Activated Inositol Phosphate, Substrate for Synthesis of Prostaglandylinositol Cyclic Phosphate (Cyclic PIP)-The Key for the Effectiveness of Inositol-Feeding.

The natural cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), is biosynthesized from prostaglandin E (PGE) and activated inositol phosphate (n-Ins-P), which is synthesized by a particulate rat-liver-enzyme from GTP and a precursor named inositol phosphate (pr-Ins-P), whose 5-ring phosphodiester structure is essential for n-Ins-P synthesis. Aortic myocytes, preincubated with [3H] myo-inositol, synthesize after angiotensin II stimulation (30 s) [3H] pr-Ins-P (65% yield), which is converted to [3H] n-Ins-P and [3H] cyclic PIP. Acid-treated (1 min) [3H] pr-Ins-P co-elutes with inositol (1,4)-bisphosphate in high performance ion chromatography, indicating that pr-Ins-P is inositol (1:2-cyclic,4)-bisphosphate. Incubation of [3H]-GTP with unlabeled pr-Ins-P gave [3H]-guanosine-labeled n-Ins-P. Cyclic PIP synthase binds the inositol (1:2-cyclic)-phosphate part of n-Ins-P to PGE and releases the [3H]-labeled guanosine as [3H]-GDP. Thus, n-Ins-P is most likely guanosine diphospho-4-inositol (1:2-cyclic)-phosphate. Inositol feeding helps patients with metabolic conditions related to insulin resistance, but explanations for this finding are missing. Cyclic PIP appears to be the key for explaining the curative effect of inositol supplementation: (1) inositol is a molecular constituent of cyclic PIP; (2) cyclic PIP triggers many of insulin's actions intracellularly; and (3) the synthesis of cyclic PIP is decreased in diabetes as shown in rodents.

The Efficacy and Safety of Myo-inositol Supplementation for the Prevention of Gestational Diabetes Mellitus in Overweight and Obese Pregnant Women: A Systematic Review and Meta-Analysis.

Background: Myo-inositol has emerged as one of the preventive therapies for the development of gestational diabetes mellitus in at-risk populations. This systematic review and meta-analysis was conducted to determine the efficacy and safety of myo-inositol in decreasing the incidence of gestational diabetes in overweight and obese pregnant women. Methodology: This meta-analysis was conducted using the standard Cochrane methodology and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) 2020 guidelines. Inclusion criteria were randomized controlled trials (RCTs) that enrolled overweight and obese pregnant women and used myo-inositol supplementation. The primary outcome was the incidence of gestational diabetes mellitus at 24-28 weeks. Secondary outcomes included cesarean section rate, the incidence of pregnancy-induced hypertension, macrosomia and preterm delivery. Risk ratios (RRs) and 95% confidence intervals (CIs) were used for dichotomous data. Results: Six RCTs were included. Compared to standard micronutrient supplementation, standard dose of myo-inositol (4 g) may reduce the incidence of GDM (RR 0.54; CI [0.30, 0.96]; n = 887 women), but the certainty of evidence is low to very low. With low-dose myo-inositol however, evidence is uncertain about its benefit on the incidence of gestational diabetes mellitus in overweight and obese women with RR 0.71; CI [0.14, 3.50]. No adverse effects were noted. For the secondary outcomes, standard dose myo-inositol appears to reduce the incidence of pregnancy-induced hypertension and preterm delivery, but the certainty of evidence is low to very low. Conclusion: Current evidence is uncertain on the potential benefit of myo-inositol supplementation in overweight and obese pregnant women. While studies show that 4 g myo-inositol per day may decrease the incidence of GDM, pregnancy-induced hypertension and pre-term birth with no associated risk of serious adverse events, the certainty of evidence is low to very low. Future high-quality trials may provide more compelling evidence to support practice recommendations.

Effect of Phytate (InsP6) and Other Inositol-Phosphates (InsP5, InsP4, InsP3, InsP2) on Crystallization of Calcium Oxalate, Brushite, and Hydroxyapatite.

