Intracellular Phosphate Levels Research Articles

The ppk-expressing transgenic rice floating bed improves P removal in slightly polluted water

With significant economic advantages, the plant floating bed has been widely utilized in the ecological remediation of eutrophic water because of the excessive phosphorus (P) and nitrogen discharge in China. Previous research has demonstrated that polyphosphate kinase (ppk)-expressing transgenic rice (Oryza sativa L. ssp. japonica) (ETR) can increase the P absorption capacity to support rice growth and boost rice yield. In this study, the floating beds of ETR with single copy line (ETRS) and double copy line (ETRD) are built to investigate their capacity to remove aqueous P in slightly polluted water. Compared with the wild type Nipponbare (WT) floating bed, the ETR floating beds greatly reduce the total P concentration in slightly polluted water though the ETR floating beds have the same removal rates of chlorophyll-a, NO3−-N, and total nitrogen in slightly polluted water. The P uptake rate of ETRD on the floating bed is 72.37% in slightly polluted water, which is higher than that of ETRS and WT on the floating beds. Polyphosphate (polyP) synthesis is a critical factor for the excessive phosphate uptake of ETR on the floating beds. The synthesis of polyP decreases the level of free intracellular phosphate (Pi) in ETR on the floating beds, simulating the phosphate starvation signaling. The OsPHR2 expression in the shoot and root of ETR on the floating bed increased, and the corresponding P metabolism gene expression in ETR was changed, which promoted Pi uptake by ETR in slightly polluted water. The Pi accumulation further promoted the growth of ETR on the floating beds. These findings highlight that the ETR floating beds, especially ETRD floating bed, have significant potential for P removal and can be exploited as a novel method for phytoremediation in slightly polluted water.

Using phosphoglucose isomerase-deficient (pgi1Δ) Saccharomyces cerevisiae to map the impact of sugar phosphate levels on d-glucose and d-xylose sensing

BackgroundDespite decades of engineering efforts, recombinant Saccharomyces cerevisiae are still less efficient at converting d-xylose sugar to ethanol compared to the preferred sugar d-glucose. Using GFP-based biosensors reporting for the three main sugar sensing routes, we recently demonstrated that the sensing response to high concentrations of d-xylose is similar to the response seen on low concentrations of d-glucose. The formation of glycolytic intermediates was hypothesized to be a potential cause of this sensing response. In order to investigate this, glycolysis was disrupted via the deletion of the phosphoglucose isomerase gene (PGI1) while intracellular sugar phosphate levels were monitored using a targeted metabolomic approach. Furthermore, the sugar sensing of the PGI1 deletants was compared to the PGI1-wildtype strains in the presence of various types and combinations of sugars.ResultsMetabolomic analysis revealed systemic changes in intracellular sugar phosphate levels after deletion of PGI1, with the expected accumulation of intermediates upstream of the Pgi1p reaction on d-glucose and downstream intermediates on d-xylose. Moreover, the analysis revealed a preferential formation of d-fructose-6-phosphate from d-xylose, as opposed to the accumulation of d-fructose-1,6-bisphosphate that is normally observed when PGI1 deletants are incubated on d-fructose. This may indicate a role of PFK27 in d-xylose sensing and utilization. Overall, the sensing response was different for the PGI1 deletants, and responses to sugars that enter the glycolysis upstream of Pgi1p (d-glucose and d-galactose) were more affected than the response to those entering downstream of the reaction (d-fructose and d-xylose). Furthermore, the simultaneous exposure to sugars that entered upstream and downstream of Pgi1p (d-glucose with d-fructose, or d-glucose with d-xylose) resulted in apparent synergetic activation and deactivation of the Snf3p/Rgt2p and cAMP/PKA pathways, respectively.ConclusionsOverall, the sensing assays indicated that the previously observed d-xylose response stems from the formation of downstream metabolic intermediates. Furthermore, our results indicate that the metabolic node around Pgi1p and the level of d-fructose-6-phosphate could represent attractive engineering targets for improved d-xylose utilization.

Loss of function of CMPK2 causes mitochondria deficiency and brain calcification

Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.

OR2AT4, an Ectopic Olfactory Receptor, Suppresses Oxidative Stress-Induced Senescence in Human Keratinocytes.

