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.

Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP6 kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast.

The inositol pyrophosphate signaling molecule 1,5-IP8 is an agonist of RNA 3'-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1, pho84, and tgp1. IP8 is synthesized from 5-IP7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1+ cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP6 kinase Kcs1 that generates 5-IP7, the immediate precursor to IP8. Transcription profiling of the kcs1 mutants in an asp1+ background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP3, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP8 governs repression of fission yeast phosphate homeostasis genes pho1, pho84, and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP8 agonism of transcription termination. It was assumed that IP8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP3 and the essential Kcs1 kinase that converts IP6 to 5-IP7, the immediate precursor of IP8.

An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor.

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the αC helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases.

Accentuating the positive and eliminating the negative: Efficacy of TiO2 as digestibility index marker for poultry nutrition studies.

Inert digestibility index markers such as titanium dioxide are universally accepted to provide simple measurement of digestive tract retention and relative digestibility in poultry feeding trials. Their use underpins industry practice: specifically dosing regimens for adjunct enzymes added to animal feed. Among these, phytases, enzymes that degrade dietary phytate, inositol hexakisphosphate, represent a billion-dollar sector in an industry that raises ca. 70 billion chickens/annum. Unbeknown to the feed enzyme sector, is the growth in cell biology of use of titanium dioxide for enrichment of inositol phosphates from extracts of cells and tissues. The adoption of titanium dioxide in cell biology arises from its affinity under acid conditions for phosphates, suggesting that in feeding trial contexts that target phytate degradation this marker may not be as inert as assumed. We show that feed grade titanium dioxide enriches a mixed population of higher and lower inositol phosphates from acid solutions. Additionally, we compared the extractable inositol phosphates in gizzard and ileal digesta of 21day old male Ross 308 broilers fed three phytase doses (0, 500 and 6000 FTU/kg feed) and one inositol dose (2g/kg feed). This experiment was performed with or without titanium dioxide added as a digestibility index marker at a level of 0.5%, with all diets fed for 21 days. Analysis yielded no significant difference in effect of phytase inclusion in the presence or absence of titanium dioxide. Thus, despite the utility of titanium dioxide for recovery of inositol phosphates from biological samples, it seems that its use as an inert marker in digestibility trials is justified-as its inclusion in mash diets does not interfere with the recovery of inositol phosphates from digesta samples.

Research Note: Influence of monocalcium phosphate and phytase in the diet on phytate degradation in cecectomized laying hens.

This study investigated the effects of phytase and monocalcium phosphate supplementation on the dephosphorylation of phytic acid [myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate); InsP6] in cecectomized laying hens using total excreta collection. Four corn-soybean meal-rapeseed meal-based diets were mixed with or without 6 g of monocalcium phosphate/kg, with or without supplementation of 1,500 FTU phytase/kg, and had the same calcium concentration at 39 g/kg of feed. Each diet was tested in 5 replicates using a row-column design with 10 cecectomized laying hens in 2 periods. The hens received 120 g/d of feed while being housed individually in metabolism units, and total excreta were collected for a period of 4 d. The monocalcium phosphate × phytase interaction was not significant for InsP6 degradation (P = 0.054). Phytase increased InsP6 disappearance from 13% to 83% (P < 0.001), whereas monocalcium phosphate had no effect. Concentrations of most of the lower inositol phosphate isomers in excreta were higher when monocalcium phosphate was added to the diets. The concentration of Ins(1,2,5,6)P4 in excreta was the highest among the studied partially dephosphorylated inositol phosphates with phytase supplementation and was higher than in diets without phytase supplementation (P < 0.001). Supplementation with phytase increased myo-inositol concentration in excreta (P = 0.002), whereas monocalcium phosphate had no effect. Phosphorus utilization ranged from 4% to 18% and was not significantly affected by the treatments. These results suggest that phytase supplementation markedly increased InsP6 degradation in laying hens. The cecectomized laying hen assay may be suitable for studying the effects of phytase supplementation on phytate dephosphorylation under dietary conditions when performance and phosphorus excretion are unlikely to be affected.

