Non‐enzymatic glycolysis and pentose phosphate pathway‐like reactions in a plausible Archean ocean

The reaction sequences of central metabolism, glycolysis and the pentose phosphate pathway provide essential precursors for nucleic acids, amino acids and lipids. However, their evolutionary origins are not yet understood. Here, we provide evidence that their structure could have been fundamentally shaped by the general chemical environments in earth's earliest oceans. We reconstructed potential scenarios for oceans of the prebiotic Archean based on the composition of early sediments. We report that the resultant reaction milieu catalyses the interconversion of metabolites that in modern organisms constitute glycolysis and the pentose phosphate pathway. The 29 observed reactions include the formation and/or interconversion of glucose, pyruvate, the nucleic acid precursor ribose‐5‐phosphate and the amino acid precursor erythrose‐4‐phosphate, antedating reactions sequences similar to that used by the metabolic pathways. Moreover, the Archean ocean mimetic increased the stability of the phosphorylated intermediates and accelerated the rate of intermediate reactions and pyruvate production. The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. The reactions were particularly sensitive to ferrous iron Fe(II), which is understood to have had high concentrations in the Archean oceans. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron‐rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.     

Regulation of the pentose phosphate pathway in cancer

Energy metabolism is significantly reprogrammed in many human cancers, and these alterations confer many advantages to cancer cells, including the promotion of biosynthesis, ATP generation, detoxification and support of rapid proliferation. The pentose phosphate pathway (PPP) is a major pathway for glucose catabolism. The PPP directs glucose flux to its oxidative branch and produces a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), an essential reductant in anabolic processes. It has become clear that the PPP plays a critical role in regulating cancer cell growth by supplying cells with not only ribose-5-phosphate but also NADPH for detoxification of intracellular reactive oxygen species, reductive biosynthesis and ribose biogenesis. Thus, alteration of the PPP contributes directly to cell proliferation, survival and senescence. Furthermore, recent studies have shown that the PPP is regulated oncogenically and/or metabolically by numerous factors, including tumor suppressors, oncoproteins and intracellular metabolites. Dysregulation of PPP flux dramatically impacts cancer growth and survival. Therefore, a better understanding of how the PPP is reprogrammed and the mechanism underlying the balance between glycolysis and PPP flux in cancer will be valuable in developing therapeutic strategies targeting this pathway.     

On the origin of biochemistry at an alkaline hydrothermal vent

A model for the origin of biochemistry at an alkaline hydrothermal vent has been developed that focuses on the acetyl-CoA (Wood-Ljungdahl) pathway of CO2 fixation and central intermediary metabolism leading to the synthesis of the constituents of purines and pyrimidines. The idea that acetogenesis and methanogenesis were the ancestral forms of energy metabolism among the first free-living eubacteria and archaebacteria, respectively, stands in the foreground. The synthesis of formyl pterins, which are essential intermediates of the Wood-Ljungdahl pathway and purine biosynthesis, is found to confront early metabolic systems with steep bioenergetic demands that would appear to link some, but not all, steps of CO2 reduction to geochemical processes in or on the Earth's crust. Inorganically catalysed prebiotic analogues of the core biochemical reactions involved in pterin-dependent methyl synthesis of the modern acetyl-CoA pathway are considered. The following compounds appear as probable candidates for central involvement in prebiotic chemistry: metal sulphides, formate, carbon monoxide, methyl sulphide, acetate, formyl phosphate, carboxy phosphate, carbamate, carbamoyl phosphate, acetyl thioesters, acetyl phosphate, possibly carbonyl sulphide and eventually pterins. Carbon might have entered early metabolism via reactions hardly different from those in the modern Wood-Ljungdahl pathway, the pyruvate synthase reaction and the incomplete reverse citric acid cycle. The key energy-rich intermediates were perhaps acetyl thioesters, with acetyl phosphate possibly serving as the universal metabolic energy currency prior to the origin of genes. Nitrogen might have entered metabolism as geochemical NH3 via two routes: the synthesis of carbamoyl phosphate and reductive transaminations of alpha-keto acids. Together with intermediates of methyl synthesis, these two routes of nitrogen assimilation would directly supply all intermediates of modern purine and pyrimidine biosynthesis. Thermodynamic considerations related to formyl pterin synthesis suggest that the ability to harness a naturally pre-existing proton gradient at the vent-ocean interface via an ATPase is older than the ability to generate a proton gradient with chemistry that is specified by genes.     