Olfactory receptors (ORs) are the largest protein superfamily in mammals. Certain ORs are ectopically expressed in extranasal tissues and regulate cell type-specific signal transduction pathways. OR2AT4 is ectopically expressed in skin cells and promotes wound healing and hair growth. As the capacities of wound healing and hair growth decline with aging, we investigated the role of OR2AT4 in the aging and senescence of human keratinocytes. OR2AT4 was functionally expressed in human keratinocytes (HaCaT) and exhibited co-expression with G-protein-coupled receptor signaling components, Golfα and adenylate cyclase 3. The OR2AT4 ligand sandalore modulates the intracellular calcium, inositol phosphate, and cyclic adenosine monophosphate (cAMP) levels. The increased calcium level induced by sandalore was attenuated in cells with OR2AT4 knockdown. OR2AT4 activation by sandalore inhibited the senescent cell phenotypes and restored cell proliferation and Ki-67 expression. Sandalore also inhibited the expression of senescence-associated β-galactosidase and increased p21 expression in senescent HaCaT cells in response to hydrogen peroxide. Additionally, sandalore activated the CaMKKβ/AMPK/mTORC1/autophagy signaling axis and promoted autophagy. OR2AT4 knockdown attenuated the increased in the intracellular calcium level, cell proliferation, and AMPK phosphorylation induced by sandalore. These findings demonstrate that the effects of sandalore are mediated by OR2AT4 activation. Our findings suggest that OR2AT4 may be a novel therapeutic target for anti-aging and anti-senescence in human keratinocytes.

Multi-omics in classical galactosemia: Evidence for the involvement of multiple metabolic pathways.

Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis.

The Global Regulator PhoU Positively Controls Growth and Butenyl-Spinosyn Biosynthesis in Saccharopolyspora pogona.

Butenyl-spinosyn, a highly effective biological insecticide, is produced by Saccharopolyspora pogona. However, its application has been severely hampered by its low yield. Recent studies have shown that PhoU plays a pivotal role in regulating cell growth, secondary metabolite biosynthesis and intracellular phosphate levels. Nevertheless, the function of PhoU remains ambiguous in S. pogona. In this study, we investigated the effects of PhoU on the growth and the butenyl-spinosyn biosynthesis of S. pogona by constructing the mutants. Overexpression of phoU increased the production of butenyl-spinosyn to 2.2-fold that of the wild-type strain. However, the phoU deletion resulted in a severe imbalance of intracellular phosphate levels, and suppression of the growth and butenyl-spinosyn biosynthesis. Quantitative Real-time PCR (qRT-PCR) analysis, distinctive protein detection and mass spectrometry revealed that PhoU widely regulated primary metabolism, energy metabolism and DNA repair, which implied that PhoU influences the growth and butenyl-spinosyn biosynthesis of S. pogona as a global regulator.

Interplay between primary familial brain calcification-associated SLC20A2 and XPR1 phosphate transporters requires inositol polyphosphates for control of cellular phosphate homeostasis

Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP-binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.

The role of sodium phosphate cotransporters in ectopic calcification.

Phosphate plays a critical role in many vital cellular processes. Deviations from normal serum phosphate levels, including alterations in the extracellular phosphate/pyrophosphate ratio, can cause severe consequences, such as ectopic calcification. Cellular phosphate levels are tightly controlled by sodium phosphate cotransporters, underscoring their importance in cellular physiology. The role of sodium phosphate cotransporters in ectopic calcification requires further elucidation, taking into account their important role in the control of intracellular phosphate levels and the synthesis of ATP, the main source of extracellular pyrophosphate (a potent endogenous inhibitor of calcification). In this review, we discuss the roles of phosphate and pyrophosphate homeostasis in ectopic calcification, with a specific focus on phosphate transporters. We concentrate on the five known sodium-dependent phosphate transporters and review their localisation and regulation by external factors, and the effects observed in knockout studies and in naturally occurring mutations.

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.

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.

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.

