPi-IIa and other proteins interacting with PDZK1 in vitro [Na(+)/H(+) exchanger (NHE3), chloride-formate exchanger (CFEX)/putative anion transporter-1 (PAT1), NHERF1] was not altered in Pdzk1(-/-) mice. The abundance of NaPi-IIa adapted to acute and chronic changes in dietary P(i) intake, but steady-state levels of NaPi-IIa were reduced in Pdzk1(-/-) under a P(i) rich diet. This was paralleled by a higher urinary fractional P(i) excretion. The abundance of the anion exchanger CFEX/PAT1 (SLC26A6) was also reduced. In contrast, NHERF1 abundance increased in the brush border membrane of Pdzk1(-/-) mice fed a high-P(i) diet. Acute regulation of NaPi-IIa by PTH in vivo and by PTH and activators of protein kinases A, C and G (PKA, PKC and PKG) in vitro (kidney slice preparation) was not altered in Pdzk1(-/-) mice. In conclusion, loss of PDZK1 did not result in major changes in proximal tubule function or NaPi-IIa regulation. However, under a P(i)-rich diet, loss of PDZK1 reduced NaPi-IIa abundance indicating that PDZK1 may play a role in the trafficking or stability of NaPi-IIa under these conditions.

Numerous studies have demonstrated various structure/function correlations at the level of transport proteins in the kidney cell membranes and various intracellular organelles. However, characterization of the lipid phase of these membranes is rare. Here, we report the differences in lipid organization and dynamics of the brush-border membranes (BBM), basolateral membranes (BLM) and endocytotic vesicles (EV), isolated from the kidney cortex of intact rats, studied with the EPR spectroscopy of the spin-labeled membrane lipids. The EPR spectra were analyzed by comparing experimentally observed line shapes with the line shapes calculated according to the theoretical model developed for liquid crystals. In the fitting procedure, three different lipid domains were assumed, which revealed clear differences in the lipid ordering and rotational correlation times, as well as in the lipid partition of these domains in each of the three types of membranes. A similar approach, used to compare the spectroscopic characteristics of BBM from control and cadmium-intoxicated rats, showed significantly changed ordering and increased molecular mobility in the lipid phase of BBM from Cd-treated animals. As tested by an established fluorescence assay, the Cd-induced changes in the lipid mobility co localized with approximately 5-fold higher conductance of BBM for potassium, with unchanged conductance for protons.

Thyroid hormone status influences calcium metabolism. To elucidate the mechanism of action of thyroid hormones on transcellular transport of calcium in rat intestine, Ca(2+) influx and efflux studies were carried out in brush border membrane vesicles (BBMV) and across the basolateral membrane (BLM) of enterocytes, respectively. Steady-state uptake of Ca(2+) into BBMV as well as Ca(2+) efflux from the BLM enterocytes was significantly increased in hyperthyroid (Hyper-T) rats and decreased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Kinetic studies revealed that increase in steady state Ca(2+) uptake into BBMV from hyper-T rats was fraternized with decrease in Michaelis Menten Constant (K(m)), indicating a conformational change in Ca(2+) transporter. Further, this finding was supported by significant changes in transition temperature and membrane fluidity. Increased Ca(2+) efflux across enterocytes was attributed to sodium-dependent Ca(2+) exchange activity which was significantly higher in Hyper-T rats and lower in Hypo-T rats as compared to Eu-T rats. However, there was no change in Ca(2+)-ATPase activity of BLMs of all groups. Kinetic studies of Na(+)/Ca(2+) exchanger revealed that alteration in Na(+)-dependent Ca(2+) efflux was directly associated with maximal velocity (V(max)) of exchanger among all the groups. cAMP, a potent activator of Na(+)/Ca(2+) exchanger, was found to be significantly higher in intestinal mucosa of Hyper-T rats as compared to Eu-T rats. Therefore, the results of this study suggest that Ca(2+) influx across BBM is possibly modulated by thyroid hormones by mediating changes in membrane fluidity. Thyroid hormones activated the Na(+)/Ca(2+) exchange in enterocytes possibly via cAMP-mediated pathway.

