Intracellular Phosphate Research Articles

Obesity causes renal mitochondrial dysfunction and energy imbalance and accelerates chronic kidney disease in mice.

Obesity and metabolic syndrome are well-known risk factors for chronic kidney disease (CKD); however, less is known about the mechanism(s) by which metabolic syndrome might accelerate kidney disease. We hypothesized that metabolic syndrome should accelerate the development of kidney disease and that it might be associated with alterations in energy metabolism. We studied the pound mouse (which develops early metabolic syndrome due to a leptin receptor deletion) and wild-type littermates and compared the level of renal injury and muscle wasting after equivalent injury with oral adenine. Renal function, histology, and biochemical analyses were performed. The presence of metabolic syndrome was associated with earlier development of renal disease (12 mo) and earlier mortality in pound mice compared with controls. After administration of adenine, kidney disease was worse in pound mice, and this was associated with greater tubular injury with a decrease in kidney mitochondria, lower tissue ATP levels, and worse oxidative stress. Pound mice with similar levels of renal function as adenine-treated wild-type mice also showed worse sarcopenia, with lower tissue ATP and intracellular phosphate levels. In summary, our data demonstrate that obesity and metabolic syndrome accelerate the progression of CKD and worsen CKD-dependent sarcopenia. Both conditions are associated with renal alterations in energy metabolism and lower tissue ATP levels secondary to mitochondrial dysfunction and reduced mitochondrial number.

Inositol Pyrophosphate InsP8 Acts as an Intracellular Phosphate Signal in Arabidopsis

The maintenance of cellular phosphate (Pi) homeostasis is of great importance in living organisms. The SPX domain-containing protein 1 (SPX1) proteins from both Arabidopsis and rice have been proposed to act as sensors of Pi status. The molecular signal indicating the cellular Pi status and regulating Pi homeostasis in plants, however, remains to be identified, as Pi itself does not bind to the SPX domain. Here, we report the identification of the inositol pyrophosphate InsP8 as a signaling molecule that regulates Pi homeostasis in Arabidopsis. Polyacrylamide gel electrophoresis profiling of InsPs revealed that InsP8 level positively correlates with cellular Pi concentration. We demonstrated that the homologs of diphosphoinositol pentakisphosphate kinase (PPIP5K), VIH1 and VIH2, function redundantly to synthesize InsP8, and that the vih1 vih2 double mutant overaccumulates Pi. SPX1 directly interacts with PHR1, the central regulator of Pi starvation responses, to inhibit its function under Pi-replete conditions. However, this interaction is compromised in the vih1 vih2 double mutant, resulting in the constitutive induction of Pi starvation-induced genes, indicating that plant cells cannot sense cellular Pi status without InsP8. Furthermore, we showed that InsP8 could directly bind to the SPX domain of SPX1 and is essential for the interaction between SPX1 and PHR1. Collectively, our study suggests that InsP8 is the intracellular Pi signaling molecule serving as the ligand of SPX1 for controlling Pi homeostasis in plants.

A composite consisting of bromine-doped carbon dots and ferric ions as a fluorescent probe for determination and intracellular imaging of phosphate.

A composite probe has been developed for fluorometric determination and imaging of phosphate in real water samples and in cells. The method is based on the use of weakly blue fluorescent bromine-doped carbon dots (C-dots) containing aromatic carbon-bromine groups and loaded with Fe3+ ions. The carboxy, phenolic hydroxy and aldehyde groups on the surface of the C-dots can coordinate with Fe3+ to form an adsorbed complex that reduces the blue fluorescence through an inner filter effect. If phosphate is added, it will capture Fe3+ on the surface of C-dots and restore fluorescence by ~88% via a displacement approach. The probe, best operated at excitation/emission maxima of 370/418nm, has a linear response in the 0.4 to 22μM phosphate concentration range and a 0.25μM of detection limit. The relative standard deviation (at a phosphate level of 8.0μM) is 3.6% (for n = 5). The method was applied to confocal imaging of phosphate in HeLa cells. Graphical abstractSchematic representation of the synthesis of bromine-doped carbon dots (C-dots) by a "one-step" approach. They are shown to be capable of (a) detecting phosphate in real water samples through the displacement approach, and (b) of imaging intracellular phosphate.