Pathological calcifications may consist of calcium oxalate (CaOx), hydroxyapatite (HAP), and brushite (BRU). The objective of this study was to evaluate the effect of phytate (inositol hexakisphosphate, InsP6), InsP6 hydrolysates, and individual lower InsPs (InsP5, InsP4, InsP3, and InsP2) on the crystallization of CaOx, HAP and BRU in artificial urine. All of the lower InsPs seem to inhibit the crystallization of calcium salts in biological fluids, although our in vitro results showed that InsP6 and InsP5 were stronger inhibitors of CaOx crystallization, and InsP5 and InsP4 were stronger inhibitors of BRU crystallization. For the specific in vitro experimental conditions we examined, the InsPs had very weak effects on HAP crystallization, although it is likely that a different mechanism is responsible for HAP crystallization in vivo. For example, calciprotein particles seem to have an important role in the formation of cardiovascular calcifications in vivo. The experimental conditions that we examined partially reproduced the in vivo conditions of CaOx and BRU crystallization, but not the in vivo conditions of HAP crystallization.

Cyclitols: From Basic Understanding to Their Association with Neurodegeneration.

One of the most common cyclitols found in eukaryotic cells-Myo-inositol (MI) and its derivatives play a key role in many cellular processes such as ion channel physiology, signal transduction, phosphate storage, cell wall formation, membrane biogenesis and osmoregulation. The aim of this paper is to characterize the possibility of neurodegenerative disorders treatment using MI and the research of other therapeutic methods linked to MI's derivatives. Based on the reviewed literature the researchers focus on the most common neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and Spinocerebellar ataxias, but there are also works describing other seldom encountered diseases. The use of MI, d-pinitol and other methods altering MI's metabolism, although research on this topic has been conducted for years, still needs much closer examination. The dietary supplementation of MI shows a promising effect on the treatment of neurodegenerative disorders and can be of great help in alleviating the accompanying depressive symptoms.

Group-specific cellular metabolism in Medulloblastoma.

BACKGROUND: Cancer metabolism influences multiple aspects of tumorigenesis and causes diversity across malignancies. Although comprehensive research has extended our knowledge of molecular subgroups in medulloblastoma (MB), discrete analysis of metabolic heterogeneity is currently lacking. This study seeks to improve our understanding of metabolic phenotypes in MB and their impact on patients' outcomes. METHODS: Data from four independent MB cohorts encompassing 1,288 patients were analysed. We explored metabolic characteristics of 902 patients (ICGC and MAGIC cohorts) on bulk RNA level. Moreover, data from 491 patients (ICGC cohort) were searched for DNA alterations in genes regulating cell metabolism. To determine the role of intratumoral metabolic differences, we examined single-cell RNA-sequencing (scRNA-seq) data from 34 additional patients. Findings on metabolic heterogeneity were correlated to clinical data. RESULTS: Established MB groups exhibit substantial differences in metabolic gene expression. By employing unsupervised analyses, we identified three clusters of group 3 and 4 samples with distinct metabolic features in ICGC and MAGIC cohorts. Analysis of scRNA-seq data confirmed our results of intertumoral heterogeneity underlying the according differences in metabolic gene expression. On DNA level, we discovered clear associations between altered regulatory genes involved in MB development and lipid metabolism. Additionally, we determined the prognostic value of metabolic gene expression in MB and showed that expression of genes involved in metabolism of inositol phosphates and nucleotides correlates with patient survival. CONCLUSION: Our research underlines the biological and clinical relevance of metabolic alterations in MB. Thus, distinct metabolic signatures presented here might be the first step towards future metabolism-targeted therapeutic options.

Myo-Inositol and D-Chiro-Inositol as Modulators of Ovary Steroidogenesis: A Narrative Review.

Myo-inositol is a natural polyol, the most abundant among the nine possible structural isomers available in living organisms. Inositol confers some distinctive traits that allow for a striking distinction between prokaryotes and eukaryotes, the basic clusters into which organisms are partitioned. Inositol cooperates in numerous biological functions where the polyol participates or by furnishing the fundamental backbone of several related derived metabolites, mostly obtained through the sequential addition of phosphate groups (inositol phosphates, phosphoinositides, and pyrophosphates). Overall myo-inositol and its phosphate metabolites display an entangled network, which is involved in the core of the biochemical processes governing critical transitions inside cells. Noticeably, experimental data have shown that myo-inositol and its most relevant epimer D-chiro-inositol are both necessary to permit a faithful transduction of insulin and of other molecular factors. This improves the complete breakdown of glucose through the citric acid cycle, especially in glucose-greedy tissues, such as the ovary. In particular, while D-chiro-inositol promotes androgen synthesis in the theca layer and down-regulates aromatase and estrogen expression in granulosa cells, myo-inositol strengthens aromatase and FSH receptor expression. Inositol effects on glucose metabolism and steroid hormone synthesis represent an intriguing area of investigation, as recent results have demonstrated that inositol-related metabolites dramatically modulate the expression of several genes. Conversely, treatments including myo-inositol and its isomers have proven to be effective in the management and symptomatic relief of a number of diseases associated with the endocrine function of the ovary, namely polycystic ovarian syndrome.