Brush border membane, basolateral membrane and endocytotic vesicles from the mammalian proximal tubule cells are structurally heterogeneous, and are associated with different roles in kidney function. Besides having different sets of transporters, these three types of membrane organelles also differ in lipid composition. In order to examine whether these differences in lipid composition could be investigated with EPR spectroscopy with the use of spin labeled fatty-acid analogues, all three types of renal organelles were isolated from the kidney cortex of intact rats by standard procedures. Their membranes were spin labeled by lipophylic spin probe, MeFASL (10, 3) for EPR analysis. The EPR spectra were taken at various temperatures ranging from 22 to 42 °C. The spectra were analyzed by comparing experimentally observed line shapes with the lines shapes calculated according to the theoretical model developed for liquid crystals. The deduced order parameters and rotational correlation times refer to the molecular ordering and mobility, respectively, in the lipid phase of the labeled membranes. In the fitting procedure three different lipid domains were assumed. The results clearly revealed differences in the lipid ordering and rotational correlation times among all three types of membrane organelles. The ratio of the lipid domains in each type of membranes and the parameters characterizing different domains will be useful quantities in future studies of various experimental pathophysiological and toxicological entities, such as diabetes mellitus and heavy metal nephrotoxicity, that may be associated with lipid abnormalities.

Oxidation of biological membranes has been suggested as a major pathological process in a variety of disease states including intestinal ischemia and inflammatory bowel disease. Previous studies on the small intestinal brush border membrane have shown that part of the decrease in the activity of the Na + -dependent glucose transporter (SGLT1) observed after oxidation could be secondary to the derangement in membrane fluidity that accompanied oxidative damage. The present study examined the relationship between oxidative-induced hemileaflet fluidity alterations and the resultant change in Na + -dependent glucose transport activity. To address this issue, in vitro oxidation of guinea pig brush border membrane vesicles was induced by incubation of the vesicles with ferrous sulfate and ascorbate. We found that oxidation decreased the fluidity of both the outer and inner hemileaflets, the decrease being greater in the outer leaflet. Moreover, the preferential alteration in hemileaflet fluidity was accompanied by a decrease in glucose transport. However, when membrane perturbing agents such as hexanol and A 2 C were used to restore membrane fluidity to levels comparable to controls, rates of glucose transport could not be interpreted in terms of variation of bulk membrane fluidity or variation in fluidity of any specific membrane leaflet. On the basis of these experiments, we propose that previous studies that reported coincidental alteration in membrane fluidity and glucose transport cannot be interpreted on the basis of bulk fluidity or hemileaflet fluidity.

The present study examined riboflavin (RF) uptake by isolated rabbit renal brush border membrane (BBM). RF uptake was linear for up to 30 s and leveled off thereafter reaching an equilibrium with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the uptake was the results of transport (61.4%) into the intravesicular space as well as binding (38.6%) to membrane surfaces. The process of RF uptake was saturable as a function of substrate concentration with an apparent Km of 25.7 +/- 7.6 microM and Vmax of 75.6 +/- 14.7 pmol/mg protein/10 s. cis-Addition of unlabeled RF and its structural analogues, lumiflavin and lumichrome, inhibited the uptake of [3H]RF significantly, indicating the involvement of a carrier-mediated process in RF uptake by renal BBM. RF uptake by renal BBM was partly Na+-dependent so that when Na+ was replaced by potassium, choline, lithium or tetramethylammonium, the RF uptake was reduced to ca. 60% of the control. This Na+-dependency was unlikely to be due to Na+-cotransport mechanism because RF uptake occurred without the characteristic 'overshoot' phenomenon as for other Na+-cotransport systems and the elimination of transmembrane Na+-gradient by preloading Na+ to the intravesicular space did not affect RF uptake. In contrast, removal of Na+ eliminated the binding component of RF uptake, suggesting the requirement of Na+ for RF binding to BBM. The RF uptake was not affected when extravesicular pH was varied within the physiological pH range of 6.5 to 8.5. No effect on BBM [3H]RF uptake was found when the transmembrane electrical potential was altered by either the presence of anions with different membrane permeability (Cl- = NO3- > SO4- > gluconate-) or by using nigericin (10 microg/mg protein) with an outwardly or inwardly directed transmembrane K+ gradient. The uptake of RF by BBM vesicles was, however, inhibited by probenecid and organic anion transport inhibitors, 4,4-diiso-thiocyanatostilbene-2,2-disulfonic acid (DIDS, 1 mM) and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mM). In summary, these results demonstrate the existence of a membrane-associated, and organic anion inhibitor-sensitive, carrier system for RF uptake by renal BBM.

Renal proximal tubular Na/phosphate cotransport is the key target in the control of renal phosphate handling. Using molecular tools for the type II Na/phosphate cotransporter, it could be documented that altered brush border membrane phosphate transport reflects a state of altered brush border expression of the specific transporter molecule. This alteration is either the consequence of specific membrane insertion/retrieval processes and/or reflects states of altered synthesisdegradation of the transporter.