Phytase dose-dependent response of kidney inositol phosphate levels in poultry.

Phytases, enzymes that degrade phytate present in feedstuffs, are widely added to the diets of monogastric animals. Many studies have correlated phytase addition with improved animal productivity and a subset of these have sought to correlate animal performance with phytase-mediated generation of inositol phosphates in different parts of the gastro-intestinal tract or with release of inositol or of phosphate, the absorbable products of phytate degradation. Remarkably, the effect of dietary phytase on tissue inositol phosphates has not been studied. The objective of this study was to determine effect of phytase supplementation on liver and kidney myo-inositol and myo-inositol phosphates in broiler chickens. For this, methods were developed to measure inositol phosphates in chicken tissues. The study comprised wheat/soy-based diets containing one of three levels of phytase (0, 500 and 6,000 FTU/kg of modified E. coli 6-phytase). Diets were provided to broilers for 21 D and on day 21 digesta were collected from the gizzard and ileum. Liver and kidney tissue were harvested. Myo-inositol and inositol phosphates were measured in diet, digesta, liver and kidney. Gizzard and ileal content inositol was increased progressively, and total inositol phosphates reduced progressively, by phytase supplementation. The predominant higher inositol phosphates detected in tissues, D-and/or L-Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5, differed from those (D-and/or L-Ins(1,2,3,4)P4, D-and/or L-Ins(1,2,5,6)P4, Ins(1,2,3,4,6)P5, D-and/or L-Ins(1,2,3,4,5)P5 and D-and/or L-Ins(1,2,4,5,6)P5) generated from phytate (InsP6) degradation by E. coli 6-phytase or endogenous feed phytase, suggesting tissue inositol phosphates are not the result of direct absorption. Kidney inositol phosphates were reduced progressively by phytase supplementation. These data suggest that tissue inositol phosphate concentrations can be influenced by dietary phytase inclusion rate and that such effects are tissue specific, though the consequences for physiology of such changes have yet to be elucidated.

Generation of 13C-Labeled Inositol and Inositol Phosphates by Stable Isotope Labeling Cell Culture for Quantitative Metabolomics.

Inositol and inositol phosphates (IPx) are central metabolites. Their accurate quantitative analysis in complex biological samples is challenging due to lengthy sample preparation procedures, sample losses by strong adsorption to surfaces, and unpredictable matrix effects. Currently, U13C-inositol and U13C-IPx are not available from commercial sources. In this study, we developed a method that is capable of generating U13C-inositol and U13C-IPx. An inositol-independent cell line L929S was cultured in inositol-free medium supplemented with U13C-glucose. Inositol contamination in FBS was observed as the critical parameter for labeling efficiency (LE). A balance between cell growth and LE was achieved by adopting a two-step labeling strategy. In the first step, a LE of 90% could be obtained by normal cell growth in the long-term. Cells were then cultured in a second step in ultra-labeling medium for improved LE for a short duration before harvesting. The generated U13Canalogs were of high isotopic purity (>99%). Utilized as internal standards spiked before sample preparation in biological applications, U13Canalogs can effectively compensate sample loss during sample preparation as well as the matrix effect during electrospray ionization. An exemplary pharmacological study was conducted with phospholipase C inhibitor and activator to document the great utility of the prepared stable isotope-labeled internal standards in elucidating the PLC-dependent IP code. U13CIPx are used as internal standards to generate quantitative profiles of IPx in HeLa cell samples after treatment with PLC inhibitor and activator. This established method generating U13Canalogs is cost-effective, robust, and reproducible, which can facilitate quantitative studies of inositol and IPx in biological scenarios.

Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers.

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling, and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologues display phosphohydrolase activity with a high specificity for the 5-ß-phosphate of inositol pyrophosphates and only minor activity against the ß-phosphates of 4-InsP7 and 6-InsP7. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-ß-phosphate-specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC.