Inactivating the lipid kinase activity of PI3KC2ß is sufficient to rescue myotubular myopathy in mice.

Phosphoinositides (PIs) are membrane lipids that regulate signal transduction and vesicular trafficking. X-linked centronuclear myopathy (XLCNM), also called myotubular myopathy, results from loss-of-function mutations in the MTM1 gene, which encodes the myotubularin phosphatidylinositol 3-phosphate (PtdIns3P) lipid phosphatase. No therapy for this disease is currently available. Previous studies showed that loss of expression of the class II phosphoinositide 3-kinase (PI3K) PI3KC2ß (PI3KC2B) protein improved the phenotypes of an XLCNM mouse model. PI3Ks are well known to have extensive scaffolding functions and the importance of the catalytic activity of this PI3K for rescue remains unclear. Here, using PI3KC2ß kinase-dead mice, we show that the selective inactivation of PI3KC2ß kinase activity is sufficient to fully prevent muscle atrophy and weakness, histopathology, and sarcomere and triad disorganization in Mtm1-knockout mice. This rescue correlates with normalization of PtdIns3P level and mTORC1 activity, a key regulator of protein synthesis and autophagy. Conversely, lack of PI3KC2ß kinase activity did not rescue the histopathology of the BIN1 autosomal CNM mouse model. Overall, these findings support the development of specific PI3KC2ß kinase inhibitors to cure myotubular myopathy.

Regulation of inositol 1,2,4,5,6-pentakisphosphate and inositol hexakisphosphate levels in Gossypium hirsutum by IPK1.

MAIN CONCLUSION: The IPK1 genes, which code for 2-kinases that can synthesize Ins(1,2,4,5,6)P5 from Ins(1,4,5,6)P4, are expressed throughout cotton plants, resulting in the highest Ins(1,2,4,5,6)P5 concentrations in young leaves and flower buds. Cotton leaves contain large amounts of Ins(1,2,4,5,6)P5 and InsP6 compared to plants not in the Malvaceae family. The inositol polyphosphate pathway has been linked to stress tolerance in numerous plant species. Accordingly, we sought to determine why cotton and other Malvaceae have such high levels of these inositol phosphates. We have quantified the levels of InsP5 and InsP6 in different tissues of cotton plants and determined the expression of IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene) in vegetative and reproductive tissues. Gossypium hirsutum was found to contain four IPK1 genes that were grouped into two pair (AB, CD) where each pair consists of very similar sequences that were measured together. More IPK1AB is expressed in leaves than in roots, whereas more IPK1CD is expressed in roots than in leaves. Leaves and flower buds have more InsP5 and InsP6 than stems and roots. Leaves and roots contain more InsP5 than InsP6, whereas flower buds and stems contain more InsP6 than InsP5. Dark-grown seedlings contain more InsP5 and InsP6 than those grown under lights, and the ratio of InsP5 to InsP6 is greater in the light-grown seedlings. During 35 days of the life cycle of the third true leaf, InsP5 and InsP6 gradually decreased by more than 50%. Silencing IPK1AB and IPK1CD with Cotton Leaf Crumple Virus-induced gene silencing (VIGS) resulted in plants with an intense viral phenotype, reduced IPK1AB expression and lowered amounts of InsP5. The results are consistent with Ins(1,2,4,5,6)P5 synthesis from Ins(1,4,5,6)P4 by IPK1. This study detailed the central role of IPK1 in cotton inositol polyphosphate metabolism, which has potential to be harnessed to improve the resistance of plants to different kinds of stress.

Phytate Hydrolysate Differently Modulates the Immune Response of Human Healthy and Cancer Colonocytes to Intestinal Bacteria.