Thyroid hormone (T-3), a known stimulator of renal proximal tubular brush border membrane Na-dependent phosphate (P-i) uptake (Na/P-i-cotransport), stimulated Na-dependent P-i transport in opossum kidney (OK) cells. Na/P-i-cotransport was stimulated in a time- and dose-dependent manner with maximal effects (57%) at 24 h and 10(-10) M T-3. This stimulation was related to an increase in the apparent capacity (V-max) of Na/P-i-cotransport. Treatment with T-3 had no effect on Na-independent transport of P-i or of L-arginine. The stimulation of Na/P-i-cotransport was paralleled by an increase in the messenger ribonucleic acid (mRNA) encoding the OK cell apical Na/P-i-cotransporter (termed NaPi-4); the mRNA levels related to the activity of Na-independent L-arginine transport (rBAT) were unaffected by T-3. Actinomycin D (10(-7)M) completely prevented the stimulatory effect of T-3 On OK cell Na/P-i-cotransport and on NaPi-4 mRNA content. In conclusion, T-3 stimulates apical Na/P-i-cotransport in OK cells most likely by enhancing its transcription

p33/41 is a Ca2+-dependent carbohydrate-binding protein and is identical to annexin IV, a member of the annexin protein family. The localization of p33/41 in bovine kidney specimens was investigated immununohistochemically by use of specific polyclonal antibodies. The most interesting finding on immunostaining was that p33/41 was highly concentrated in the apical plasma membrane of the epithelial cells in the proximal tubules contrary to the distribution throughout the cytoplasm in the papillary ducts and papilla epithelium. The enrichment of p33/41 m the apical membrane was confirmed by immunoblotting of the brush border membrane fraction prepared from a kidney homogenate. Sequential extraction with EDTA and Triton X-100, and a partition experiment with Triton X-114 revealed that most p33/41 associates with the renal brush border membrane in a Ca2+-independent manner and is integrated into the membrane like intrinsic membrane proteins.

Meprins are oligomeric cell-surface metalloproteinases that are expressed at high concentrations in the renal brush border membranes of mice. Meprins consist of two types of subunits, α and β, which are the products of two different genes. The β subunit cDNA was cloned and sequenced from mouse kidneys, and the membrane association and the in vivo proteolytic processing of mouse meprin subunits were investigated. The primary translation product of the meprin β subunit is composed of 704 amino acids, and contains several domains, including a signal sequence at the NH₂-terminus, a prosequence, a protease domain, an adhesion domain (MAM domain), an epidermal growth factor-like domain, a potential transmembrane-spanning domain, and a short cytoplasmic tail. The B subunit is evolutionarily related to the α subunit. The α and β subunit share about 42% overall sequence identity, and have a similar arrangement of functional domains; however, a 56 amino acid segment near the COOH-terminus of a is missing in β, and the signal sequences, transmembrane and cytoplasmic domains share no significant sequence similarity. The protease domains of o and B are 55% identical. NH₂-terminal protein sequencing of detergent-solubilized meprin subunits from mouse kidneys showed that the prosequence in a is removed in the mature subunit. By contrast, only the signal sequence is removed from the mature β subunit NH₂-terminus, and the β subunit retains the prosequence. Further, the mature α subunit, but not the β subunit, is proteolytically processed at the COOH-terminus and does not contain the transmembrane and the EGF-like domains encoded by the meprin α cDNA. The β subunit is a type I integral membrane protein. By contrast, α does not transverse the membrane, and its membrane association depends on disulfide bonds. The oligomeric organization of membrane-bound meprins was analyzed by SDSPAGE under non-reducing conditions, and by isoelectric focusing. ICR mouse kidneys express αβ heterodimers and α₂ homodimers; C3H/He mice contain β₂ dimers. Transfection of COS-1 cells with the full-length meprin α subunit cDNA resulted in the secretion of meprin dimers into the culture medium, indicating that the COOH-terminal transmembrane domain of meprin α subunits is posttranslationally removed from the protein in COS-1 cells, as it is in mouse kidney cells. Replacement of the COOH-terminal 137 amino acids of a with the COOH-terminus of B, or deletion of the 56 amino acid inserted domain in α, resulted in mutant proteins that were not secreted into the medium, but rather were membrane-bound, indicating that the inserted domain of α is essential for proteolytic cleavage and secretion. Deletion of the COOH-terminal 133 residues of a did not affect meprin α dimerization or intracellular transport. The meprin a subunits secreted from transfected COS-1 cells were catalytically inactive, but could be activated by limited proteolysis with trypsin. Thus, processing at the NH,-terminus differed in COS-1 cells and in mouse kidney. COS-1 cells did not remove the prosequence from the α subunit protein, and removal of the prosequence was essential for catalytic activity of the a subunit. These results have implications for the biosynthesis and regulation of a cell surface proteinase, and thus relate to the elucidation of the mechanisms by which biological events at the cell surface are regulated. Ph. D.

