Brush Border Membrane Research Articles

Intestinal Epithelial Cell Brush Border Membrane Cl:HCO3 Exchanger Regulation by Mast Cells in Chronic Ileitis

Malabsorption of NaCl is the primary cause of diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption occurs via the dual operation of Na:H and Cl:HCO3 exchange in the brush border membrane (BBM) of villus cells. Cl:HCO3 exchange is mediated by BBM transporters DRA (downregulated in adenoma) and PAT1 (putative anion transporter 1) in the mammalian small intestine. DRA/PAT1-mediated Cl:HCO3 exchange was significantly downregulated in the BBM of villus cells in a rabbit model of chronic ileitis, while Na:H exchange was unaffected. The inhibition of Cl:HCO3 exchange was restored in the rabbits when treated with a broad-spectrum immunomodulator, i.e. a glucocorticoid, indicating that the downregulation of DRA/PAT1 is likely to be immune-mediated during chronic enteritis. Mucosal mast cells are one type of key immune cells that are known to proliferate and release immune inflammatory mediators, thus playing a significant role in the pathogenesis of IBD. However, how mast cells may regulate DRA- and PAT1-mediated Cl:HCO3 exchange in a rabbit model of chronic ileitis is unknown. In this study, treatment of rabbits with chronic intestinal inflammation with the mast cell stabilizer ketotifen did not affect the mucosal architecture of the inflamed intestine. However, ketotifen treatment reversed the inhibition of Cl:HCO3 activity in the BBM of villus cells. This restoration of Cl:HCO3 activity to normal levels by ketotifen was found to be secondary to restoring the affinity of the exchangers for its substrate chloride. This observation was consistent with molecular studies, where the mRNA and BBM protein expressions of DRA and PAT1 remained unaffected in the villus cells under all experimental conditions. Thus, this study indicates that mast cells mediated the inhibition of coupled NaCl absorption by inhibiting Cl:HCO3 exchange in a rabbit model of chronic enteritis.

Effects of dietary succinic acid supplementation on growth performance, digestive ability, intestinal development and immunity of large yellow croaker (Larimichthys crocea) larvae

The application of artificial micro-diet is an effective way to improve and standardize the quality of aquatic animal larvae. However, the widespread adoption of micro-diet faces a bottleneck due to the limited utilization capacity of the larvae. A 30-day feeding experiment was carried out to investigate the effect of dietary succinic acid (SA) on the growth performance, digestive ability, intestinal development, and immunity of large yellow croaker larvae (initial body weight 11.33 ± 0.57 mg). Four isonitrogenous and isolipidic diets were formulated, incorporating 0.00%, 0.01%, 0.02% and 0.03% SA separately. The results showed that a diet with 0.02% SA significantly increased both the final body weight and the specific growth rate of the larvae. Regarding digestive ability, 0.01% SA supplementation significantly enhanced trypsin activity in both intestinal and pancreatic segments. In addition, 0.01% SA supplementation notably improved amylase activity in the intestinal segment, while diets with 0.01%-0.02% SA significantly improved lipase activity in the pancreatic segment. In terms of intestinal development, 0.01% SA supplementation remarkably boosted the activities of alkaline-phosphatase and leucine-aminopeptidase on brush border membrane in intestine. Furthermore, 0.03% SA supplementation significantly increased the expression of occludin. In terms of immunity, larvae fed diets with 0.01%-0.02% SA exhibited significantly higher lysozyme activity compared to the control group. Supplementation with 0.01% SA also significantly increased both iNOS activity and NO content. In summary, the findings of this study suggested that adding 0.02% SA can improve the growth performance of large yellow croaker larvae by improving digestive enzymes activities, promoting intestinal development, and enhancing nonspecific immunity.

Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers.

In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another.

Downregulation of APN1 and ABCC2 mutation in Bt Cry1Ac-resistant Trichoplusia ni are genetically independent.

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni, has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6, shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.