Cooperation between two strains of Enterobacter and Klebsiella in the simultaneous nitrogen removal and phosphate accumulation processes

Two strains, Enterobacter sp. Z1 and Klebsiella sp. Z2, were exhibited great capacities for heterotrophic nitrification-aerobic denitrification (HNAD) and intracellular phosphate accumulation. Strikingly, the co-cultured strains enhanced the removal efficiency of total nitrogen and phosphate, with removal efficiencies of ammonia, nitrate, nitrite and soluble phosphate of 99.64%, 99.85%, 96.94% and 66.7% respectively. Furthermore, high removal efficiencies from wastewaters with high concentrations of ammonia (over 1000 mg/L) were achieved by inoculation with the co-strains, which left residual ammonia of less than 1 mg/L within 10 h. To elucidate the mechanism of HNAD in co-strains, quantitative PCR was carried out to examine the expression levels of hydroxylamine oxidase (Hao), nitrate reductase (NapA and NarG), nitrite reductase (NirS) and polyphosphate kinase (Ppk), and the results showed that the napA2, narG and ppk genes in the strains were significantly upregulated under the co-cultured conditions and provided an explanation for the nitrogen and phosphate removal.

The inositol hexakisphosphate kinases IP6K1 and -2 regulate human cellular phosphate homeostasis, including XPR1-mediated phosphate export

Phosphate's central role in most biochemical reactions in a living organism requires carefully maintained homeostasis. Although phosphate homeostasis in mammals has long been studied at the organismal level, the intracellular mechanisms controlling phosphate metabolism are not well-understood. Inositol pyrophosphates have emerged as important regulatory elements controlling yeast phosphate homeostasis. To verify whether inositol pyrophosphates also regulate mammalian cellular phosphate homeostasis, here we knocked out inositol hexakisphosphate kinase (IP6K) 1 and IP6K2 to generate human HCT116 cells devoid of any inositol pyrophosphates. Using PAGE and HPLC analysis, we observed that the IP6K1/2-knockout cells have nondetectable levels of the IP6-derived IP7 and IP8 and also exhibit reduced synthesis of the IP5-derived PP-IP4. Nucleotide analysis showed that the knockout cells contain increased amounts of ATP, whereas the Malachite green assay found elevated levels of free intracellular phosphate. Furthermore, [32Pi] pulse labeling experiments uncovered alterations in phosphate flux, with both import and export of phosphate being decreased in the knockout cells. Functional analysis of the phosphate exporter xenotropic and polytropic retrovirus receptor 1 (XPR1) revealed that it is regulated by inositol pyrophosphates, which can bind to its SPX domain. We conclude that IP6K1 and -2 together control inositol pyrophosphate metabolism and thereby physiologically regulate phosphate export and other aspects of mammalian cellular phosphate homeostasis.

Rapid Aldosterone-Mediated Signaling in the DCT Increases Activity of the Thiazide-Sensitive NaCl Cotransporter.