The phosphate esters of myo-inositol (Ins) occur ubiquitously in biology. These molecules exist as soluble or membrane-resident derivatives and regulate a plethora of cellular functions including phosphate homeostasis, DNA repair, vesicle trafficking, metabolism, cell polarity, tip-directed growth, and membrane morphogenesis. Phosphorylation of all inositol hydroxyl groups generates phytic acid (InsP6), the most abundant inositol phosphate present in eukaryotic cells. However, phytic acid is not the most highly phosphorylated naturally occurring inositol phosphate. Specialized small molecule kinases catalyze the formation of the so-called myo-inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8. These molecules are characterized by one or several "high-energy" diphosphate moieties and are ubiquitous in eukaryotic cells. In plants, PP-InsPs play critical roles in immune responses and nutrient sensing. The detection of inositol derivatives in plants is challenging. This is particularly the case for inositol pyrophosphates because diphospho bonds are labile in plant cell extracts due to high amounts of acid phosphatase activity. We present two steady-state inositol labeling-based techniques coupled with strong anion exchange (SAX)-HPLC analyses that allow robust detection and quantification of soluble and membrane-resident inositol polyphosphates in plant extracts. These techniques will be instrumental to uncover the cellular and physiological processes controlled by these intriguing regulatory molecules in plants.

Fermentation with Edible Rhizopus Strains to Enhance the Bioactive Potential of Hull-Less Pumpkin Oil Cake.

Solid-state fermentation with food-grade fungal strains can be applied to enhance the bioactive parameters of agro-industrial by-products. Tempe-type fermentation can be adapted to various substrates, but the key factor is the appropriate strain selection. The aim of this study was to compare the potential of Rhizopus strains for obtaining products of improved antioxidant activity from pumpkin oil cake. For this purpose, substances reacting with the Folin-Ciocalteu reagent, with free radical scavenging potential, as well as reducing power were assessed. The effect of the fermentation on the phytate level and inositol phosphate profile in the material was also monitored. The fermentation resulted in the significant enhancement of the antioxidant potential of pumpkin oil cake in the case of all the strains tested, but the most efficient one was R. oligosporus ATCC 64063. During the course of fermentation, the level of phytate in the material decreased (the highest reduction rate was observed in the oil cake fermented with R. oryzae CBS 372.63), while peptides and fungal glucosamine were accumulated. Tempe-type fermentation can be considered as an alternative way of improving the bioactive parameters of pumpkin oil cake and, thanks to the various activities of different Rhizopus strains, it is possible to obtain products of desired parameters.

Rescue of mutant gonadotropin-releasing hormone receptor function independent of cognate receptor activity.

Molecules that correct the folding of protein mutants, restoring their functional trafficking, are called pharmacoperones. Most are clinically irrelevant and possess intrinsic antagonist or agonist activity. Here, we identify compounds capable of rescuing the activity of mutant gonadotropin-releasing hormone receptor or GnRHR which, is sequestered within the cell and if dysfunctional leads to Hypogonadotropic Hypogonadism. To do this we screened the E90K GnRHR mutant vs. a library of 645,000 compounds using a cell-based calcium detection system. Ultimately, we identified 399 compounds with EC50 ≤ 5 µM with no effect in counterscreen assays. Medicinal chemistry efforts confirmed activity of 70 pure samples and mode of action studies, including radioligand binding, inositol phosphate, and toxicity assays, proved that we have a series of tractable compounds that can be categorized into structural clusters. These early lead molecules rescue mutant GnRHR function and are neither agonist nor antagonists of the GnRHR cognate receptor, a feature required for potential clinical utility.

Phytase supplementation effects on amino acid digestibility depend on the protein source in the diet but are not related to InsP6 degradation in broiler chickens.