323 Advances in using membrane vesicles for studying intestinal functions

Abstract Enterocytes are the major cell lineage of the small intestinal epithelia along the crypt-villus axis in gut mucosa. Enterocytes participate in the gut terminal nutrient digestion and absorption; and have essential roles in nutrient and metabolite recycling and efflux, nutrient and hormonal sensing, interactions with symbiotic microbiome and binding to infectious viruses, thus influencing the host homeostasis and whole-body physiological status. Intestinal membrane vesicles have been widely used for studying various gut functions in the past several decades. Villus enterocytic brush border membrane and the apical membrane vesicles along the crypt-villus axis in gut mucosa can be prepared and isolated by Mg2+- and Ca2+-precipitation and differential centrifugation procedures. Whereas, gut mucosal basolateral membrane vesicles can be prepared, fractionated and differentiated from other intracellular organelles such as the mitochondrial inner membrane by the Percoll gradient ultracentrifugation. Nutrient transport and exchange activities in the membrane vesicles in vitro are conducted often by rapid hand filtration, and in excess, in an automated fast sampling apparatus. Nutrient transport and exchange activity kinetics are derived according to the well-established classic Michaelis-Menten and the tracer inhibitory kinetic models with a simple linear diffusional component. Physiological and biochemical properties associated with various nutrient uptake systems such as pH optimum, requirements for co-transported ions (e.g., Na+, K+, Cl-), transporter affinity and substrate specificity can be effectively investigated in vitro with the membrane vesicles by altering the compositions of uptake buffers and intra-vesicular pre-loading buffers. Some of the fundamental gut functions are established via using the membrane vesicles in different mammalian species. And these include H+-peptide cotransport, Na+-cotransport and Na+-independent systems of hexose uptake across the apical membrane and the low-affinity Na+-independent hexose bidirectional uptake across the basolateral membrane that were characterized in rodents; Na+-dependent cotransport and Na+-independent systems of amino acid (AA) transport across the apical membrane that were revealed in rodents and pigs; as well as the Na+-hexose and AA cotransport and the co-expression of the coronavirus SARS-CoV-2 host epithelial apical membrane binding receptor, i.e., the angiotensin-converting enzyme-2 (ACE2), along the gut crypt-villus axis that were identified in liquid formula-fed piglets. In the post genomic and metagenomic sequencing era, further research efforts need to pursue understanding of expression and various functionality of the gut terminal hydrolases, nutrient transporters, nutrient exchangers, nutrient sensors, substrate and particle receptors, taste receptors, viral binding receptors such as ACE2 and hormonal receptors and their interactions with luminal factors such as nutrient, metabolites, symbiotic bacterial outer membrane vesicles etc., along the longitudinal and crypt-villus axes, through using the mammalian primary enterocytic apical and the basolateral membrane vesicles under various research settings.

Utility of Cry1Ja for Transgenic Insect Control.

Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development.

Dietary supplementation of microencapsulated botanicals and organic acids enhances the expression and function of intestine epithelial digestive enzymes and nutrient transporters in broiler chickens

Organic acids and botanicals have shown protective effects on gut barrier and against inflammation in broilers. However, their effects on intestinal digestive enzymes and nutrients transporters expression and functions have not been fully studied. The objective of this study was to understand how a microencapsulated blend of botanicals and organic acids affected intestinal enzyme activities and nutrient transporters expression and functions in broilers. A total of 288 birds were assigned to a commercial control diet or diet supplemented with 500 g/MT (metric ton) of the microencapsulated additive. Growth performance was recorded weekly. At d 21 and d 42, jejunum and ileum were isolated for enzyme (maltase, sucrase, and aminopeptidase) and transporter (SGLT1, GLUT2, GLUT1, EAAT3, B0AT1, and PepT1) analyses. Jejunum specific nutrients (glucose, alanine, and glutamate) transport activities were evaluated by Ussing chamber. Protein expression of nutrient transporters in small intestine were measured in mucosa and brush-border membrane (BBM) samples by western blot. Intestinal gene expression of the transporters was determined by RT-PCR. Statistical analysis was performed using Student's t-test comparing the supplemented diet to the control. The feed efficiency was significantly improved through the study period in the supplemented group (P ≤ 0.05). Significant changes of intestinal histology were shown in both jejunum (P ≤ 0.10) and ileum (P ≤ 0.05) after 21 d of treatment. At d21, jejunal maltase activity was upregulated (P ≤ 0.10). The Ussing chamber transport of glucose and alanine was increased, which was in line with increased gene expression (GLUT2, GLUT1, EAAT3, and B0AT1) (P ≤ 0.10 and P ≤ 0.05, respectively) and BBMV protein levels (B0AT1, P < 0.10). At d21, ileal sucrase and maltase activities were upregulated (P ≤ 0.05). Increased expressions of GLUT1, EAAT3, and B0AT1 were observed in both mRNA and protein levels (P ≤ 0.05). Similar pattern of changes was also shown at d42 of age. Our results suggest that feeding microencapsulated additives improves intestinal nutrient digestion and transporter expression and function in broilers, thereby enhancing feed efficiency.