The NaCl cotransporter NCC in the kidney distal convoluted tubule (DCT) regulates urinary NaCl excretion and BP. Aldosterone increases NaCl reabsorption via NCC over the long-term by altering gene expression. But the acute effects of aldosterone in the DCT are less well understood. Proteomics, bioinformatics, and cell biology approaches were combined with animal models and gene-targeted mice. Aldosterone significantly increases NCC activity within minutes in vivo or ex vivo. These effects were independent of transcription and translation, but were absent in the presence of high potassium. In vitro, aldosterone rapidly increased intracellular cAMP and inositol phosphate accumulation, and altered phosphorylation of various kinases/kinase substrates within the MAPK/ERK, PI3K/AKT, and cAMP/PKA pathways. Inhibiting GPR30, a membrane-associated receptor, limited aldosterone's effects on NCC activity ex vivo, and NCC phosphorylation was reduced in GPR30 knockout mice. Phosphoproteomics, network analysis, and in vitro studies determined that aldosterone activates EGFR-dependent signaling. The EGFR immunolocalized to the DCT and EGFR tyrosine kinase inhibition decreased NCC activity ex vivo and in vivo. Aldosterone acutely activates NCC to modulate renal NaCl excretion.

Health outcomes of a high fructose intake: the importance of physical activity.

Fructose metabolism is generally held to occur essentially in cells of the small bowel, the liver, and the kidneys expressing fructolytic enzymes (fructokinase, aldolase B and a triokinase). In these cells, fructose uptake and fructolysis are unregulated processes, resulting in the generation of intracellular triose phosphates proportionate to fructose intake. Triose phosphates are then processed into lactate, glucose and fatty acids to serve as metabolic substrates in other cells of the body. With small oral loads, fructose is mainly metabolized in the small bowel, while with larger loads fructose reaches the portal circulation and is largely extracted by the liver. A small portion, however, escapes liver extraction and is metabolized either in the kidneys or in other tissues through yet unspecified pathways. In sedentary subjects, consumption of a fructose‐rich diet for several days stimulates hepatic de novo lipogenesis, increases intrahepatic fat and blood triglyceride concentrations, and impairs insulin effects on hepatic glucose production. All these effects can be prevented when high fructose intake is associated with increased levels of physical activity. There is also evidence that, during exercise, fructose carbons are efficiently transferred to skeletal muscle as glucose and lactate to be used for energy production. Glucose and lactate formed from fructose can also contribute to the re‐synthesis of muscle glycogen after exercise. We therefore propose that the deleterious health effects of fructose are tightly related to an imbalance between fructose energy intake on one hand, and whole‐body energy output related to a low physical activity on the other hand.

Phosphate Assay Kit in One Cell for Electrochemical Detection of Intracellular Phosphate Ions at Single Cells

In this paper, phosphate assay kit in one cell is realized for the electrochemical detection of intracellular phosphate ions at single cells. The components of the phosphate assay kit, including maltose phosphorylase, maltose, mutarotase, and glucose oxidase, are electrochemically injected into a living cell through a nanometer-sized capillary with the ring electrode at the tip. These components react with phosphate ions inside the cell to generate hydrogen peroxide that is electrochemically oxidized at the ring electrode for the qualification of intracellular phosphate ions. An average 1.7 nA charge was collected from eight individual cells, suggesting an intracellular phosphate concentration of 2.1 mM. The establishment in the electrochemical measurement of phosphate ions provides a special strategy to monitor the fluctuation of intracellular phosphate at single cells, which is significant for the future investigation of phosphate signal transduction pathway.

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290 Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that β-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.

Phosphate solubilizing bacteria and fungi in desert soils: species, limitations and mechanisms

ABSTRACT Desert soils are infertile, and the ability to improve them by P-fertilization is limited by the solubility of phosphate. We aimed to understand the function of phosphate solubilizing bacteria and the mechanisms behind phosphate solubilization in desert soils. Vegetated and barren desert soils, mine spoil and a fertile temperate grassland loam were sampled. Bacteria and fungi were isolated and identified, and their phosphate-solubilizing abilities were measured in vitro. The release of plant available PO4, SO4, NO3 and NH4 from desert soils did not compare with that of a grassland soil. Desert soils had substantially lower solubilization than grassland, 162 and 99–121 µg PO4-P g−1 dry soil, respectively. Phosphate-solubilizing bacteria and fungi were inhabiting the soils. Si addition increased phosphate solubilization of fungi by 50%. The isolated microbes were shown, using 31P nuclear magnetic resonance (NMR) analysis, to rapidly take-up both intracellular and extracellular phosphate during the phosphate solubilizing process. Desert soil had potentially active microbial populations that are capable to solubilize inorganic phosphorus; S and Si as the limiting factors. Acidification as the main mechanism to solubilize mineral phosphate was not as evident in our desert soils as in former studies dealing more fertile soils.

