High Activity Of Pentose Phosphate Pathway Research Articles

Regulation of Erythritol Metabolism, a Biomarker of Cardiometabolic Disease

Abstract Objectives Serum erythritol is a predictive biomarker of chronic disease development and complications. In cohort studies, elevated serum erythritol at baseline was predictive of central adiposity gain, T2DM, and cardiovascular disease development up to 20 years later. Erythritol is a polyol that has been well characterized as a non-nutritive sweetener. Erythritol was recently discovered to be synthesized endogenously in humans from glucose through the pentose phosphate pathway (PPP). The regulation of erythritol synthesis in mammals, however, is not characterized. The purpose of this study was to assess stimuli that modulate intracellular erythritol levels and the distribution of erythritol in key metabolic tissues. Methods First, we fed 8-week-old C57BL/6J mice a defined diet (AIN93G) for two weeks, after which plasma and tissue erythritol were measured by GC-MS. To further evaluate the cellular regulation of erythritol synthesis, we used A549 cells for their high PPP activity. Using siRNA, we knocked down SORD (a potential erythritol-synthesizing enzyme) and glucose-6-phosphate dehydrogenase (G6PD) under normal (5 mM) and high (25 mM) glucose conditions and measured intracellular erythritol. We also exposed A549’s to hydrogen peroxide to assess if erythritol synthesis is elevated by oxidative stress, an additional stimulator of the PPP. Results Plasma erythritol ranged from 0.44 uM to 0.73 uM in young, healthy mice. Erythritol differed significantly based on tissue type (p < 0.0001). The liver and kidney had the highest relative erythritol, followed by quadriceps, whereas white adipose tissue contained the least erythritol. SORD knockdown significantly reduced erythritol synthesis under high glucose, but not normal glucose conditions (p < 0.01). Knockdown of G6PD did not impact erythritol synthesis at either glucose concentration. Finally, treatment with hydrogen peroxide significantly increased intracellular erythritol (p < 0.0001). Conclusions In conclusion, erythritol is present in mouse plasma, SORD-expressing tissues, and human cells. Erythritol was elevated by high glucose and oxidative stress–two key factors in the pathogenesis of cardiometabolic diseases. Together, these data suggest that erythritol synthesis occurs parallel to elevated PPP flux in response to metabolic stressors. Funding Sources N/A

Regulation and Distribution of Endogenous Erythritol, a Biomarker of Cardiometabolic Disease

Serum erythritol is a novel predictive biomarker of chronic disease development and complications. In cohort studies, elevated serum erythritol at baseline was predictive of central adiposity gain, T2DM, and cardiovascular disease development. Higher erythritol has also been associated with complications of these chronic diseases such as diabetic retinopathy and nephropathy. Erythritol is a 4-carbon polyol that has been well characterized as a non-nutritive sweetener. More recently, erythritol was discovered to be synthesized endogenously in humans from glucose through the pentose phosphate pathway (PPP). The PPP generates NADPH for anabolic reactions and to combat oxidative stress. We identified two human enzymes that catalyze the conversion of erythrose-4-phosphate to erythritol: sorbitol dehydrogenase (SORD) and alcohol dehydrogenase 1 (ADH1). SORD knockdown significantly decreases erythritol synthesis in vitro. The regulation of erythritol synthesis in mammals is not characterized. The purpose of this study was to assess stimuli that modulate intracellular erythritol levels and the distribution of erythritol in key metabolic tissues. We hypothesize that erythritol synthesis will be induced by stimuli that increase PPP activity, and that erythritol will be present in tissues with high SORD expression. First, we fed 8-week-old C57BL/6J mice a defined diet (AIN93G) for two weeks. After two weeks, plasma and tissue erythritol including liver, kidney, white adipose, and skeletal muscle, were measured by GC-MS. To further evaluate the cellular regulation of erythritol synthesis, we used A549 cells due to their high PPP activity. Using siRNA, we knocked down SORD and glucose-6-phosphate dehydrogenase (G6PD) under normal (5mM) and high (25mM) glucose conditions, then measured intracellular erythritol. We also exposed A549’s to hydrogen peroxide to assess if erythritol synthesis is elevated by oxidative stress. Plasma erythritol ranged from 0.44 uM to 0.73 uM in young, healthy mice. Erythritol was significantly different based on tissue type (p < 0.0001, one-way ANOVA). The liver and kidney had the highest relative erythritol, followed by quadriceps, whereas white adipose tissue contained the least erythritol. Interestingly, SORD knockdown significantly reduced erythritol synthesis under high glucose, but not normal glucose conditions (p < 0.01, Sidak's multiple comparisons test). Knockdown of G6PD did not impact erythritol synthesis at either glucose concentration. Finally, treatment with hydrogen peroxide significantly increased intracellular erythritol (p < 0.0001, student's t-test). In mice, erythritol was highest in tissues known to have high SORD expression. Unexpectedly, erythritol was lowest in adipose, which is known to have high PPP activity. This finding is supported by the reduction of intracellular erythritol induced by SORD, but not G6PD knockdown. Erythritol synthesis was also elevated by two factors known to stimulate the PPP: high glucose and oxidative stress. Together, these data suggest that erythritol synthesis is linked to PPP flux, but is regulated downstream of the oxidative phase of the PPP.