Profiling of dynamic changes in hypermetabolic livers

The liver plays an important role in the overall negative nitrogen balance leading to muscle wasting commonly observed in patients following many conditions, including severe injury, cancer, and diabetes. In order to study changes in liver metabolism during the establishment of such catabolic states, we used a rat skin burn injury model that induces hypermetabolism and muscle wasting. At various times during the first week following the injury, livers were isolated and perfused in a recirculating system under well-defined conditions. We applied a steady-state metabolic flux analysis model of liver metabolism and then used k-means clustering to objectively group together reaction flux time profiles. We identified six distinct groups of reactions that were differentially responsive: (1) pentose phosphate pathway (PPP); (2) amino acid oxidation reactions leading to the formation of tricarboxylic acid (TCA) cycle intermediates; (3) gluconeogenesis; (4) TCA-cycle and mitochondrial oxidation; (5) lipolysis, beta-oxidation, and ketone body formation; and (6) urea-cycle. Burn injury sequentially upregulated the urea-cycle, the PPP, and the TCA-cycle, in order, while beta-oxidation and gluconeogenesis remained unchanged. The upregulation of the PPP was transient, whereas the rise in urea- and TCA-cycle fluxes was sustained. An ATP balance predicted an increased production of ATP and energy expenditure starting on day 3 post-burn, which correlated with the induction of the oxidative phosphorylation uncoupler uncoupling protein-2. We conclude that metabolic profiling using flux analysis and clustering analysis is a useful methodology to characterize the differential activation of metabolic pathways in perfused organs and to identify specific key pathways that are sensitive to a stimulus or insult without making a priori assumptions.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Megacell phenotype and its relation to metabolic alterations in transketolase deficient strain of Bacillus pumilus

Fermentation with transketolase (tkt) deficient strain of Bacillus is the only reported industrially viable process for production of D ‐ribose, a commercially important pentose sugar. In addition to direct effects of tkt deficiency, the mutation in non‐oxidative part of pentose phosphate pathway (PPP) is known to display several unexpected physiological characteristics such as decreased ability to utilize D ‐glucose, altered carbon catabolite repression, lack of motility, etc. Here we demonstrate the morphological plasticity of tkt deficient strain of Bacillus pumilus ATCC 21951 and its possible relation with D ‐ribose productivity, a measure of carbon flux through PPP. The bacilli divide normally in nutrient rich media such as Luria–Bertani (LB) broth while showing cell elongation of up to 20‐fold without a visible septum accompanied by moderate to high extracellular D ‐ribose accumulation in glucose‐rich media. The cells stained with DAPI (4′‐6‐diamidino‐2‐phenylindole) and anti FtsZ antibody showed nucleoid separation and Z‐ring formation in LB broth but not in glucose‐rich media. FtsZ protein is known to localize at the future division site forming a ring, called Z‐ring, at an early stage in cytokinesis. The strain experiences inhibition or delay in Z‐ring formation resulting in cell elongation, possibly due to its altered cell membrane composition resulting from tkt deficiency. We hypothesize that the lack of PPP intermediates may have two effects on the strain: (i) altered the cell membrane leading to delay in Z‐ring formation and cell elongation; and (ii) induction of genes of the oxidative part of PPP resulting in D ‐ribose accumulation. Nutrient rich media such as LB broth may alleviate these metabolite deficiencies thereby restoring normal cell division and inhibiting excessive D ‐ribose accumulation. The D ‐ribose productivity and cell elongation may therefore be co‐morbid. The results have implications in designing optimal media and monitoring strategy based on morphological analysis. Biotechnol. Bioeng. 2009;102: 1387–1397. © 2008 Wiley Periodicals, Inc.     

Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production.     

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等.葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中.本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用.     

Amyloid beta peptide (1–42)‐mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate‐reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1–42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O₂ pressure, in Abeta‐treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O₂ pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta‐treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post‐translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Biosynthesis of ribose-5-phosphate and erythrose-4-phosphate in archaea: a phylogenetic analysis of archaeal genomes

A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP), the ribulose monophosphate (RuMP) pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium) produce ribose-5-phosphate via the nonoxidative PPP (NOPPP), whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1) lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP), the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P) among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii) probably does not synthesize aromatic amino acids at all.     

High salt diet induces metabolic alterations in multiple biological processes of Dahl salt-sensitive rats

High salt induced renal disease is a condition resulting from the interactions of genetic and dietary factors causing multiple complications. To understand the metabolic alterations associated with renal disease, we comprehensively analyzed the metabonomic changes induced by high salt intake in Dahl salt-sensitive (SS) rats using GC-MS technology and biochemical analyses. Physiological features, serum chemistry, and histopathological data were obtained as complementary information. Our results showed that high salt (HS) intake for 16 weeks caused significant metabolic alterations in both the renal medulla and cortex involving a variety pathways involved in the metabolism of organic acids, amino acids, fatty acids, and purines. In addition, HS enhanced glycolysis (hexokinase, phosphofructokinase and pyruvate kinase) and amino acid metabolism and suppressed the TCA (citrate synthase and aconitase) cycle. Finally, HS intake caused up-regulation of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the ratio of NADPH/NADP⁺, NADPH oxidase activity and ROS production, suggesting that increased oxidative stress was associated with an altered PPP pathway. The metabolic pathways identified may serve as potential targets for the treatment of renal damage. Our findings provide comprehensive biochemical details about the metabolic responses to a high salt diet, which may contribute to the understanding of renal disease and salt-induced hypertension in SS rats.     