Impairment of chemical hypoxia-induced sphingosine kinase-1 expression and activation in rheumatoid arthritis synovial fibroblasts: A signature of exhaustion?

Sphingosine kinase 1 (SphK1) and 2 (SphK2) have been shown contribute to synovial inflammation in animal models of arthritis. However, low levels of intracellular sphingosine-1 phosphate (S1P) were reported in fibroblast-like synoviocytes (FLS) from patients in the end stage of rheumatoid arthritis (RA) compared to normal FLS. Moreover, the S1P receptor-mediated chemokine synthesis was altered in RAFLS in response to chemical hypoxia. Since the mechanisms responsible for low levels of intracellular S1P in RAFLS are not fully identified, we evaluated the contribution of SphKs to the S1P-induced synthesis of chemokines under conditions of chemical hypoxia. Our results show that a chemical hypoxia mimetic cobalt chloride (CoCl2) increased SphK1 expression and activation in normal FLS but not in RAFLS. Using selective inhibitors of SphKs and gene silencing approaches, we provide evidence that both SphK1 and SphK2 are involved in hypoxia-induced chemokine production in normal FLS. In contrast, only SphK2 mediates hypoxia-induced chemokine production in RAFLS. Moreover, CoCl2 increased S1P2 and S1P3 receptor mRNA levels in normal FLS but not in RAFLS. The data suggest that altered expression and/or activation of SphK1 combined with reduced induction of S1P receptor expression by CoCl2 impaired the CoCl2-mediated autocrine S1P receptor signaling loop and chemokine production in RAFLS.

Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae

IntroductionPhosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized.ResultsXfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses.ConclusionOur study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.

A dual functional MOF-based fluorescent sensor for intracellular phosphate and extracellular 4-nitrobenzaldehyde.

Herein, we present the successful synthesis, characterization and sensing application of a hydrazine functionalized Zr(iv)-based UiO-66 metal-organic framework (MOF) called Zr-UiO-66-N2H3 (1). The guest-free material 1' shows selective and sensitive fluorimetric detection (turn-on mechanism) of phosphate (PO43-) anions in HEPES buffer (10 mM, pH = 7.4) and aqueous medium. It can also fluorimetrically detect (turn-off mechanism) 4-nitrobenzaldehyde (4-NB) in a HEPES : DMSO (9 : 1, v/v) medium with high selectivity and sensitivity. The selectivity for both analytes is retained in the presence of other potentially competitive analytes. The detection limit for PO43- ions is 0.196 μM, which is far below the PO43- level present in the aqueous environment. MOF 1' can detect intracellular phosphate and it also has the capacity to exhibit differences in the intracellular phosphate level. Furthermore, the probe is capable of sensing PO43- ions in real samples such as tap water, lake water, human urine and human blood serum. The sensitivity of the probe for sensing 4-NB is very high (detection limit = 4.7 μM). The possible mechanisms for sensing PO43- ions and 4-NB have been explored in detail by experimental techniques.

Y2O3 Nanoparticles Caused Bone Tissue Damage by Breaking the Intracellular Phosphate Balance in Bone Marrow Stromal Cells.