Metabolic constraints drive self-organization of specialized cell groups.

How phenotypically distinct states in isogenic cell populations appear and stably co-exist remains unresolved. We find that within a mature, clonal yeast colony developing in low glucose, cells arrange into metabolically disparate cell groups. Using this system, we model and experimentally identify metabolic constraints sufficient to drive such self-assembly. Beginning in a uniformly gluconeogenic state, cells exhibiting a contrary, high pentose phosphate pathway activity state, spontaneously appear and proliferate, in a spatially constrained manner. Gluconeogenic cells in the colony produce and provide a resource, which we identify as trehalose. Above threshold concentrations of external trehalose, cells switch to the new metabolic state and proliferate. A self-organized system establishes, where cells in this new state are sustained by trehalose consumption, which thereby restrains other cells in the trehalose producing, gluconeogenic state. Our work suggests simple physico-chemical principles that determine how isogenic cells spontaneously self-organize into structured assemblies in complimentary, specialized states.

Metabolic characterization of the chitinolytic bacterium Serratia marcescens using a genome-scale metabolic model

BackgroundSerratia marcescens is a chitinolytic bacterium that can potentially be used for consolidated bioprocessing to convert chitin to value-added chemicals. Currently, S. marcescens is poorly characterized and studies on intracellular metabolic and regulatory mechanisms would expedite development of bioprocessing applications.ResultsIn this study, our goal was to characterize the metabolic profile of S. marcescens to provide insight for metabolic engineering applications and fundamental biological studies. Hereby, we constructed a constraint-based genome-scale metabolic model (iSR929) including 929 genes, 1185 reactions and 1164 metabolites based on genomic annotation of S. marcescens Db11. The model was tested by comparing model predictions with experimental data and analyzed to identify essential aspects of the metabolic network (e.g. 138 essential genes predicted). The model iSR929 was refined by integrating RNAseq data of S. marcescens growth on three different carbon sources (glucose, N-acetylglucosamine, and glycerol). Significant differences in TCA cycle utilization were found for growth on the different carbon substrates, For example, for growth on N-acetylglucosamine, S. marcescens exhibits high pentose phosphate pathway activity and nucleotide synthesis but low activity of the TCA cycle.ConclusionsOur results show that S. marcescens model iSR929 can provide reasonable predictions and can be constrained to fit with experimental values. Thus, our model may be used to guide strain designs for metabolic engineering to produce chemicals such as 2,3-butanediol, N-acetylneuraminic acid, and n-butanol using S. marcescens.

Modulation of Glycolysis and the Pentose Phosphate Pathway Influences Porcine Oocyte In Vitro Maturation

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p<0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p<0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.