Receptor interactions of protoxin and activated Vip3Aa structural conformations in Spodoptera exigua.

The Vip3Aa insecticidal protein, produced by Bacillus thuringiensis, has been effectively used in commercial Bt-crops to manage lepidopteran pests. Upon ingestion by larvae, the protoxin is processed by midgut proteases into the activated protein and binds specifically to its receptors in the midgut, leading to insect mortality. Cryo-EM resolution of the trypsin-processed Vip3Aa protein unveiled structural remodelling of the N-terminal region during the transition from protoxin to activated protein. This conformational change has been demonstrated to be crucial for toxicity against Spodoptera exigua larvae, a major global lepidopteran pest. In this study, we investigated the relevance of the structural remodelling for the specific binding to midgut receptors. We conducted in vitro binding assays with radiolabelled proteins and brush border membrane vesicles (BBMV) from S. exigua, employing structural mutants that lock the protein in either its protoxin or its activated conformation. Our results indicate that both structural stages of the protein share binding sites in the midgut epithelium. Moreover, in vivo competition assays revealed that Vip3Aa is able to bind to functional receptors in S. exigua larvae both as protoxin and as activated protein. Altogether, our findings point to both structural conformations contributing to receptor binding. In vivo, either spontaneous structural shift upon proteolytic cleavage or receptor-mediated remodelling could be occurring. However, the timing and context in which the conformational change occurs could influence membrane insertion and toxicity. Our results show the complex interplay between proteolytic processing, protein structure and receptor interactions in Vip3Aa's toxicity. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification of Antioxidant Methyl Derivatives of Ortho-Carbonyl Hydroquinones That Reduce Caco-2 Cell Energetic Metabolism and Alpha-Glucosidase Activity.

α-glucosidase, a pharmacological target for type 2 diabetes mellitus (T2DM), is present in the intestinal brush border membrane and catalyzes the hydrolysis of sugar linkages during carbohydrate digestion. Since α-glucosidase inhibitors (AGIs) modulate intestinal metabolism, they may influence oxidative stress and glycolysis inhibition, potentially addressing intestinal dysfunction associated with T2DM. Herein, we report on a study of an ortho-carbonyl substituted hydroquinone series, whose members differ only in the number and position of methyl groups on a common scaffold, on radical-scavenging activities (ORAC assay) and correlate them with some parameters obtained by density functional theory (DFT) analysis. These compounds' effect on enzymatic activity, their molecular modeling on α-glucosidase, and their impact on the mitochondrial respiration and glycolysis of the intestinal Caco-2 cell line were evaluated. Three groups of compounds, according their effects on the Caco-2 cells metabolism, were characterized: group A (compounds 2, 3, 5, 8, 9, and 10) reduces the glycolysis, group B (compounds 1 and 6) reduces the basal mitochondrial oxygen consumption rate (OCR) and increases the extracellular acidification rate (ECAR), suggesting that it induces a metabolic remodeling toward glycolysis, and group C (compounds 4 and 7) increases the glycolysis lacking effect on OCR. Compounds 5 and 10 were more potent as α-glucosidase inhibitors (AGIs) than acarbose, a well-known AGI with clinical use. Moreover, compound 5 was an OCR/ECAR inhibitor, and compound 10 was a dual agent, increasing the proton leak-driven OCR and inhibiting the maximal electron transport flux. Additionally, menadione-induced ROS production was prevented by compound 5 in Caco-2 cells. These results reveal that slight structural variations in a hydroquinone scaffold led to diverse antioxidant capability, α-glucosidase inhibition, and the regulation of mitochondrial bioenergetics in Caco-2 cells, which may be useful in the design of new drugs for T2DM and metabolic syndrome.

