Alteration of a Cry1A Shared Binding Site in a Cry1Ab-Selected Colony of Ostrinia furnacalis

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.

Berberine Decreases Intestinal GLUT2 Translocation and Reduces Intestinal Glucose Absorption in Mice

Postprandial hyperglycemia is an important causative factor of type 2 diabetes mellitus, and permanent localization of intestinal GLUT2 in the brush border membrane is an important reason of postprandial hyperglycemia. Berberine, a small molecule derived from Coptidis rhizome, has been found to be potent at lowering blood glucose, but how berberine lowers postprandial blood glucose is still elusive. Here, we investigated the effect of berberine on intestinal glucose transporter 2 (GLUT2) translocation and intestinal glucose absorption in type 2 diabetes mouse model. Type 2 diabetes was induced by feeding of a high-fat diet and injection of streptozotocin and diabetic mice were treated with berberine for 6 weeks. The effects of berberine on intestinal glucose transport and GLUT2 translocation were accessed in isolated intestines and intestinal epithelial cells (IEC-6), respectively. We found that berberine treatment improved glucose tolerance and systemic insulin sensitivity in diabetic mice. Furthermore, berberine decreased intestinal glucose transport and inhibited GLUT2 translocation from cytoplasm to brush border membrane in intestinal epithelial cells. Mechanistically, berberine inhibited intestinal insulin-like growth factor 1 (IGF-1R) phosphorylation and thus reduced localization of PLC-β2 in the membrane, leading to decreased GLUT2 translocation. These results suggest that berberine reduces intestinal glucose absorption through inhibiting IGF-1R-PLC-β2-GLUT2 signal pathway.

Changes in the Expression of Renal Brush Border Membrane N-Glycome in Model Rats with Chronic Kidney Diseases

Chronic kidney disease (CKD) is defined by a reduced renal function i.e., glomerular filtration rate (GFR), and the presence of kidney damage is determined by measurement of proteinuria or albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush-border membranes (BBMs) on PT cells play an important role in maintaining the stability of PT functions. The PT BBM, a highly dynamic, organized, specialized membrane, contains a variety of glycoproteins required for the functions of PT. Since protein glycosylation regulates many protein functions, the alteration of glycosylation due to the glycan changes has attracted more interests for a variety of disease studies recently. In this work, liquid chromatography-tandem mass spectrometry was utilized to analyze the abundances of permethylated glycans from rats under control to mild CKD, severe CKD, and diabetic conditions. The most significant differences were observed in sialylation level with the highest present in the severe CKD and diabetic groups. Moreover, high mannose N-glycans was enriched in the CKD BBMs. Characterization of all the BBM N-glycan changes supports that these changes are likely to impact the functional properties of the dynamic PT BBM. Further, these changes may lead to the potential discovery of glycan biomarkers for improved CKD diagnosis and new avenues for therapeutic treatments.

Critical domains in the specific binding of radiolabelled Vip3Af insecticidal protein to brush border membrane vesicles from Spodoptera spp. and cultured insect cells

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis ). In this study, we have set up the conditions to analyze the specific binding of 125 I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but that Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine-mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125 I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding, and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125 I-DI-III. In addition, the truncated protein 125 I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo. IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for the ex vivo toxicity to Sf21 cells.

Sub-fractions from Carica Papaya Seed Extracts Can Prevent Potassium Bromate- induced Changes in Activities of Renal Brush Border Membrane Enzymes and Some Enzymes of Carbohydrate Metabolism in the Kidney of Rats