Y2O3 nanoparticles (NPs) have become great promising products for numerous applications in nanoscience especially for biomedical application, therefore increasing the probability of human exposure and gaining wide attention in biosecurity. It is well known that rare earth (RE) materials are deposited in the bone and excreted very slowly. Nevertheless, the effect of Y2O3-based NPs on bone metabolism has not been exactly known yet. In the present study, the effects of Y2O3 NPs on bone marrow stromal cells (BMSCs) and bone metabolism in mice after intravenous injection were studied. The results demonstrated that Y2O3 NPs could be taken up into BMSCs and localized in acidifying intracellular lysosomes and underwent dissolution and transformation from Y2O3 to YPO4, which could lead to a break in the intracellular phosphate balance and induce lysosomal- and mitochondrial-dependent apoptosis pathways. Furthermore, after being administered to mice, a higher concentration of yttrium occurred in bone, which caused the apoptosis of bone cells and induced the destruction of bone structure. However, the formation of a YPO4 coating on the surface of Y2O3 NPs by pretreatment of Y2O3 NPs in lysosome-simulated body fluid could observably decrease the toxicity in vivo and in vitro. This study may be useful for practical application of Y2O3 NPs in the biomedical field.

Knockout of the Hmt1p Arginine Methyltransferase in Saccharomyces cerevisiae Leads to the Dysregulation of Phosphate-associated Genes and Processes

Hmt1p is the predominant arginine methyltransferase in Saccharomyces cerevisiae Its substrate proteins are involved in transcription, transcriptional regulation, nucleocytoplasmic transport and RNA splicing. Hmt1p-catalyzed methylation can also modulate protein-protein interactions. Hmt1p is conserved from unicellular eukaryotes through to mammals where its ortholog, PRMT1, is lethal upon knockout. In yeast, however, the effect of knockout on the transcriptome and proteome has not been described. Transcriptome analysis revealed downregulation of phosphate-responsive genes in hmt1Δ, including acid phosphatases PHO5, PHO11, and PHO12, phosphate transporters PHO84 and PHO89 and the vacuolar transporter chaperone VTC3 Analysis of the hmt1Δ proteome revealed decreased abundance of phosphate-associated proteins including phosphate transporter Pho84p, vacuolar alkaline phosphatase Pho8p, acid phosphatase Pho3p and subunits of the vacuolar transporter chaperone complex Vtc1p, Vtc3p and Vtc4p. Consistent with this, phosphate homeostasis was dysregulated in hmt1Δ cells, showing decreased extracellular phosphatase levels and decreased total Pi in phosphate-depleted medium. In vitro, we showed that transcription factor Pho4p can be methylated at Arg-241, which could explain phosphate dysregulation in hmt1Δ if interplay exists with phosphorylation at Ser-242 or Ser-243, or if Arg-241 methylation affects the capacity of Pho4p to homodimerize or interact with Pho2p. However, the Arg-241 methylation site was not validated in vivo and the localization of a Pho4p-GFP fusion in hmt1Δ was not different from wild type. To our knowledge, this is the first study to reveal an association between Hmt1p and phosphate homeostasis and one which suggests a regulatory link between S-adenosyl methionine and intracellular phosphate.

A novel GNRHR gene mutation causing congenital hypogonadotrophic hypogonadism in a Brazilian kindred.

Congenital hypogonadotrophic hypogonadism (CHH) is a challenging inherited endocrine disorder characterised by absent or incomplete pubertal development and infertility as a result of the low action/secretion of the hypothalamic gonadotrophin-releasing hormone (GnRH). Given a growing list of gene mutations accounting for CHH, the application of massively parallel sequencing comprises an excellent molecular diagnostic approach because it enables the simultaneous evaluation of many genes. The present study proposes the use of whole exome sequencing (WES) to identify causative and modifying mutations based on a phenotype-genotype CHH analysis using an in-house exome pipeline. Based on 44 known genes related to CHH in humans, we were able to identify a novel homozygous gonadotrophin-releasing hormone receptor (GNRHR) p.Thr269Met mutant, which segregates with the CHH kindred and was predicted to be deleterious by in silico analysis. A functional study measuring intracellular inositol phosphate (IP) when stimulated with GnRH on COS-7 cells confirmed that the p.Thr269Met GnRHR mutant performed greatly diminished IP accumulation relative to the transfected wild-type GnRHR. Additionally, the proband carries three heterozygous variants in CCDC141 and one homozygous in SEMA3A gene, although their effects with respect to modifying the phenotype are uncertain. Because they do not segregate with reproductive phenotype in family members, we advocate they do not contribute to CHH oligogenicity. WES proved to be useful for CHH molecular diagnosis and reinforced its benefit with respect to identifying heterogeneous genetic disorders. Our findings expand the GnRHR mutation spectrum and phenotype-genotype correlation in CHH.