Effect of supplemental milk replacer and liquid starter diet for 4 and 11 days postweaning on intestinal parameters of weaned piglets and growth to slaughter

Reduced piglet feed intake immediately postweaning (pw) leads to disruption of small intestine structure and function and reduced growth. Our objective was to evaluate the effect of providing supplemental milk or liquid starter diet for either 4 or 11 days pw, on intestinal parameters of newly weaned piglets and growth to slaughter. At weaning (28 ± 0.6 days old), five hundred and eighty−seven piglets ((Large White × Landrace) × Duroc) were divided into 59 pen groups, each containing 9–10 same sex (entire male or female) piglets. The pen groups were blocked by sex and weaning weight and provided with ad-libitum access to one of five dietary treatments: (1) Dry pelleted starter diet (control; CON); (2) CON+liquid milk replacer for 4 days pw (M4); (3) CON+liquid milk replacer for 11 days pw (M11); (4) CON+liquid starter diet for 4 days pw (S4) and (5) CON+liquid starter diet for 11 days pw (S11). Pen groups were weighed at weaning, days 11, 20, 28, and 47 pw and at target sale weight. Feed disappearance (on a DM basis) was recorded on each weighing day. On day 7 pw, 10 piglets per treatment were euthanised to collect small intestine tissue samples for determination of villus height (VH), crypt depth and brush-border membrane enzyme activity. Data were analysed using SAS-version 9.4. Between days 0 and 11 pw, M11 increased average daily feed intake by 48% and average daily gain (ADG) by 57% compared to CON (P < 0.05), and increased ADG by 54% (P < 0.05) compared to S4. Piglets on M11 also had improved feed conversion efficiency compared with CON piglets between days 0 and 11 pw. Treatment did not affect growth performance after day 28 pw, or carcass parameters at slaughter. At day 7 pw, M11 piglets had 37% higher jejunal VH than CON piglets (P < 0.05) and S11 piglets had 28% higher ileal VH than S4 piglets (P < 0.05). M11 piglets had up to 150% higher ileal sucrase activity than M4, S4 and S11 piglets (P < 0.05) and 180% higher ileal maltase activity than S4 piglets (P < 0.05). In conclusion, M11 reduced the immediate negative effects of weaning, as it was associated with increased feed intake, growth, brush-border membrane enzyme activity and improved intestinal structure early pw. However, there were no carryover effects of any of the liquid supplements on growth or feed efficiency or carcass weight at slaughter.

Composition and abundance of midgut plasma membrane proteins in two major hemipteran vectors of plant viruses, Bemisia tabaci and Myzus persicae.

Multiple species within the order Hemiptera cause severe agricultural losses on a global scale. Aphids and whiteflies are of particular importance due to their role as vectors for hundreds of plant viruses, many of which enter the insect via the gut. To facilitate the identification of novel targets for disruption of plant virus transmission, we compared the relative abundance and composition of the gut plasma membrane proteomes of adult Bemisia tabaci (Hemiptera: Aleyrodidae) and Myzus persicae (Hemiptera: Aphididae), representing the first study comparing the gut plasma membrane proteomes of two different insect species. Brush border membrane vesicles were prepared from dissected guts, and proteins extracted, identified and quantified from triplicate samples via timsTOF mass spectrometry. A total of 1699 B. tabaci and 1175 M. persicae proteins were identified. Following bioinformatics analysis and manual curation, 151 B. tabaci and 115 M. persicae proteins were predicted to localize to the plasma membrane of the gut microvilli. These proteins were further categorized based on molecular function and biological process according to Gene Ontology terms. The most abundant gut plasma membrane proteins were identified. The ten plasma membrane proteins that differed in abundance between the two insect species were associated with the terms "protein binding" and "viral processes." In addition to providing insight into the gut physiology of hemipteran insects, these gut plasma membrane proteomes provide context for appropriate identification of plant virus receptors based on a combination of bioinformatic prediction and protein localization on the surface of the insect gut.

Effects of Pea (Pisum sativum) Prebiotics on Intestinal Iron-Related Proteins and Microbial Populations In Vivo (Gallus gallus).