Abstract 4697: Metabolic regulation of caspase-2

Abstract Caspase-2 has been shown to act as an apical caspase, upstream of cytochrome c release, in a variety of cell death settings including chemotherapy-induced apoptosis. However, the mechanism governing caspase-2 activation in response to chemotherapeutics is poorly understood. We previously demonstrated that production of NADPH via the pentose phosphate pathway (PPP) stimulates caspase-2 suppression via a CaMKII-dependent phosphorylation on Ser135 (Nutt et al., 2005), and that as PPP activity declines, caspase-2 becomes dephosphorylated and activated to promote cell death. Our current efforts are focused on identifying the cellular factors that link metabolism to caspase-2 activation, with the hope that such factors could be exploited therapeutically in cancer. In this study we show that inhibition of Sirt1 deacetylase overrides the anti-apoptotic effect of high PPP activity (stimulated by G6P) to cause caspase-2 activation and cell death. In an effort to more precisely understand the role of Sirt1 in caspase-2 activation, we developed a novel biotin-switch-based proteomics approach to identify Sirt1 substrates in an unbiased manner. This approach yielded a variety of high-confidence Sirt1 substrates, many of which are cytoplasmic proteins that primarily group into the areas of glycolysis/metabolism, oxidative stress, cytoskeletal dynamics, and apoptosis; all areas where Sirt1 has been implicated. We had previously shown that one of these substrates, 14-3-3ζ, binds to caspase-2 in a phosphorylation-dependent manner and protects Ser135 from dephosphorylation (Nutt et al., 2009). We show here that acetylation of 14-3-3ζ occurs in response to a drop in PPP activity and triggers 14-3-3ζ release from caspase-2, leading to caspase-2 activation. Conversely, we show that high PPP levels promote Sirt1 activity, which, in turn, promotes 14-3-3ζ binding to caspase-2 by antagonizing the acetylation of 14-3-3ζ. We have extended these results to a panel of breast tumor cell lines, in which inhibition of Sirt1 induces the rapid dissociation of caspase-2 from 14-3-3ζ, and results in a marked enhancement of the cells’ sensitivity to paclitaxel. Interestingly, we observe cytoplasmic localization of Sirt1 at high levels in these cells with very little to none in the nucleus, in contrast to a more nuclear Sirt1 distribution in normal breast tissue. In conclusion, these data support a model in which Sirt1 promotes the pro-survival effect of high metabolic activity by directly regulating 14-3-3ζ-mediated inhibition of caspase-2, and suggest that as PPP activity declines, a consequent decrease in Sirt1 activity triggers caspase-2 activation. As Sirt1 is known to also modulate numerous transcription factors, its suppressive effect on caspase-2 may play a particularly key role under conditions in which transcription is not ongoing (e.g., paclitaxel-induced mitotic arrest) or when Sirt1 is excluded from the nucleus (breast and other tumor cell lines). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4697. doi:10.1158/1538-7445.AM2011-4697

Glucose catabolic pathways in Lentinula edodes determined with radiorespirometry and enzymic analysis

Carbohydrate metabolism in different tissues was studied using radiorespirometry and enzymic analysis. The 14 C-1/ 14 C-6 ratios in CO 2 respired from specifically labelled glucose fed to Lentinula edodes tissues grown on a chemically defined medium ranged from 2·5 (vegetative mycelium) to 14·9 (young lamellae). This reflects the relative activity of the pentose phosphate pathway (PPP), very high in basidiome but low in mycelium, the highest ratio being recorded in tissues which are biosynthetically most active (young lamellae), requiring the reducing power of NADPH generated through the PPP. Extensive conversion of 14 C-3, 4-labelled glucose to 14 CO 2 in the mycelium underlined the important role of the Embden-Meyerhof-Parnas pathway (EMP) in that tissue. A 14 C-1/ 14 C-6 ratio of 14·8 for young lamellae grown on woodchips indicates that the growth medium did not influence the pathway used in the basidiome. Ratios for the young pileipellis and stipe were 8·0 and 10·4 respectively. A ratio of 3·6 for Coprinus cinereus gills confirms the comparatively lesser importance of the PPP in this organism. Enzymic determination corroborated the metabolic pattern deduced from radiolabelling. Activity of PPP enzymes (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were three times as high in the basidiome compared with the mycelium, highest activity being in the young pileus. EMP enzymes (fructose 1,6-bisphosphate aldolase and glucose 6-phosphate isomerase) were more active in mycelium than in fruit body, and also more active than PPP enzymes within the mycelium itself. Within the basidiome, specific activities of EMP and PPP enzymes were about the same. Enzymic activity in mature pileus of C. cinereus resembled the pattern in the mycelium rather than the basidiome of L. edodes ; EMP enzymes were very much more active than PPP. The positive correlation between high PPP activity and accumulation of large amounts of mannitol in the basidiome is discussed.

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

In sycamore cells grown on nitrate as opposed to glutamate there is a higher pentose phosphate pathway carbon flux relative to glycolysis in the early stages of cell growth when nitrate assimilation is most active. The high pentose phosphate pathway activity compared with glycolysis in nitrate grown cells is accompanied by enhanced levels of hexokinase, pyruvate kinase, glucose-6-phosphate de-hydrogenase, 6-phosphogluconate dehydrogenase and transketolase. There is no significant increase in activity of the solely glycolytic enzyme, phosphofructokinase. It is suggested that the increased pentose phosphate pathway activity in nitrate grown cells is correlated with a demand by nitrite assimilation for NADPH.