The objective was to determine phytase effects on prececal amino acid (AA) digestibility and phytate (InsP6) breakdown when different oilseed meals were used in broiler chicken diets. The study included 14 diets: a corn-soybean meal (SBM) basal diet and 6 diets that contained SBM, rapeseed meal (RSM), and sunflower meal (SFM) with 2 inclusion levels at the expense of corn starch (150 and 300 g/kg SBM or SFM, or 100 and 200 g/kg RSM). Each diet was mixed with or without a phytase supplement of 1,500 FTU/kg. Diets were provided to broilers for 5 D. Digesta from the posterior half of the ileum were collected on day 21. The average essential AA digestibility, calculated by a regression approach, without and with phytase was 84 and 85% (SBM), 74 and 77% (SFM), and 66 and 73% (RSM), respectively. In the diets, phytase effects on AA digestibility were lower owing to other protein sources also present in the diet, but significant. Prececal InsP6 disappearance was significantly affected by interactions between oilseed meal, inclusion level, and phytase supplementation. Overall, prececal InsP6 disappearance was higher in SBM diets (52%) than in SFM diets (38%) and intermediate in RSM diets (43%). Across diets, phytase supplementation effects on prececal InsP6 degradation linearly increased with the InsP6 concentration of the diet up to 12 g/kg DM. The only exception from linearity was the diet with the high inclusion of SFM, which contained 15.9 g InsP6/kg DM. In the ileal content, the concentration of myo-inositol was significantly increased by phytase supplementation, and this effect was highest in the diets that contained SBM as the only oilseed meal. Concentrations of lower inositol phosphates were increased by phytase supplementation, and this effect was most remarkable for Ins(1,2,3,4)P4 and inositol tetrakisphosphates. The study showed that phytase effects on AA digestibility varied among the 3 tested oilseed meals, but these differences were not detectable in the diets containing these meals. Although phytase effects on ileal content of InsP6 and its degradation products were substantial, they were not related to the effects on AA digestibility.

Banxia Xiexin Decoction () Treats Diabetic Gastroparesis through PLC-IP3-Ca2+/NO-cGMP-PKG Signal Pathway.

OBJECTIVE: To test the effect of Banxia Xiexin Decoction (, BXD) on the contraction and relaxation of gastric smooth muscle (SM) in diabetic gastroparesis (DGP) model rats, and to explore the mechanism of BXD in the prevention and treatment of DGP through experiments of signal pathway both in vivo and in vitro. METHODS: Sixty Sprague-Dawley rats were divided into 6 groups according to a random number table: control group, model group, high-, medium- and low-dose BXD groups (9.2, 4.6 and 1.8 g/(kg·d), respectively), and domperidone group (10 mg/(kg·d)), 10 rats per group. DGP model was established initially by a single intraperitoneal injection of streptozotocin (STZ), and was confirmed by recording gastric emptying, intestinal transport velocity and gastric myoelectric activity of rats after 2 months. Each group was treated with a corresponding drug for 4 weeks. The mRNA and protein expressions of phospholipase C (PLC), inositol triphosphate (IP3), neuronal nitric oxide synthase (nNOS), and cyclic guanosine monophosphate (cGMP) dependent protein kinase G (PKG) were detected by reverse transcription-polymerase chain reaction and Western blot, respectively, while nitric oxide (NO) and cGMP expressions were detected by enzyme-linked immunosorbent assay. Gastric tissues were obtained from rats for primary cell culture preparation. Gastric SM cells were treated with 0.8 µmol/L of STZ or STZ plus 1,000, 500 and 200 µg/mL of BXD or STZ plus 2.5 µmol/mL of domperidone for 24, 48, 72 or 96 h, respectively. The length of gastric SM cells and intracellular Ca2+ concentration ([Ca2+]i) before and after BXD treatment was measured. RESULTS: Compared with the model group, high- and medium-dose BXD and domperidone significantly increased the expressions of PLC, IP3, NO, nNOS, cGMP and PKG in rat's gastric tissue (P<0.01). Gastric SM cells treated with BXD showed a time- and dose-dependent increase in cell viability (P<0.01). The treatment with high- and medium-dose BXD and domperidone inhibited the increase in gastric SM cells length and increased [Ca2+]i compared with the model cells (P<0.01). CONCLUSIONS: Treatment with high- and medium-dose BXD significantly attenuated STZ-induced experimental DGP in rats. The therapeutic effect of BXD on DGP rats might be associated with the PLC-IP3-Ca2+/NO-cGMP-PKG signal pathway.