TDAG51 is a key modulator of vascular calcification and osteogenic transdifferentiation of arterial smooth muscle cells

Cardiovascular disease (CVD) is the primary cause of morbidity and mortality in patients with chronic kidney disease (CKD). More than half of all deaths from patients with end-stage renal disease (ESRD) requiring dialysis can be attributed to CVD. Although traditional risk factors for CVD are prevalent in ESRD, they cannot fully explain the high mortality rates. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced CKD and is now recognized as an important predictor and an independent risk factor for cardiovascular and all-cause and mortality in these patients. VC in CKD is triggered by vascular smooth muscle cells (VSMCs) transforming into osteoblasts, a consequence of elevated circulating phosphate levels due to poor kidney function. Understanding and targeting this SMC trans-differentiation and subsequent VC represents a viable therapeutic avenue for managing CKD complications. Similar to bone formation, VC is a highly regulated process that draws many parallels with osteogenic differentiation. We have recently observed that T-cell Death Associated Gene 51 (TDAG51), a protein upregulated by oxidative and endoplasmic reticulum stress, is induced in SMCs under conditions of hyperphosphatemia and is expressed in the medial layer of calcified human vessels. We have found that ablation of TDAG51 reduces SMC trans-differentiation and VC, both in human SMCs treated with TDAG51 siRNA and in mouse VSMCs isolated from TDAG51−/− aortas. To explain these findings, we observed that TDAG−/− VSMCs express reduced levels of the of the Pit-1, a solute transporter responsible for cellular phosphate uptake. Consistent with this observation, reduced Pit-1 expression was associated with reduced intracellular phosphate levels and reduced phosphate-induced apoptosis. Further, TDAG−/− VSMCs exhibit significantly reduced transcriptional activity of a phosphate-inducible transcription factor, and well-established driver of VC, known as Runx2. Support or Funding Information This work was supported in part by research grants to Richard C. Austin from the Heart and Stroke Foundation of Ontario (T-6146), the Heart and Stroke Foundation of Canada (G-13-0003064 and G-15-0009389) and the Canadian Institutes of Health Research (74477). TDAG51 is involved in the development and progression of VC through the regulation of intracellular phosphate content and Runx2 transcriptional activity. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The galactose-induced decrease in phosphate levels leads to toxicity in yeast models of galactosemia

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations in the GALT gene. A number of evidences indicate that the galactose-1-phosphate accumulation observed in patient cells is a cause of toxicity in this disease. Nevertheless, the consequent molecular events caused by the galactose-1-phosphate accumulation remain elusive. Here we show that intracellular inorganic phosphate levels decreased when yeast models of classic galactosemia were exposed to galactose. The decrease in phosphate levels is probably due to the trapping of phosphate in the accumulated galactose-1-phosphate since the deletion of the galactokinase encoding gene GAL1 suppressed this phenotype. Galactose-induced phosphate depletion caused an increase in glycogen content, an expected result since glycogen breakdown by the enzyme glycogen phosphorylase is dependent on inorganic phosphate. Accordingly, an increase in intracellular phosphate levels suppressed the galactose effect on glycogen content and conferred galactose tolerance to yeast models of galactosemia. These results support the hypothesis that the galactose-induced decrease in phosphate levels leads to toxicity in galactosemia and opens new possibilities for the development of better treatments for this disease.

Renal Fanconi Syndrome and Hypophosphatemic Rickets in the Absence of Xenotropic and Polytropic Retroviral Receptor in the Nephron.

Tight control of extracellular and intracellular inorganic phosphate (Pi) levels is critical to most biochemical and physiologic processes. Urinary Pi is freely filtered at the kidney glomerulus and is reabsorbed in the renal tubule by the action of the apical sodium-dependent phosphate transporters, NaPi-IIa/NaPi-IIc/Pit2. However, the molecular identity of the protein(s) participating in the basolateral Pi efflux remains unknown. Evidence has suggested that xenotropic and polytropic retroviral receptor 1 (XPR1) might be involved in this process. Here, we show that conditional inactivation of Xpr1 in the renal tubule in mice resulted in impaired renal Pi reabsorption. Analysis of Pi transport in primary cultures of proximal tubular cells or in freshly isolated renal tubules revealed that this Xpr1 deficiency significantly affected Pi efflux. Further, mice with conditional inactivation of Xpr1 in the renal tubule exhibited generalized proximal tubular dysfunction indicative of Fanconi syndrome, characterized by glycosuria, aminoaciduria, calciuria, and albuminuria. Dramatic alterations in the renal transcriptome, including a significant reduction in NaPi-IIa/NaPi-IIc expression, accompanied these functional changes. Additionally, Xpr1-deficient mice developed hypophosphatemic rickets secondary to renal dysfunction. These results identify XPR1 as a major regulator of Pi homeostasis and as a potential therapeutic target in bone and kidney disorders.

Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine.

In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 (+)) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 (+) expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 (+) expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 (+) and some of the regulation acts through pho7 (+).

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1(-/-);Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.

Uric acid-dependent inhibition of AMP kinase induces hepatic glucose production in diabetes and starvation: evolutionary implications of the uricase loss in hominids.

Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes.

Expression Patterns of Phosphate Transporter Gene ECGPT Associated with the Salt Stress Response in Perennial Eucalypt Tree Hybrid Clones

Salinity effects on plant can often be related to mineral ion content alteration, including phosphate. Under saline conditions, phosphate levels were reported to decrease in plants. Such effects could indirectly affect intracellular phosphate levels, leading to phosphate deficiency, which in turn leads to increased activities of phosphate uptake mechanisms. This research was aimed to investigate the effects of salinity on the expression changes of phosphate transporter genes isolated from E. camaldulensis x E. globulus hybrid clones subjected to salt concentrations of 0 (control), 50, 100, and 150 mMNaCl. Fragments of genes (1164bp long and encodes 304 amino acid polypeptides) known to be involved in phosphate uptake were identified and isolated by RACE from these hybrids, encoding a Phosphate Transporter ( Ecg PT). Expression studies using Northern-Blot analysis revealed that the expression of Ecg PT was found to be affected by salt, suggesting a direct effect of salinity on phosphate uptake. Ecg PT was expressed differently in different clones, indicating different degrees of phosphate transporter activation in order to tolerate salt stress . Molecular data are discussed in relation to measurements of ion levels in different organs of different clones and under various salt regimes during the course of the hydroponic experiment.

Effects of supplementary butyrate on butanol production and the metabolic switch in Clostridium beijerinckii NCIMB 8052: genome-wide transcriptional analysis with RNA-Seq

BackgroundButanol (n-butanol) has high values as a promising fuel source and chemical feedstock. Biobutanol is usually produced by the solventogenic clostridia through a typical biphasic (acidogenesis and solventogenesis phases) acetone-butanol-ethanol (ABE) fermentation process. It is well known that the acids produced in the acidogenic phase are significant and play important roles in the switch to solventogenesis. However, the mechanism that triggers the metabolic switch is still not clear.ResultsSodium butyrate (40 mM) was supplemented into the medium for the ABE fermentation with Clostridium beijerinckii NCIMB 8052. With butyrate addition (reactor R1), solvent production was triggered early in the mid-exponential phase and completed quickly in < 50 h, while in the control (reactor R2), solventogenesis was initiated during the late exponential phase and took > 90 h to complete. Butyrate supplementation led to 31% improvement in final butanol titer, 58% improvement in sugar-based yield, and 133% improvement in butanol productivity, respectively. The butanol/acetone ratio was 2.4 versus 1.8 in the control, indicating a metabolic shift towards butanol production due to butyrate addition. Genome-wide transcriptional dynamics was investigated with RNA-Seq analysis. In reactor R1, gene expression related to solventogenesis was induced about 10 hours earlier when compared to that in reactor R2. Although the early sporulation genes were induced after the onset of solventogenesis in reactor R1 (mid-exponential phase), the sporulation events were delayed and uncoupled from the solventogenesis. In contrast, in reactor R2, sporulation genes were induced at the onset of solventogenesis, and highly expressed through the solventogenesis phase. The motility genes were generally down-regulated to lower levels prior to stationary phase in both reactors. However, in reactor R2 this took much longer and gene expression was maintained at comparatively higher levels after entering stationary phase.ConclusionsSupplemented butyrate provided feedback inhibition to butyrate formation and may be re-assimilated through the reversed butyrate formation pathway, thus resulting in an elevated level of intracellular butyryl phosphate, which may act as a phosphate donor to Spo0A and then trigger solventogenesis and sporulation events. High-resolution genome-wide transcriptional analysis with RNA-Seq revealed detailed insights into the biochemical effects of butyrate on solventogenesis related-events at the gene regulation level.

Counteracting Roles of AMP Deaminase and AMP Kinase in the Development of Fatty Liver

Fatty liver (hepatic steatosis) is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP). It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK) is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation –either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.