Phosphate and carbohydrate facilitate the formation of filamentous Salmonella enterica during osmotic stress.

Salmonella enterica is a human pathogen that can produce filamentous cells in response to environmental stress. The molecular mediators and biosynthetic pathways that contribute to the formation of filamentous cells (>10 µm in length) during osmotic stress are mostly unknown. The comparison of filamentous and non-filamentous cells in this study was aided by the use of a filtration step to separate cell types. Osmotic stress caused an efflux of phosphate from cells, and the addition of phosphate and a carbohydrate to Luria broth with 7 % NaCl (LB-7NaCl) significantly increased the proportion of filamentous cells in the population (58 %). In addition to direct measurements of intracellular and extracellular phosphate concentrations, the relative abundance of the iraP transcript that is induced by phosphate limitation was monitored. Non-filamentous cells had a greater relative abundance of iraP transcript than filamentous cells. IraP also affects the stability of RpoS, which regulates the general stress regulon, and was detected in non-filamentous cells but not filamentous cells. Markers of metabolic pathways for the production of acetyl-CoA (pflB, encoding for pyruvate formate lyase) and fatty acids (fabH) that are essential to membrane biosynthesis were found in greater abundance in filamentous cells than non-filamentous cells. There were no differences in the DNA, protein and biomass levels in filamentous and non-filamentous cells after 48 h of incubation, although the filamentous cells produced significantly (P<0.05) more acetate. This study found that phosphate and carbohydrate enhanced the formation of filamentous cells during osmotic stress, and there were differences in key regulatory elements and markers of metabolic pathways in filamentous and non-filamentous S. enterica.

PO-104 The shifted balance of high-energy phosphates drives the AMPK dephosphorylation and reduced slow myosin expression during unloading

Objective Mechanical unloading of postural muscles results in both muscle atrophy and a slow to fast fibre type transition. The cause of such changes is a reduction in slow-type MyHC isoform expression and an increase in fast-type MyHC isoform expression. It is believed that calcineurin/NFAT signalling pathway and AMP-activated protein kinase (AMPK) are involved in the regulation of slow-type MyHC isoform. Previously we showed that AMPK phosphorylation is significantly decreased in rat soleus at the early stage of mechanical unloading. We found, that stimulation of AMPK activity at the early stage of gravitational unloading prevents a decline of slow MyHC expression. We assumed that a decrease in AMPK activity in rat soleus at the early stage of gravitational unloading could be associated with changes in the ratio of intracellular high-energy phosphates (ATP/ADP). The aim of the study was to investigate the impact of high-energy phosphates ratio changes on AMPK activity and slow-type MyHC isoform expression in rat soleus muscle at the early stages of gravitational unloading.&#x0D; Methods To verify the hypothesis, we used administration of β-guanidinopropionic acid (βGPA), before (6 day) and during 24-h hindlimb suspension. The content of phospho-AMPK, phospho-ACC, phospho-PKD, HDAC4/5 in rat soleus was determined by Western-blotting. The amount of MyHCI(β) pre-mRNA and mRNA was evaluated by RT-PCR. &#x0D; Results After 24-h HS we observed a decrease (p&lt;0.05) in phospho-AMPK content vs. control group, but in HS+ βGPA group didn’t differ from the control. After 24-h unloading we found a significant increase in the content of nuclear HDAC4 in the HS group, but in the HS+ βGPA group the content of nuclear HDAC4 didn’t differ from the control group. 24-h unloading resulted in a decrease in MyHCI(β) pre-mRNA and mRNA expression vs. the control group. The expression level of MyHCI(β) pre-mRNA and mRNA in HS+ βGPA group didn’t differ from the control.&#x0D; Conclusions Thus βGPA administration prevents a decline in AMPK phosphorylation. Therefore, we can conclude that at the early stage of gravitational unloading an accumulation of high-energy phosphates (ATP, ADP and creatine phosphate) takes place and leads to reduced AMPK activity and a slow to fast myosin fibre type transition. The study was supported by Russian Science Foundation grant # 18-15-00107.