Aim: To investigate the effect of the chromatographic fractions of Carica papaya seed on KBrO3 –induced reduction in the activities of renal brush border membrane (BBM) marker enzymes and the changes in activities of some enzymes of carbohydrate metabolism in the kidney of rats. Study Design: twenty male Wistar rats were divided into four groups, five rats per group; normal control, KBrO3 control, papaya fraction control and KBrO3 group administered with 126mg/kg body weight of the most active fraction of partially purified methanol extract of C. papaya for 48 hours. Place and Duration of Study: Department of Biochemistry Laboratory, Faculty of Basic Medical Sciences, Bayero University Kano, Nigeria. Methodology: The activities of renal BBM marker enzymes: γ-glutamyl transferase, alkaline phosphatase, maltase and leucine aminopeptidase were assayed in homogenates of renal cortex and medulla, and in brush border membrane vesicle (BBMV) isolated from cortex using standard methods. Furthermore, activities of the following enzymes representing different pathways of carbohydrate metabolism were determined in renal homogenates: hexokinase (HK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose 6-phosphatase (G6P), fructose 1,6-bisphosphatase (FBP), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME). Results: KBrO3 administration significantly (P<0.05) decreases the activities of all the BBM marker enzymes in renal homogenates and BBMV. It also decreases the activities of MDH, G6P, FBP and G6PD, and significantly increases (P<0.05) that of HK, LDH and ME in renal homogenates however co-administration of most active fraction of C. papaya seed prevented all the KBrO3 -induced changes in these biochemical parameters. Conclusion: Chromatographic fractions of C. papaya seed extract possesses potent phytochemicals that could prevent KBrO3 –induced reduction in activities of renal BBM marker enzymes and the changes in enzymes of carbohydrate metabolism studied and therefore could be analyzed further to isolate the bioactive compounds.

Quinoa Fiber and Quercetin Alter the Composition and Function of the Cecal Microbiome and Improve Brush Border Membrane Functionality and Morphology In-Vivo (Gallus gallus)

Objectives Assessment and comparison of the effects of various concentrations of soluble extracts of quinoa fiber (Chenopodium quinoa Willd.) and quercetin-3-glucoside on the zinc and iron status, brush border membrane (BBM) functionality, intestinal morphology, and cecal bacterial populations in-vivo (Gallus gallus). Methods The study utilized Gallus gallus intra-amniotic feeding, a clinically validated method to assess the effects of quinoa, quercetin, and control using seven groups (no injection, 18 Ω H2O, 5% inulin, 1% quercetin 3-glucoside, 2.5% quinoa fiber, 5% quinoa fiber, 1% quercetin 3-glucoside + 5% quinoa fiber). Upon hatch, the cecum, duodenum, pectoral muscle, liver, and blood samples were collected for the estimation of the relative abundance of the gut microbiome, mRNA gene expression Zn and Fe-related transporter proteins and brush border membrane functionality and morphology, glycogen, relative expression of lipid-related genes and hemoglobin levels, respectively. Results The results demonstrated an increase (P &lt; 0.05) in villi height, weight, and surface area in the groups administered with quercetin, and a dose-dependent increase was observed with quinoa soluble fiber treatment. Additionally, an increase in ferroportin and duodenal cytochrome B (DcytB) was observed in the group injected with both quinoa and quercetin. Similarly, zinc transporter 7 (ZnT7) and sucrose-isomaltase (SI) gene expression was upregulated in this group. Further, the administration of quinoa soluble fiber altered the composition and function of the cecal microbiome. Conclusions The evidence suggests that quinoa and quercetin have a synergistic effect, together they are found to improve BBM morphology and functionality, affect the intestinal microbiome, increase short-chain fatty acid production, and thereby improving mineral solubility. Quinoa fibers, a polyphenol-rich superfood, may help fight micronutrient deficiencies in target populations. Funding Sources N/A.

FC 004PHOSPHATE FACILITATES PHOSPHATURIA BY PIT-2-MEDIATED ACTIVATION OF ERK1/2 SIGNALLING INTERNALIZING NAPI-2A FROM THE APICAL BRUSH BORDER MEMBRANE INDEPENDENT OF FGF23