(1) Phytic acid (PA) is a component of cereal seeds and legumes, therefore its consumption is much higher in a vegan and vegetarian diet compared to a conventional diet. The diet is the main driver of metabolic activity of gut microbiota, therefore, the ability to degrade phytates by the microbiota of vegans significantly exceeds that of the gut microbiota of omnivores. The aim of the study was to investigate the early phase of the immune response of colonocytes treated with an enzymatic hydrolysate of phytic acid (hPA120) and gut bacteria. (2) Cell lines derived from healthy (NCM460D) and cancer (HCT116) colonic tissue and fecal bacteria from vegan (V) and omnivorous (O) donors were investigated. Fecal bacteria were grown in mucin and phytic acid supplemented medium. Cultured bacteria (BM) were loaded onto colonocytes alone (V BM and O BM) or in combination with the phytate hydrolysate (V BM + hPA120 and O BM + hPA120). After a treatment of 2 h, bacterial adhesion, secretion of cytokines, and the expression of genes and proteins important for immune response were determined. (3) All bacteria-treated colonocytes increased the expression of IL8 compared to controls. The significant increase of the secreted IL-8 (p < 0.01) in both cell lines was observed for O BM and O BM + hPA120. The increase of TNF, IL-1ß, and IL-10 secretion in healthy colonocytes (V BM alone and with hPA120 treatments; p < 0.05) and for TNF and IL-10 in cancer cells (treatments except O BM + hPA120 and V BM, respectively; p > 0.05) were stated. A comparison of solely the effect of hPA120 on bacteria-treated colonocytes (BM vs. BM + hPA120) showed that hPA120 decreased expression of NFkB1 and TNFR (p < 0.001) in healthy colonocytes. In cancer colonocytes, the expression of TLR4 and IL1R increased after BM + hPA120 treatment, whereas the secretion of IL-8 and MYD88 and TNFR expression decreased (p < 0.01). (4) The investigated hPA120 showed a differentiated modulatory activity on the immune response of healthy and cancer human colonocytes. Especially when analyzed independently on the gut bacteria origin, it reduced the proinflammatory response of HCT116 cells to gut bacteria, while being neutral for the bacteria-treated healthy colonocytes.

Phytate Dephosphorylation Products Also Act as Potent Inhibitors of Calcium Oxalate Crystallization.

Phytate has been classified as an anti-nutrient, but there are no adverse effects from the consumption of a balanced diet with 1 to 2 g of daily phytate (inositol-hexaphosphate, InsP6) as a calcium magnesium salt, the form naturally present in grains. Furthermore, recent research has shown that phytate consumption may prevent pathological calcifications, such as kidney stones and cardiovascular calcifications. However, many endogenous and exogenous enzymes can hydrolyze phytate to lower inositol phosphates (InsPs) that also have biological activity. We performed a controlled hydrolysis of phytate and identified the products (InsPs) using tandem mass spectrometry (MS/MS). The total level of all InsPs was measured using a non-specific methodology. In addition, we evaluated the effects of the InsP6 hydrolysates on calcium oxalate crystallization using scanning electron microscopy and measuring the time needed for the induction of crystallization. Our results indicate that InsP6 and its hydrolysis products functioned as effective inhibitors of calcium oxalate crystallization. Thus, even though InsP6 is hydrolyzed after consumption, the enzymatic products also have the potential to reduce pathological calcifications. Finally, although it is useful to measure the overall level of InsPs in biological fluids, such as urine, there is a need to develop simple analytical methods to quantify the level of individual InsPs.

A SPX domain vacuolar transporter links phosphate sensing to homeostasis in Arabidopsis.

Excess phosphate (Pi) is stored into the vacuole through Pi transporters so that cytoplasmic Pi levels remain stable in plant cells. We hypothesized that the vacuolar Pi transporters may harbor a Pi-sensing mechanism so that they are activated to deliver Pi into the vacuole only when cytosolic Pi reaches a threshold high level. We tested this hypothesis using Vacuolar Phosphate Transporter 1 (VPT1), a SPX domain-containing vacuolar Pi transporter, as a model. Recent studies have defined SPX as a Pi-sensing module that binds inositol polyphosphate signaling molecules (InsPs) produced at high cellular Pi status. We showed here that Pi-deficient conditions or mutation of the SPX domain severely impaired the transport activity of VPT1. We further identified an auto-inhibitory domain in VPT1 that suppresses its transport activity. Taking together the results from detailed structure-function analyses, our study suggests that VPT1 is in the auto-inhibitory state when Pi status is low, whereas at high cellular Pi status InsPs are produced and bind SPX domain to switch on VPT1 activity to deliver Pi into the vacuole. This thus provides an auto-regulatory mechanism for VPT1-mediated Pi sensing and homeostasis in plant cells.