Genetic analysis of suppressor mutants of a pho84 disruptant in the search for genes involved in intracellular inorganic phosphate sensing in Saccharomyces cerevisiae.

To achieve inorganic phosphate (Pi) homeostasis, cells must be able to sense intracellular and extracellular Pi concentrations. In the Pi signaling (PHO) pathway in Saccharomyces cerevisiae, high Pi represses genes involved in Pi uptake (e.g., PHO84) and Pi utilization (PHO5); conversely, the cyclin-dependent kinase inhibitor Pho81 inhibits the activity of the Pho80-Pho85 cyclin-cyclin dependent kinase complex in low-Pi conditions, leading to induction of these genes. However, how yeast senses Pi availability remains unresolved. To identify factors involved in Pi sensing upstream of the Pho81-Pho80-Pho85 complex, we generated and screened suppressor mutants of a Δpho84 strain that shows constitutive PHO5 expression. By a series of genetic tests, including dominance-recessiveness, complementation and tetrad analyses, three sef (suppressor of pho84 [pho eighty-four]) mutants (sef8, sef9 and sef10) were shown to contain a novel single mutation. The sef mutants suppressed the phenotype of constitutive PHO5 expression at the transcriptional level, but did not show restored Pi uptake capacity. An epistasis-hypostasis test revealed that the sef mutations were hypostatic to pho80 mutation, indicating that their gene products function upstream of the Pho81-Pho80-Pho85 complex in the PHO pathway. The sef mutations identified are associated with gene(s) that may be involved in the homeostasis of an intracellular Pi level-sensing mechanism in S. cerevisiae.

Nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant results in albuminuria and mesangial expansion.

Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in the onset and progression of diseases. The presence of cellular vacuoles-observed in many histologic analyses of renal diseases-is a macroscopic hint for disturbed intracellular trafficking processes. However, how vacuoles develop and which intracellular pathways are directly affected remain largely unknown. Previous studies showed that in some cases, vacuolization is linked to malfunction of the Vac14 complex. This complex, including the scaffold protein Vac14, the lipid kinase PIKfyve, and its counteracting lipid phosphatase Fig4, regulates intracellular phosphatidylinositol phosphate levels, which in turn, control the maturation of early-into-late endosomes, as well as the processing of autophagosomes into autophagolysosomes. Here, we analyzed the role of Vac14 in mice and observed that the nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant led to albuminuria, accompanied by mesangial expansion, increased glomerular size, and an elevated expression of several kidney injury markers. Overexpression of this Vac14 variant in podocytes did not reveal a strong in vivo phenotype, indicating that Vac14-dependent trafficking processes are more important for tubular than for glomerular processes in the kidney. In vitro overexpression of Vac14L156R in Madin-Darby canine kidney cells had no impact on apico-basal polarity defects but resulted in a faster reassembly of junctional structures after Ca2+ depletion and delayed endo- and transcytosis rates. Taken together, our data suggest that increased albuminuria of Vac14L156R-overexpressing mice is a consequence of a lowered endo- and transcytosis of albumin in renal tubules.