In vitro digestion effect on mineral bioaccessibility and antioxidant bioactive compounds of plant-based beverages.

Consumption of plant-based beverages (PBB) is a growing trend; and have been used as viable substitutes for dairy based products. To date, no study has comparatively analyzed mineral composition and effect of in vitro digestion on the bioaccessibility of different PBB. The aim of this research was to investigate the content of essential minerals (calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn)) and to estimate the effect of in vitro digestion in plant-based beverages, and their antioxidant bioactive compounds (phenolic compounds and antioxidant capacity). Moreover, the presence of antinutritional factors, such as myo-inositol phosphates fractions, were evaluated. Samples of PBB (rice, cashew nut, almond, peanut, coconut, oat, soy, blended or not with another ingredients, fortified with minerals or naturally present) and milk for comparison were evaluated. TPC ranged from 0.2 mg GAEq/L for coconut to 12.4 mg GAEq/L for rice and, the antioxidant capacity (DPPH) ranged from 3.1 to 306.5 µmol TE/L for samples containing peanut and oat, respectively. Only a few samples presented myo-inositol phosphates fractions in their composition, mostly IP5 and IP6, especially cashew nut beverages. Mineral content showed a wide range for Ca, ranging from 10 to 1697.33 mg/L for rice and coconut, respectively. The Mg content ranged from 6.29 to 251.23-268.43 mg/L for rice and cashew nut beverages, respectively. Fe content ranged from 0.76 mg/L to 12.89 mg/L for the samples of rice. Zinc content ranged from 0.57 mg/L to 8.13 mg/L for samples of oat and soy, respectively. Significant variation was observed for Ca (8.2-306.6 mg/L) and Mg (1.9-107.4 mg/L) dialyzed between the beverages, with lower concentrations of Fe (1.0 mg/L) and Zn (0.5 mg/L) in dialyzed fractions. This study provides at least 975 analytically determined laboratory results, providing important information for characterization and comparison of different plant-based beverages.

The Impact of Phytases on the Release of Bioactive Inositols, the Profile of Inositol Phosphates, and the Release of Selected Minerals in the Technology of Buckwheat Beer Production.

A relatively high concentration of phytate in buckwheat malt, and the low activity of endogenous buckwheat phytases, both of which limit the effective use of substrates (starch, proteins, minerals) for fermentation and yeast metabolism, gives rise to the potential for application of phytases in beer production. This study aims at obtaining a 100% buckwheat wort with high bioactive cyclitols (myo-inositol and D-chiro-inositol) concentrations released by exogenous phytases and acid phosphatases. Two mashing programs were used in the study, i.e., (1) typical for basic raw materials, namely the well-established Congress method, and (2) optimized for phytase activity. The results indicated a nearly 50% increase in the level of bioactive myo-inositol and an 80% degradation of phytate in the wort as a result of simultaneous application of phytase and phosphatase enzymes in the mashing of buckwheat malt. In addition, high D-chiro-inositol concentrations were released from malt to the buckwheat wort. The concerted action of the two phytases significantly increased (19-44%) Zn2+ concentrations in wort. This may be of great importance during mash fermentation by Saccharomyces cerevisiae yeasts. There is a potential to develop technology for buckwheat beer production, which, in addition to being free from gluten, comprises high levels of bioactive myo- and D-chiro-inositols.