The kinetics of uptake of radioactive label from [U-14C]Gly, L-[4,5-3H]Leu and the dipeptide [14C]Gly—L-[4,5-3H]Leu by the brush border membrane vesicles of porcine small intestine have been studied. The effect of aminopeptidase N inhibitors and leucine-binding protein on accumulation rates has also been tested. Comparison of the kinetic parameters for uptake and hydrolysis of Gly—L-Leu makes it possible to conclude that the dipeptide transfer includes two conjugated steps, viz., hydrolysis catalysed by aminopeptidase N and transport of the resultant free amino acids by a specific carrier.

The uptake of free and esterified cholesterol at the brush border membrane is protein-mediated. Here we show that this sterol uptake is effectively inhibited by exchangeable serum apolipoproteins. Binding of the apolipoprotein to the brush border membrane mediates the inhibitory effect. Evidence is presented to show that the structural motif responsible for the inhibition is the amphipathic α-helix.

Effects of phosphate intake on distribution of type II Na/P i cotransporter mRNA in rat kidney. Background Renal phosphate (Pi) reabsorption is regulated by dietary Pi intake, as well as in other ways. Changes in Pi reabsorption are associated with the modulation of sodium/Pi cotransporter type II (NaPi-2) protein abundance in the brush border membrane (BBM) of proximal tubules (PTs) and of renal NaPi-2 mRNA levels. In this study, we address whether the NaPi-2 protein and NaPi-2 mRNA distribution patterns in the renal cortex vary in parallel with changes of dietary Pi intake. Methods We investigated in cryosections of perfusion-fixed rat kidneys by in situ hybridization (ISH) and immunohistochemistry (IHC) the distribution patterns of NaPi-2 mRNA and of NaPi-2 protein one week, two hours, and four hours after changes in dietary Pi intake. Results NaPi-2 mRNA and NaPi-2 protein were present in PTs exclusively. In rats adapted to one week of high Pi intake, signals for NaPi-2 mRNA and NaPi-2 protein in cortical PTs were weak, except in the convoluted parts of PTs of juxtamedullary nephrons. After one week of low Pi intake, the ISH and IHC signals for NaPi-2 were high in PT segments in all cortical levels. The switch from a chronic high to a low Pi intake within two and four hours induced no increase and a slight increase, respectively, in the NaPi-2 mRNA signal in PTs of midcortical and of superficial nephrons, whereas in the BBM of these nephrons, NaPi-2 protein was markedly up-regulated. Two and four hours after switching from low to high Pi intake, the overall high ISH signal for NaPi-2 mRNA was unchanged, whereas NaPi-2 protein staining was drastically down-regulated in the BBM of PTs from superficial and midcortical nephrons. Conclusions The marked changes in NaPi-2 protein abundance in the BBM, following altered dietary Pi intake, precede corresponding changes at the RNA level by several hours. Thus, the early adaptation to altered Pi intake involves mRNA-independent mechanisms. The up- or down-regulation of NaPi-2 protein abundance in the BBM and NaPi-2 mRNA in PT affects mainly midcortical and superficial nephrons.

The brush border membrane of the insect midgut is an initial site for interaction of insecticidal proteins. We have investigated the possibility that it may contain a target site for two insecticidal fungal toxins, destruxin and efrapeptin, both of which are ATPase inhibitors. We have studied the effects of the toxins on the hydrolytic activity of a vacuolar type ATPase (V-ATPase) that we have identified from Galleria mellonella midgut columnar cell brush border membrane vesicles (BBMV) by its cation and pH dependence, sensitivity to proton pump inhibitors and K(m) (0.49 mM ATP). Efrapeptin strongly inhibited the BBMV V-ATPase but destruxin had little effect. We compared the effects of the inhibitors on known plant membrane hydrolytic enzymes, and although the vacuolar pyrophosphatase and plasma membrane ATPase were not inhibited by the toxins, the V-ATPase from mung bean, but not barley, was inhibited (50%) by 10 microM concentrations of both compounds. Different forms of the toxins were tested on the ATPases and destruxin B and efrapeptin F were the most effective. Kinetic analysis showed that the purified forms of both compounds inhibited the V-ATPases uncompetitively and modelling of data for inhibition of the BBMV V-ATPase by efrapeptin at concentrations of 0.06--12 microM yielded a K(i) of 0.125 microM.