Background and Aims Increased phosphate load stimulates the secretion of fibroblast growth factor (FGF) 23 in the bone leading to decreased phosphate reabsorption in the kidney. FGF23 activates FGFR1/Klotho/ERK1/2 signalling in proximal tubule cells to suppress type II sodium phosphate transporters NaPi-2a and NaPi-2c in the apical brush border membrane (BBM) resulting in lower serum phosphate levels. The type III sodium-dependent phosphate transporters PiT-1 and PiT-2 are expressed in key organs of phosphate regulation and were shown to activate ERK1/2 in osseous cells in the presence of high extracellular phosphate. Furthermore, PiT-2 was shown to be responsible for the phosphate-dependent FGF23 secretion in bone cells. Whether phosphate itself can be sensed by kidney cells and stimulate its own excretion remains unknown. The aim of our study was to examine the molecular mechanism regulating renal phosphate transport in the setting of chronic oral phosphate loading in mice and to analyse phosphate sensing as well as phosphaturic actions of phosphate itself independent of FGF23. Method First, eight-week-old male C57BL/6 wildtype mice were fed a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD). Mice were sacrificed after six months and blood and urine were collected to determine parameters of phosphate homeostasis. Kidneys were isolated to evaluate the HPD-induced regulation of phosphate transporters by qPCR, immunoblot and histological analyses. Second, murine proximal tubule (mPT) cells were stimulated with either phosphate or FGF23 in the presence or absence of Foscarnet, as an inhibitor of phosphate transporters, to verify the molecular mechanism of phosphate sensing. Results Although, HPD caused significantly elevated circulating levels of intact FGF23 which resulted in hyperphosphaturia, serum phosphate levels were still enhanced compared to NPD-fed mice. Renal Klotho protein expression was significantly reduced in HPD mice and histological staining demonstrated lower Klotho accumulation in proximal and distal tubule cells, while FGFR1 was not altered. The FGF23/Klotho/FGFR1 downstream pathway revealed neither a clear activation of the ERK1/2 signalling pathway nor induction of the transcription factor Egr-1 due to HPD. Nevertheless, NaPi-2a mRNA expression was significantly reduced in HPD-fed mice compared to NPD group and NaPi-2c was unchanged. The amount of NaPi-2a protein in isolated BBM vesicles of HPD-fed mice was lower compared to NPD and immunofluorescent staining confirmed the internalisation of NaPi-2a from the apical BBM. Among the type III sodium-dependent phosphate cotransporters, renal PiT-1 mRNA expression was not altered in HPD-fed mice, but PiT-2 was significantly increased compared to NPD group and immunofluorescent staining revealed an enhanced localization of PiT-2 on the basolateral membrane of proximal tubule cells. Stimulation of mPTs with phosphate or FGF23 increased the expression of PiT-2, induced the phosphorylation of ERK1/2 and decreased NaPi-2a in vitro. The pre-treatment with Foscarnet blunted the phosphate-mediated activation of ERK1/2 signalling pathway, but not the FGF23-induced effects, suggesting a direct phosphate transporter-regulating mechanism of high phosphate in renal proximal tubule cells. Conclusion A chronic high dietary intake of phosphate results in downregulation of renal Klotho causing hyperphosphatemia, suggesting in part a renal resistance of FGF23/Klotho signalling pathway. However, HPD-induced internalization NaPi-2a from the apical BBM pointing to an FGF23-independent mechanism regulating phosphate reabsorption. Our data indicate that in the settings of high phosphate-mediated renal resistance of FGF23, phosphate itself may stimulate its urinary secretion via PiT-2-mediated activation of ERK1/2 signalling pathway which results in NaPi-2a downregulation and hyperphosphaturia independent of FGF23.

CD36 Senses Dietary Lipids and Regulates Lipids Homeostasis in the Intestine

Dietary lipids absorbed in the intestine are closely related to the development of metabolic syndrome. CD36 is a multi-functional scavenger receptor with multiple ligands, which plays important roles in developing hyperlipidemia, insulin resistance, and metabolic syndrome. In the intestine, CD36 is abundant on the brush border membrane of the enterocytes mainly localized in proximal intestine. This review recapitulates the update and current advances on the importance of intestinal CD36 in sensing dietary lipids and regulating intestinal lipids uptake, synthesis and transport, and regulating intestinal hormones secretion. However, further studies are still needed to demonstrate the complex interactions between intestinal CD36 and dietary lipids, as well as its importance in diet associated metabolic syndrome.

Evaluation of the Relative Available Energy of Cyclic Nigerosylnigerose (CNN) using breath hydrogen excretion in healthy humans

Cyclic nigerosylnigerose (CNN) is a cyclic tetrasaccharide with properties distinct from those of other conventional cyclodextrins. We investigated the relative available energy of CNN in healthy humans. CNN digestibility was determined using brush border membrane vesicles from the small intestines of rats. CNN was not hydrolyzed by rat intestinal enzymes. To investigate breath hydrogen excretion, thirteen human subjects were included in a double-blind cross-over, randomized, placebo-controlled study. The effects of CNN on hydrogen excretion were compared with those of a typical nondigestible, fermentable fructooligosaccharide (FOS). In the study participants, hydrogen excretion hardly increased upon CNN and was remarkably lower than for FOS. The available energy value was determined using the fermentability based on breath hydrogen excretion and was evaluated as 0 kcal/g for CNN. CNN was hardly metabolized and hence may be used as a low-energy dietary fiber.