Cells overexpressing fructose-2,6-bisphosphatase showed enhanced pentose phosphate pathway flux and resistance to oxidative stress

Changes in the content of fructose-2,6-bisphosphate, a modulator of glycolytic flux, also affect other metabolic fluxes such as the non-oxidative pentose phosphate pathway. Since this is the main source of precursors for biosynthesis in proliferating cells, PFK-2/FBPase-2 has been proposed as a potential target for neoplastic treatments. Here we provide evidence that cells with a low content of fructose-2,6-bisphosphate have a lower energy status than controls, but they are also less sensitive to oxidative stress. This feature is related to the activation of the oxidative branch of the pentose phosphate pathway and the increased production of NADPH.     

Metabolic flux analysis in Escherichia coli by integrating isotopic dynamic and isotopic stationary 13C labeling data

The novel concept of isotopic dynamic 13C metabolic flux analysis (ID-13C MFA) enables integrated analysis of isotopomer data from isotopic transient and/or isotopic stationary phase of a 13C labeling experiment, short-time experiments, and an extended range of applications of 13C MFA. In the presented work, an experimental and computational framework consisting of short-time 13C labeling, an integrated rapid sampling procedure, a LC-MS analytical method, numerical integration of the system of isotopomer differential equations, and estimation of metabolic fluxes was developed and applied to determine intracellular fluxes in glycolysis, pentose phosphate pathway (PPP), and citric acid cycle (TCA) in Escherichia coli grown in aerobic, glucose-limited chemostat culture at a dilution rate of D = 0.10 h(-1). Intracellular steady state concentrations were quantified for 12 metabolic intermediates. A total of 90 LC-MS mass isotopomers were quantified at sampling times t = 0, 91, 226, 346, 589 s and at isotopic stationary conditions. Isotopic stationarity was reached within 10 min in glycolytic and PPP metabolites. Consistent flux solutions were obtained by ID-13C MFA using isotopic dynamic and isotopic stationary 13C labeling data and by isotopic stationary 13C MFA (IS-13C MFA) using solely isotopic stationary data. It is demonstrated that integration of dynamic 13C labeling data increases the sensitivity of flux estimation, particularly at the glucose-6-phosphate branch point. The identified split ratio between glycolysis and PPP was 55%:44%. These results were confirmed by IS-13C MFA additionally using labeling data in proteinogenic amino acids (GC-MS) obtained after 5 h from sampled biomass.     

Metabolic Reprogramming for Producing Energy and Reducing Power in Fumarate Hydratase Null Cells from Hereditary Leiomyomatosis Renal Cell Carcinoma

Fumarate hydratase (FH)-deficient kidney cancer undergoes metabolic remodeling, with changes in mitochondrial respiration, glucose, and glutamine metabolism. These changes represent multiple biochemical adaptations in glucose and fatty acid metabolism that supports malignant proliferation. However, the metabolic linkages between altered mitochondrial function, nucleotide biosynthesis and NADPH production required for proliferation and survival have not been elucidated. To characterize the alterations in glycolysis, the Krebs cycle and the pentose phosphate pathways (PPP) that either generate NADPH (oxidative) or do not (non-oxidative), we utilized [U-¹³C]-glucose, [U-¹³C,¹⁵N]-glutamine, and [1,2- ¹³C₂]-glucose tracers with mass spectrometry and NMR detection to track these pathways, and measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of growing cell lines. This metabolic reprogramming in the FH null cells was compared to cells in which FH has been restored. The FH null cells showed a substantial metabolic reorganization of their intracellular metabolic fluxes to fulfill their high ATP demand, as observed by a high rate of glucose uptake, increased glucose turnover via glycolysis, high production of glucose-derived lactate, and low entry of glucose carbon into the Krebs cycle. Despite the truncation of the Krebs cycle associated with inactivation of fumarate hydratase, there was a small but persistent level of mitochondrial respiration, which was coupled to ATP production from oxidation of glutamine-derived α–ketoglutarate through to fumarate. [1,2- ¹³C₂]-glucose tracer experiments demonstrated that the oxidative branch of PPP initiated by glucose-6-phosphate dehydrogenase activity is preferentially utilized for ribose production (56-66%) that produces increased amounts of ribose necessary for growth and NADPH. Increased NADPH is required to drive reductive carboxylation of α-ketoglutarate and fatty acid synthesis for rapid proliferation and is essential for defense against increased oxidative stress. This increased NADPH producing PPP activity was shown to be a strong consistent feature in both fumarate hydratase deficient tumors and cell line models.     

Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress

Glucose‐6‐phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re‐expression of wild‐type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2‐dependent manner. The SIRT2‐mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress.     

Arginine methylation of ribose-5-phosphate isomerase A senses glucose to promote human colorectal cancer cell survival

Cancer cells remodel their metabolic network to adapt to variable nutrient availability. Pentose phosphate pathway(PPP) plays protective and biosynthetic roles by oxidizing glucose to generate reducing power and ribose. How cancer cells modulate PPP activity in response to glucose supply remains unclear. Here we show that ribose-5-phosphate isomerase A(RPIA), an enzyme in PPP, directly interacts with co-activator associated arginine methyltransferase 1(CARM1) and is methylated at arginine 42(R42). R42 methylation up-regulates the catalytic activity of RPIA. Furthermore, glucose deprivation strengthens the binding of CARM1 with RPIA to induce R42 hypermethylation. Insufficient glucose supply links to RPIA hypermethylation at R42, which increases oxidative PPP flux. RPIA methylation supports ROS clearance by enhancing NADPH production and fuels nucleic acid synthesis by increasing ribose supply. Importantly, RPIA methylation at R42 significantly potentiates colorectal cancer cell survival under glucose starvation. Collectively, RPIA methylation connects glucose availability to nucleotide synthesis and redox homeostasis.     

A possible role for the pentose phosphate pathway of spermatozoa in gamete fusion in the mouse

Glucose metabolism is essential for successful gamete fusion in the mouse. Although the metabolic activity of the oocyte does not appear to play a significant role in the fusion step, the metabolic role of the spermatozoon is not known. The aim of this study was therefore to characterize the role of glucose metabolism in mouse spermatozoa. Initially, the high-affinity glucose transporter GLUT3 was identified in mouse sperm. In characterizing the glucose metabolism of mouse sperm, we have shown 1) that mouse epididymal spermatozoa have a functional pentose phosphate pathway (PPP), implying that they produce NADPH, which is required for reducing reactions, and ribose 5-phosphate, which is required for nucleic acid synthesis; and 2) that sperm are able to fuse with the oocyte when NADPH is substituted for glucose, suggesting that sperm need to produce NADPH via the PPP in order to be able to achieve fertilization. The existence of an NADPH-regulated event that influences the ability of the sperm to fuse with the oocyte is envisaged.     

Metabolic implications of stress-induced proline accumulation in plants

In many plants, free proline accumulates in response to the imposition of a wide range of biotic and abiotic stresses. Controversy has surrounded the extent to which this shift in nitrogen metabolism benefits plants under adverse environmental conditions. Most attempts to account for the phenomenon have focused on the ability of proline to mediate osmotic adjustment, stabilise subcellular structures and scavenge free radicals. However, often the cytoplasmic pool of free proline even after the imposition of stress is insufficient size to account for pronounced biophysical effects.Alternatively, selective preservation of this stress-induced response may relate to endpoints other than simply augmenting the cellular pool of free proline. Proline accumulation may reduce stress-induced cellular acidification or prime oxidative respiration to provide energy needed for recovery. High levels of proline synthesis during stress may maintain NAD(P)+/NAD(P)H ratios at values compatible with metabolism under normal conditions. Consideration of the cofactor preference of plant 1-pyrroline-5-carboxylate (P5C) reductase as well as the in vivo concentrations of the two pyridine nucleotide cofactors and their respective redox ratios suggests that even a small increase in proline biosynthesis might have a large impact on the level of reduction of the cellular NADP pool. The increased NADP+/NADPH ratio mediated by proline biosynthesis is likely to enhance activity of the oxidative pentose phosphate pathway. This would provide precursors to support the demand for increased secondary metabolite production during stress as well as nucleotide synthesis accompanying the accelerated rate of cell division upon relief from stress, when oxidation of proline is likely to provide an important energy source for ADP phosphorylation. Thus, the extreme sensitivity of the metabolic processes of proline synthesis and degradation themselves may be of benefit by regulating metabolic processes adversely affected by stress. This viewpoint is supported by consideration of other physiological phenomena not directly related to stress responses, but in which proline metabolism may also play a regulatory role.A mechanism is proposed whereby the interconversions of proline and P5C in different cell types and the associated transfer of redox potential between tissues may constitute a form of metabolic signalling within higher plants. Stress-related alterations in proline metabolism may impinge on systems of redox control of plant gene expression.