Effect of phytase on intestinal phytate breakdown, plasma inositol concentrations, and glucose transporter type 4 abundance in muscle membranes of weanling pigs1.

The objective of this present study was to determine the effects of phytase dosing on growth performance, mineral digestibility, phytate breakdown, and the level of glucose transporter type 4 (GLUT4) in muscle plasma membranes of weanling pigs. A total of 160 barrows were used in a randomized completely block design and assigned to 4 treatments for a 7-wk study. Depending on the feeding phase, diets differed in dietary calcium (Ca) and phosphorus (P) levels (positive control [PC]: 8 to 6.8g/kg Ca; 7.3 to 6.3 g/kg P; negative control [NC]: 5.5 to 5.2 g/kg Ca; 5.4 to 4.7 g/kg P). NC diets were supplemented with phytase at 0 (NC); 500 (NC + 500 FTU); or 2,000 FTU/kg (NC + 2,000 FTU) phytase units/kg. Blood was collected after fasting (day 48) or feeding (day 49) for measurement of plasma inositol concentrations. On day 49, 2 pigs per pen were euthanized, and duodenal and ileal digesta samples were collected to determine inositol phosphates (InsP6-2) concentrations. High phytase supplementation increased BW on days 21, 35, and 49 (P < 0.05). Over the entire feeding period, ADG, ADFI, and feed efficiency were increased by NC + 2,000 FTU compared with the other treatments (P < 0.05). Postprandial plasma inositol concentration was increased in NC + 2,000 (P < 0.01), but there was only a tendency (P = 0.06) of a higher fasting plasma inositol concentration in this group. Inositol concentrations in the portal vein plasma (day 49) were not different among treatments. Duodenal digesta InsP5 and InsP6 concentrations were similar in PC and NC, but higher in these 2 treatments (P < 0.05) than those supplemented with phytase. Phytase supplementation decreased InsP6-4, resulting in increased InsP3-2 and myo-inositol concentrations. Similar effects were found in ileal contents. Compared with NC, phytase supplementation resulted in greater cumulative InsP6-2 disappearance (93.6% vs. 72.8% vs. 25.0%, for NC + 2,000 FTU, NC + 500 FTU and NC, respectively, P < 0.01) till the distal ileum. Longissimus dorsi muscle plasma membrane GLUT4 concentration was increased by NC + 2,000 FTU (P < 0.01) compared with NC. In summary, high phytase supplementation increased growth performance of nursery pigs. The higher myo-inositol release from phytate could contribute to the increased expression of GLUT4 in muscle plasma membranes. Further investigation is needed to determine whether this is associated with enhanced cellular glucose uptake and utilization.

The efficacy of 2 phytases on inositol phosphate degradation in different segments of the gastrointestinal tract, calcium and phosphorus digestibility, and bone quality of broilers.

The anti-nutritional effects of dietary inositol phosphates (IP6 through IP3) have been recognized in broiler chickens; however, inositol hexaphosphate (IP6) is more potent than the lower IP esters. The efficacies of 2 commercial phytases, a Buttiauxella sp. phytase (BSP) and a Citrobacter braakii phytase (CBP) at 500 and 1,000 FTU/kg, were studied on IP6-3 concentrations in the crop, proventriculus + gizzard, and distal ileum digesta, and ileal IP6 disappearance in broilers at day 22. Apparent ileal P and Ca digestibility, and bone quality at days 22 and 33 were also measured. Female Ross 308 broilers (n = 1,890; 30 birds × 7 diets × 9 replicates) were fed corn-soy-based crumbled diets. The 7 diets included a primary breeder recommendation-based positive control diet (PC); the PC marginally reduced in available P by 0.146% and Ca by 0.134% of the diet, (NC1) or moderately reduced by 0.174 and 0.159% of the diet, respectively (NC2). Other diets were the NC1 + BSP or CBP at 500 FTU/kg (NC1+500BSP and NC1+500CBP) and the NC2 + BSP or CBP at 1,000 FTU/kg (NC2+1,000BSP and NC2+1,000CBP). Each of the NC1 and NC2 had distal ileum IP6 disappearance similar to that of PC, but each had lower P digestibility and the majority of measured bone quality parameters than the PC. The ileal IP6 levels were decreased by 52.0 and 32.7% for NC1+500BSP and NC1+500CBP, respectively, relative to NC1 and by 73.6 and 50.9% for NC2+1,000BSP and NC2+1,000CBP, respectively, relative to NC2 (P < 0.001), with a similar effect for distal ileum IP6 disappearance. Overall, phytase in the NC diets increased P digestibility, and femur breaking strength and cortical bone mineral density at days 22 and 33. Overall, each of the phytases at each dose degraded IP6-3 across the gastrointestinal tract segments to increase P digestibility and the P and Ca utilization in bone. However, dietary BSP at 1,000 FTU/kg was most effective. Supplemental phytase degrades phytate to decrease the anti-nutritional effects in a dose- and phytase-dependent manner.