Kinetics of phytate hydrolysis during storage of red kidney beans and the implication in hard-to-cook development.

In this study, the kinetics of phytate (inositol hexaphosphate, InsP6) hydrolysis by endogenous phytase in red kidney beans stored at varying conditions of temperature (25-42 °C) and moisture content (6.9-14.5%) was determined and the potential role in hard-to-cook (HTC) development was evaluated. In addition, the concept of glass transition temperature (Tg) was assessed and correlated against the rate of phytate hydrolysis. Under the conditions studied, phytate hydrolysis during storage was mainly influenced by storage temperature and time with limited influence of storage moisture content whereby the highest and lowest storage temperatures (42 °C and 25 °C) resulted in the highest and lowest hydrolysis rates (0.058 ± 0.003 and 0.003 ± 0.001 week-1). Hydrolysis of phytate resulted in formation of lower inositol phosphates, inositol pentaphosphate (InsP5) representing an intermediate whose concentration increased with storage time and temperature. The relationship between the rate of InsP6 hydrolysis and storage above the overall Tg (T - Tg) was moisture content dependent implying that this difference did not fully explain InsP6 hydrolysis. Nevertheless, for each moisture content, the rates of InsP6 hydrolysis during storage were strongly correlated (r > 0.98, p < 0.05) with rates of HTC development signifying that InsP6 hydrolysis facilitates HTC development in beans.

Nutritional properties and amino acid profile of buckwheat bread.

The effect of replacement of wheat flour with buckwheat flour at levels of 10, 20, 30, 40, and 50% on nutritional, texture, and physicochemical characteristics of bread was studied. Among others, parameters such as amino acid profile, antioxidant properties, and inositol phosphate content were determined. Amino acid score was calculated in order to evaluate the biological value of the bread protein. The breads with buckwheat flour were characterized by significantly lower whiteness of the crumb, compared to wheat bread. A positive effect of 10, 20, and 30% buckwheat flour content on the reduction of the crumb hardness, gumminess, chewiness was observed in comparison to other bread samples. A positive effect of buckwheat flour in the amount of 10-30% on the texture parameters and slowing down the process of bread staling was observed. The antioxidant properties and inositol phosphates increased with the share of buckwheat flour in the formula. A significant increase in protein was observed in bread from 20% share of buckwheat flour. The limiting amino acid of the protein of the tested flours and breads was lysine. For wheat bread, the amino acid score was 44.71% and for those with buckwheat flour it ranged from 45.67 to 75.38%.

Ruminal and post-ruminal phytate degradation of diets containing rapeseed meal or soybean meal.