Iron deficiency remains a public health challenge globally. Prebiotics have the potential to improve iron bioavailability by modulating intestinal bacterial population, increasing SCFA production, and stimulating expression of brush border membrane (BBM) iron transport proteins among iron-deficient populations. This study intended to investigate the potential effects of soluble extracts from the cotyledon and seed coat of three pea (Pisum sativum) varieties (CDC Striker, CDC Dakota, and CDC Meadow) on the expression of BBM iron-related proteins (DCYTB and DMT1) and populations of beneficial intestinal bacteria in vivo using the Gallus gallus model by oral gavage (one day old chicks) with 1 mL of 50 mg/mL pea soluble extract solutions. The seed coat treatment groups increased the relative abundance of Bifidobacterium compared to the cotyledon treatment groups, with CDC Dakota seed coat (dark brown pigmented) recording the highest relative abundance of Bifidobacterium. In contrast, CDC Striker Cotyledon (dark-green-pigmented) significantly increased the relative abundance of Lactobacillus (p < 0.05). Subsequently, the two dark-pigmented treatment groups (CDC Striker Cotyledon and CDC Dakota seed coats) recorded the highest expression of DCYTB. Our study suggests that soluble extracts from the pea seed coat and dark-pigmented pea cotyledon may improve iron bioavailability by affecting intestinal bacterial populations.

Type 1 diabetes human enteroid studies reveal major changes in the intestinal epithelial compartment

Lack of understanding of the pathophysiology of gastrointestinal (GI) complications in type 1 diabetes (T1D), including altered intestinal transcriptomes and protein expression represents a major gap in the management of these patients. Human enteroids have emerged as a physiologically relevant model of the intestinal epithelium but establishing enteroids from individuals with long-standing T1D has proven difficult. We successfully established duodenal enteroids using endoscopic biopsies from pediatric T1D patients and compared them with aged-matched enteroids from healthy subjects (HS) using bulk RNA sequencing (RNA-seq), and functional analyses of ion transport processes. RNA-seq analysis showed significant differences in genes and pathways associated with cell differentiation and proliferation, cell fate commitment, and brush border membrane. Further validation of these results showed higher expression of enteroendocrine cells, and the proliferating cell marker Ki-67, significantly lower expression of NHE3, lower epithelial barrier integrity, and higher fluid secretion in response to cAMP and elevated calcium in T1D enteroids. Enteroids established from pediatric T1D duodenum identify characteristics of an abnormal intestinal epithelium and are distinct from HS. Our data supports the use of pediatric enteroids as an ex-vivo model to advance studies of GI complications and drug discovery in T1D patients.

#1448 Regulation of renal phosphate excretion independent of FGF23 and PTH

Abstract Background and Aims The phosphaturic hormones fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) promote renal phosphate excretion through FGFR1/Klotho and PTH1R-mediated ERK1/2 activation, respectively, leading to inhibition of the sodium-phosphate cotransporters NPT2a (SLC34A1) and NPT2c (SLC34A3) in proximal tubule (PT) cells. In bone cells, high extracellular phosphate can directly activate ERK1/2 via the sodium-phosphate cotransporters PiT-1 (SLC20A1) and PiT-2 (SLC20A2). To date, it is unclear to what extent phosphate can also regulate its own excretion in the kidney independently of FGF23 and PTH. Method To further investigate the role of phosphate in the regulation of renal phosphate homeostasis, male C57BL/6 mice were placed on a 0.8% control phosphate diet (CPD) or a 2% high phosphate diet (HPD) for 6 months. After four months, one HPD group was additionally treated with a calcimimetic (etelcalcetide) to lower FGF23 and PTH. In vitro, PT cells were stimulated with phosphate, FGF23 or PTH ± the phosphate transporter inhibitor foscarnet. Results HPD resulted in increased plasma concentrations of FGF23 and PTH, which were associated with reduced tubular phosphate reabsorption and increased fractional phosphate excretion. RNAseq analyses from isolated PT cells showed a reduction of Fgfr1, Kl, Slc34a1 and Slc34a3 and an induction of Slc20a2 in the HPD group compared to CPD. qPCR and immunoblot analyses of whole kidney tissue confirmed the reduction of the FGF23 co-receptor Klotho in the HPD group, while Fgfr1 was not significantly altered. Protein expression of NPT2a was decreased in isolated brush border membrane vesicles from HPD-fed mice, and immunofluorescence staining showed internalization of NPT2a from the apical brush border membrane. Treatment with etelcalcetide resulted in a reduction of circulating FGF23 and PTH, whereas urinary phosphate excretion in HPD mice remained elevated. In cultured PT cells, high phosphate induced phosphorylation of ERK1/2 and mRNA expression of Slc20a2, while Slc34a1 was suppressed. Phosphate-mediated PiT-2/ERK1/2 activation could be blocked by simultaneous treatment with foscarnet. Conclusion Our data suggest that high phosphate leads to internalization of NPT2a via direct activation of the PiT-2/ERK1/2 signaling cascade, independent of FGF23 and PTH, resulting in increased renal phosphate excretion.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa’s different domains could potentially be helpful in the rational design of improved toxins.In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