Interactive effects of phosphorus, calcium, and phytase supplements on products of phytate degradation in the digestive tract of broiler chickens.

This study aimed to distinguish between the single and interactive effects of phosphorus (P), calcium (Ca), and phytase on products of phytate degradation, including the disappearance of myo-inositol (MI), P, Ca, and amino acids (AA) in different segments of the digestive tract in broiler chickens. Additionally, all dephosphorylation steps from myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) to MI were investigated in the digesta of the terminal ileum. Unsexed Ross 308 broiler chickens were allocated to 56 pens with 19 birds per pen, and assigned to one of 8 dietary treatments. The dietary treatments included diets without (P-, 4.1 g/kg DM) or with (P+, 6.9 g/kg DM) monosodium phosphate supplementation, without (Ca-, 6.2 g/kg DM) or with (Ca+, 10.3 g/kg DM) additional fine limestone supplementation, and without or with 1,500 FTU phytase/kg feed in a factorial design. Adding Ca or P had no effect on InsP6 disappearance in the crop when phytase was added. InsP6 disappearance up to the terminal ileum (P-Ca- 56%) was decreased in P+Ca- (40%), and even more so in P+Ca+ (21%), when no phytase was added. Adding phytase removed all effects of P and Ca (77 to 87%); however, P+Ca+ increased the concentrations of lower InsP esters and reduced free MI in the ileum, even in the presence of phytase. These results indicate that mineral supplements, especially P and Ca combined, reduce the efficacy of endogenous microbial or epithelial phosphatases. Supplementation with phytase increased, while supplementation with Ca decreased the concentration of MI in all segments of the digestive tract and in blood plasma, demonstrating the ability of broilers to fully degrade phytate and absorb released MI. While AA disappearance was not affected by P or Ca, or an interaction among P, Ca, and phytase, it increased with the addition of phytase by 2 to 6%. This demonstrates the potential of the phytase used to increase AA digestibility, likely independent of P and Ca supply.

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic ß-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity.

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP7, is at a high level in pancreatic ß-cells and is important for insulin exocytosis. To better understand IP7 regulation in ß-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high Km for ATP makes IP7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic ß-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP7's role in cellular signal transduction is likely to have been underestimated.

Novel signals in the regulation of Pi starvation responses in plants: facts and promises.

Plants have evolved numerous adaptive developmental and metabolic responses to cope with growth in conditions of limited phosphate (Pi). Regulation of these Pi starvation responses (PSR) at the organism level involves not only cellular Pi perception in different organs, but also inter-organ communication of Pi levels via systemic signaling. Here we summarize recent discoveries on Pi starvation sensing and signaling, with special emphasis on structure-function studies that showed a role for inositol polyphosphates (InsP) as intracellular Pi signals, and on genomic studies that identified a large number of mRNAs with inter-organ mobility, which provide an immense source of potential systemic signals in the control of PSR and other responses.