This study aimed to investigate ruminal and post-ruminal degradation of phytic acid (InsP6) in diets containing either rapeseed meal (RSM) or soybean meal (SBM). In Experiment 1, the effective degradability of crude protein (CPED) and InsP6 (InsP6ED) was evaluated by incubating RSM and SBM in situ in three rumen-fistulated lactating Jersey cows for 2, 4, 6, 8, 16, 24, 48 and 72 h, and calculating effective degradability at rumen passage rates of 2% and 5%/h. In Experiment 2, eight wethers were assigned for 8 weeks to two dietary treatments (Diet RSM and Diet SBM) containing 150 g of either meal and 100 g of maize silage per feeding time and had free access to hay and water. Titanium dioxide (TiO2) was added to the diets for the last 5 days of the study. The wethers were then stunned, exsanguinated and digesta from the reticulo-rumen, omasum, abomasum, jejunum, colon, and rectum were sampled. In Experiment 1, the InsP6ED of RSM (InsP6ED2: 83%; InsP6ED5: 64%) decreased almost identically to that of CPED with increasing passage rate (CPED2: 78%; CPED5: 63%) and was significantly lower than that of SBM (InsP6ED2: 93%; InsP6ED5: 85%). In Experiment 2, ruminal InsP6 disappearance was significantly higher in wethers fed Diet SBM (89%) than in those fed Diet RSM (76%). Total post-ruminal InsP6 degradation was 6% for Diet RSM and 4% for Diet SBM (p = 0.186). The total tract InsP6 disappearance was higher in Diet SBM (93%) than in Diet RSM (82%). Considering higher InsP6 contents in RSM, Diet RSM resulted in significantly higher amounts of ruminally (Diet RSM: 4.5 g/d; Diet SBM: 3.4 g/d) and total tract (Diet RSM: 4.9 g/d; Diet SBM: 3.5 g/d) degraded InsP6. InsP5 was quantified in most of the digesta samples after feeding Diet RSM but was not detectable in the majority of digesta samples for Diet SBM. Concentrations of myo-inositol (MI) tended to be higher (p = 0.060) in the blood plasma of wethers fed Diet RSM. The consistency between ruminal InsP6 disappearance in wethers and in situ calculated InsP6ED2, along with the very low extent of post-ruminal InsP6 degradation, suggests that at a low rumen passage rate, InsP6-P from the feed becoming available to ruminants is almost entirely from InsP6 degradation in the rumen.

Assessment of crop usage in ad libitum fed birds and short-term phytase efficiency as affected by acid addition.

1. A field assessment was performed to map the extent of crop usage and thus retention time in broiler chickens. In addition, a broiler experiment was carried out to study the short-term effect of acid addition on phytase efficacy in the crop.2. In the field assessment, the crop content of 40 ad libitum fed broiler chickens from 4 different farms were sampled at 10, 20 and 30 d of age. The dry matter (DM) content varied from 0 to 32 g.3. From 11 d of age, 120 individually caged chickens were intermittently fed a high phytate-P diet with either no addition or 500 FYT C. braakii-derived phytase added or both phytase and 1.4% formic acid added. Excreta were collected for assessment of phosphorus (P) retention. At 20 and 21 d of age, starved birds were fed for 1 h, and thereafter crop and gizzard contents were collected every 20 min until 140 min after start of the feeding. At 60 and 140 min, the contents from the jejunum and ileum were collected.4. All diets reduced the concentration of phytate in the crop, however the combination of acid and phytase resulted in a higher degradation (P < 0.05) than the other diets from 20 min after the start of feeding. Simultaneously, the concentration of the smaller inositol phosphate isomers, such as inositol-5-phosphate, increased (P < 0.05). Phytase increased (P < 0.05) P retention, and the combination of acid and phytase increased jejunal P digestibility (P < 0.05) compared to the other diets.5. The results indicated that lower pH in the crop due to acid addition improved phytase efficacy and increased P digestibility in the anterior digestive tract, even with short retention times.

Phosphorus digestibility and phytate degradation in pigs fed wheat-based diets with different intrinsic phytase activity and added microbial phytase.

The objective of this study was to investigate the effect of variation in wheat-derived phytase activity on myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) degradation, inositol phosphate (InsP) isomer concentration and phosphorus (P) digestibility in pigs fed wheat-based diets. Additional effects of a microbial phytase supplementation were also studied. Three wheat genotypes (W1-W3) with an analysed phytase activity between 2760 and 3700 FTU/kg were used to formulate four experimental diets that included soybean meal and rapeseed meal but did not contain a mineral P supplement. DietW1-DietW3 only differed in the included wheat genotypes (W1-W3) at an inclusion level of 400 g/kg. DietW3+ contained W3 and a commercial 6-phytase supplementation at 500 FTU/kg diet. Eight barrows with an initial body weight of 27 kg were fitted with a simple T-cannula at the distal ileum and assigned to the four dietary treatments in a completely randomised row column design. The experiment included four periods of 12 d each. The first 5 d of each period were for diet adaptation, followed by collection of faeces (4 d), ileal digesta (2 d), and blood (last day). In DietW1-DietW3, the mean precaecal (pc) InsP6 disappearance was 48% and the mean pc P digestibility was 37% without a significant effect of the wheat genotype. The InsP6 disappearance measured in the faeces was close to complete in all treatments, and faecal P digestibility was not significantly affected by the wheat genotype (36% overall). The addition of microbial phytase caused a significant increase in pc InsP6 degradation (to 79%) and pc and total tract P digestibility (to 53% and 52%, respectively). The concentration of InsP6 degradation products in ileal digesta was not significantly affected by the wheat genotype, except for that of Ins(1,2,3,4,6)P5 and myo-inositol, which were higher in DietW3 than in DietW1 and DietW2. The added microbial phytase significantly reduced the concentration of InsP5 isomers in the ileal digesta and increased the concentrations of lower InsP isomers and myo-inositol. There were no significant effects of the added microbial phytase on pc amino acid digestibility; however, the wheat genotype exerted significant effects on the pc digestibility of Cys, Gly and Val. It was concluded that an increase in the intrinsic phytase activity of wheat achieved by crossbreeding was not reflected in InsP6 degradation and P digestibility in pigs fed wheat-based diets.