In Vivo and In Vitro Interactions between Exopolysaccharides from Bacillus thuringensis HD270 and Vip3Aa11 Protein.

Bacillus thuringiensis (Bt) secretes the nutritional insecticidal protein Vip3Aa11, which exhibits high toxicity against the fall armyworm (Spodoptera frugiperda). The Bt HD270 extracellular polysaccharide (EPS) enhances the toxicity of Vip3Aa11 protoxin against S. frugiperda by enhancing the attachment of brush border membrane vesicles (BBMVs). However, how EPS-HD270 interacts with Vip3Aa11 protoxin in vivo and the effect of EPS-HD270 on the toxicity of activated Vip3Aa11 toxin are not yet clear. Our results indicated that there is an interaction between mannose, a monosaccharide that composes EPS-HD270, and Vip3Aa11 protoxin, with a dissociation constant of Kd = 16.75 ± 0.95 mmol/L. When EPS-HD270 and Vip3Aa11 protoxin were simultaneously fed to third-instar larvae, laser confocal microscopy observations revealed the co-localization of the two compounds near the midgut wall, which aggravated the damage to BBMVs. EPS-HD270 did not have a synergistic insecticidal effect on the activated Vip3Aa11 protein against S. frugiperda. The activated Vip3Aa11 toxin demonstrated a significantly reduced binding capacity (548.73 ± 82.87 nmol/L) towards EPS-HD270 in comparison to the protoxin (34.96 ± 9.00 nmol/L). Furthermore, this activation diminished the affinity of EPS-HD270 for BBMVs. This study provides important evidence for further elucidating the synergistic insecticidal mechanism between extracellular polysaccharides and Vip3Aa11 protein both in vivo and in vitro.