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis.

In plants, phosphate (Pi) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. In this study, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by Pi and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to Pi than lower inositol phosphates, a response conserved across kingdoms. Using the capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) we could separate different InsP7 isomers in Arabidopsis and rice, and identify 4/6-InsP7 and a PP-InsP4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP7, 5-InsP7, and InsP8 increase several fold in shoots after Pi resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP6 compartmentalization. Notably, ITPK1 is critical for Pi-dependent 5-InsP7 and InsP8 synthesis in planta and its activity regulates Pi starvation responses in a PHR-dependent manner. Furthermore, we demonstrated that ITPK1-mediated conversion of InsP6 to 5-InsP7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP7 under low ATP. Collectively, our study provides new insights into Pi-dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates.

Loss of diacylglycerol kinase ε causes thrombotic microangiopathy by impairing endothelial VEGFA signaling.

Loss of function of the lipid kinase diacylglycerol kinase ε (DGKε), encoded by the gene DGKE, causes a form of atypical hemolytic uremic syndrome that is not related to abnormalities of the alternative pathway of the complement, by mechanisms that are not understood. By generating a potentially novel endothelial specific Dgke-knockout mouse, we demonstrate that loss of Dgke in the endothelium results in impaired signaling downstream of VEGFR2 due to cellular shortage of phosphatidylinositol 4,5-biphosphate. Mechanistically, we found that, in the absence of DGKε in the endothelium, Akt fails to be activated upon VEGFR2 stimulation, resulting in defective induction of the enzyme cyclooxygenase 2 and production of prostaglandin E2 (PGE2). Treating the endothelial specific Dgke-knockout mice with a stable PGE2 analog was sufficient to reverse the clinical manifestations of thrombotic microangiopathy and proteinuria, possibly by suppressing the expression of matrix metalloproteinase 2 through PGE2-dependent upregulation of the chemokine receptor CXCR4. Our study reveals a complex array of autocrine signaling events downstream of VEGFR2 that are mediated by PGE2, that control endothelial activation and thrombogenic state, and that result in abnormalities of the glomerular filtration barrier.

A dansyl-derivatized phytic acid analogue as a fluorescent substrate for phytases: experimental and computational approach.

A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases. Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared to their natural substrate.

Preparation of pure lower-order myo-Inositol phosphates on laboratory scale for physiological and enzymatic studies.

A simple method for the preparative production of lower-order myo-inositol phosphates was developed. Enzymatic phytate dephosphorylation was applied, because phytate-degrading enzymes generate usually predominantly one single myo-inositol phosphate isomer with five, four, three, two and one phosphate residue(s) bound to the myo-inositol ring in a regio- and stereoselective manner. The relative concentrations of the different lower-order myo-inositol phosphates in the reaction mixture were controlled by adjusting incubation time at 37 °C and a fixed phytate concentration and phytase activity. Purification of the individual lower-order myo-inositol phosphates was realized by anion-exchange chromatography on Q-Sepharose using a stepwise elution with ammonium formate:formic acid pH 2.5. Ethanol precipitation was successfully used to concentrate the pure lower-order myo-inositol phosphates. In a single approach 2-3 mg of pure myo-inositol tetrakis- or -trisphosphate isomers were obtained. About 60% of the initially applied phytate were converted into pure lower-order myo-inositol phosphates. The purified myo-inositol phosphate isomers were virtually free of other myo-inositol phosphate esters and could be used for enzymatic and physiological studies.