Role of Leptin in Regulating Renal Phosphate Transport

Elevated plasma phosphate (Pi) levels, known as hyperphosphatemia, are associated with an increased risk of cardiovascular complications and mortality. Although studies have suggested a correlation between obesity and hyperphosphatemia/phosphate toxicity, the root cause of this observation has not been established. Since hyperleptinemia was also found to be associated with abnormalities in bone structure, we hypothesized that effects on renal Pi transport are responsible for this observation. To address this question, we studied hyperleptinemic mice (db/db, n=14) and compared them to wild-type mice (WT, n=10). Blood and urine samples, as well as kidney tissues, were collected and analyzed. Body weight was approximately twice as high in db/db compared to WT mice (59±3 vs. 30±2 g, P&lt;0.05). Plasma Pi levels in db/db mice were significantly greater (~1.5-fold) compared to WT mice (2.6±0.1 vs. 1.7±0.1 mmol/L, P&lt;0.05). However, urinary Pi /creatinine ratios were similar between genotypes. Plasma Ca2+ levels in db/db mice were significantly higher compared to WT mice (2.68±0.03 vs. 2.48±0.03 mmol/L, P&lt;0.05) alongside urinary Ca2+/creatinine ratios being significantly higher in db/db mice compared to WT mice (1.6±0.4 vs. 0.3±0.1 mmol/mmol, P&lt;0.05). Plasma parathyroid hormone (PTH, regulating Pi and Ca2+) levels were similar in both genotypes. The phosphaturic hormone fibroblast growth factor 23 (FGF23), which is produced and released from bone, was slightly but significantly elevated in db/db compared to WT mice (254±12 vs. 227±18 pg/mL, P&lt;0.05). Bone formation markers (osteocalcin and procollagen type I N-propeptide) and bone resorption markers (tartrate-resistant acid phosphatase 5b and C-terminal telopeptide of type I collagen) were similar between genotypes, indicating that Pi and Ca2+ release from bone may not be causing these mineral differences. To determine the role of the kidney for the development of hyperphosphatemia, we performed 32P radiotracer studies in acutely isolated renal brush border membrane vesicles. Sodium-dependent 32P transport was not significantly different between genotypes. To determine the ambient in vivo contribution of the Na+-Pi cotransporter Npt2a, we employed acute pharmacological inhibition with PF-06869206 (Npt2a inhibitor, 30 mg/kg b.w, n=7) or vehicle (n=11) and compared plasma Pi before and 2 hours after administration in db/db mice. Vehicle treatment slightly increased plasma Pi levels compared to baseline (2.6±0.1 vs. 2.1±0.1 mmol/L, P&lt;0.05); in contrast, the Npt2a inhibitor significantly reduced (~50%) plasma Pi levels compared to baseline (1.3±0.1 vs. 2.5±0.1 mmol/L, P&lt;0.05). In summary, our study demonstrates that hyperphosphatemia in db/db mice is not caused by changes in renal Pi transport or bone turnover, suggesting that possibly intestinal mechanisms are responsible for increased Pi, and possibly Ca2+, absorption. Of note, Npt2a inhibition was effective in reducing hyperphosphatemia in db/db mice, indicating a potential therapeutic strategy. This work was supported by a VA Merit Review Award IBX004968A (to Dr. Rieg) and a Pilot Project from the USF Microbiomes Institute (to T.R. and J.D.R). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Changes in growth, morphology, and levels of digestive enzymes and growth-related hormones in early ontogeny of black scraper, Thamnaconus modestus

IntroductionThe black scraper, Thamnaconus modestus, is a highly valued marine fish species, but its output has gradually decreased in recent years, which may be due to its low survival rate during early ontogenesis.MethodsTherefore, in this study, we assessed the changes in growth, morphology, digestive enzymes, and hormone levels in T. modestus from 0–60 days post-hatching (dph) and revealed growth turning points by morphological measurement and determination of digestive enzyme activities and hormone levels. We found that ontogenesis could be divided into the larval (0–20 dph) and juvenile (20–60 dph) stages. Acid and alkaline protease activity significantly increased and decreased, respectively, from 12–25 dph, likely due to the development of stomach and gastric glands. Acid phosphatase levels significantly increased at 0 and 4 dph, which may be related to the regulation of metabolism and immune protection. A sharp increase in alkaline phosphatase levels at 20 and 25 dph was observed and was likely due to the development of the brush border membrane of enterocytes. The amylase level was significantly higher at 25, 30, and 35 dph, possibly due to better digestion and absorption during the transition from consuming Artemia to compound feed. In newly hatched larvae, the level of thyroid hormones triiodothyronine (T3) and thyroxine (T4) gradually increased and peaked at 35 dph, highlighting the importance of these hormones during the development of T. modestus. Growth hormone (GH) levels first increased from 0–8 dph, with a plateau at 8–20 dph, and then increased at 25–30–35 dph. For insulin-like growth factor 1 (IGF-1), a significant increase with a subsequent plateau was observed between 8 and 20 dph, followed by a substantial decrease between 30 and 35 dph. These results suggest that the regulating functions of GH and IGF-1 are synchronised. Digestive enzyme activity and hormone levels of abnormal fry at 30 dph were lower than those of normal fish, highlighting the importance of specific hormones, especially T4 and IGF-1, in the development of T. modestus.

Infant milk formula, produced by membrane filtration, promotes mucus production in the upper small intestine of young pigs

Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut.

In vitro and in vivo digestibility of prebiotic galactooligosacharides synthesized by β-galactosidase from Lactobacillus delbruecki subsp. bulgaricus CRL450.

The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non-digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by β-galactosidase (β-gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450-β-gal) from lactose, lactulose and lactitol. In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450-β-gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush-border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose-oligosaccharides were the most resistant to the physiological conditions of mice. In general β (1→6) linked products showed higher resistance compared to β (1→3) oligosaccharides. In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.