Glycolytic Intermediates Research Articles

Eugenol and its liposome-based nano carrier reduce anxiety by inhibiting glyoxylase-1 expression in mice.

The most common form of psycho-social dysfunction is anxiety with depression being related closely without any age bar. They are present with combined state of sadness, confusion, stress, fear etc. Glyoxalase system contains enzyme named glyoxalase 1 (GLO1).It is a metabolic pathway which detoxifies alpha-oxo-aldehydes, particularly methylglyoxal (MG). Methylglyoxal is mainly made by the breakdown of the glycolytic intermediates, glyceraldehyde-3-phosphates and dihydroxyacetone phosphate. Glyoxylase-1 expression is also related with anxiety behavior. A casual role or GLO-1 in anxiety behavior by using viral vectors for over expression in the anterior cingulate cortex was found and it was found that local GLO-1 over expression increased anxiety behavior. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of GLO-1 was analyzed by real time RT-PCR. Moreover, the GLO-1 protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating GLO-1 protein expression in mice.

AMPK is required for recovery from metabolic stress induced by ultrasound microbubble treatment

Ultrasound-stimulated microbubble (USMB) treatment is a promising strategy for cancer therapy. USMB promotes drug delivery by sonoporation and enhanced endocytosis, and also impairs cell viability. However, USMB elicits heterogeneous effects on cell viability, with apparently minimal effects on a subset of cells. This suggests that mechanisms of adaptation following USMB allow some cells to survive and/or proliferate. Herein, we used several triple negative breast cancer cells to identify the molecular mechanisms of adaptation to USMB-induced stress. We found that USMB alters steady-state levels of amino acids, glycolytic intermediates, and citric acid cycle intermediates, suggesting that USMB imposes metabolic stress on cells. USMB treatment acutely reduces ATP levels and stimulates the phosphorylation and activation of AMP-activated protein kinase (AMPK). AMPK is required to restore ATP levels and support cell proliferation post-USMB treatment. These results suggest that AMPK and metabolic perturbations are likely determinants of the antineoplastic efficacy of USMB treatment.

Dark accumulation of downstream glycolytic intermediates initiates robust photosynthesis in cyanobacteria.

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies have shown that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid dynamic changes (on a time scale of seconds) in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enabled estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicated that downstream glycolytic intermediates, including phosphoglycerate and phosphoenolpyruvate, accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant decreases in the initial oxygen evolution rate were observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathways. Accordingly, the degree of decrease in the initial oxygen evolution rate was proportional to the accumulated pool size of glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System.

Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography−tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.

Investigation of the Exometabolomic Profiles of Rat Islets of Langerhans Cultured in Microfluidic Biochip.

Diabetes mellitus (DM) is a complex disease with high prevalence of comorbidity and mortality. DM is predicted to reach more than 700 million people by 2045. In recent years, several advanced in vitro models and analytical tools were developed to investigate the pancreatic tissue response to pathological situations and identify therapeutic solutions. Of all the in vitro promising models, cell culture in microfluidic biochip allows the reproduction of in-vivo-like micro-environments. Here, we cultured rat islets of Langerhans using dynamic cultures in microfluidic biochips. The dynamic cultures were compared to static islets cultures in Petri. The islets' exometabolomic signatures, with and without GLP1 and isradipine treatments, were characterized by GC-MS. Compared to Petri, biochip culture contributes to maintaining high secretions of insulin, C-peptide and glucagon. The exometabolomic profiling revealed 22 and 18 metabolites differentially expressed between Petri and biochip on Day 3 and 5. These metabolites illustrated the increase in lipid metabolism, the perturbation of the pentose phosphate pathway and the TCA cycle in biochip. After drug stimulations, the exometabolome of biochip culture appeared more perturbed than the Petri exometabolome. The GLP1 contributed to the increase in the levels of glycolysis, pentose phosphate and glutathione pathways intermediates, whereas isradipine led to reduced levels of lipids and carbohydrates.

The regulatory mechanism of the yeast osmoresponse under different glucose concentrations

Cells constantly respond to environmental changes by modulating gene expression programs. These responses may demand substantial costs and, thus, affect cell growth. Understanding the regulation of these processes represents a key question in biology and biotechnology. Here, we studied the responses to osmotic stress in glucose-limited environments. By analyzing seventeen osmotic stress-induced genes and stress-activated protein kinase Hog1, we found that cells exhibited stronger osmotic gene expression response and larger integral of Hog1 nuclear localization during adaptation to osmotic stress under glucose-limited conditions than under glucose-rich conditions. We proposed and verified that in glucose-limited environment, glycolysis intermediates (representing "reserve flux") were limited, which required cells to express more glycerol-production enzymes for stress adaptation. Consequently, the regulatory mechanism of osmoresponse was derived in the presence and absence of such reserve flux. Further experiments suggested that this reserve flux-dependent stress-defense strategy may be a general principle under nutrient-limited environments.

Regulation of Normal and Neoplastic Proliferation and Metabolism by the Extended Myc Network.

The Myc Network, comprising a small assemblage of bHLH-ZIP transcription factors, regulates many hundreds to thousands of genes involved in proliferation, energy metabolism, translation and other activities. A structurally and functionally related set of factors known as the Mlx Network also supervises some of these same functions via the regulation of a more limited but overlapping transcriptional repertoire. Target gene co-regulation by these two Networks is the result of their sharing of three members that suppress target gene expression as well as by the ability of both Network's members to cross-bind one another's consensus DNA sites. The two Networks also differ in that the Mlx Network's control over transcription is positively regulated by several glycolytic pathway intermediates and other metabolites. These distinctive properties, functions and tissue expression patterns potentially allow for sensitive control of gene regulation in ways that are differentially responsive to environmental and metabolic cues while allowing for them to be both rapid and of limited duration. This review explores how such control might occur. It further discusses how the actual functional dependencies of the Myc and Mlx Networks rely upon cellular context and how they may differ between normal and neoplastic cells. Finally, consideration is given to how future studies may permit a more refined understanding of the functional interrelationships between the two Networks.

Glycolysis‐derived L‐serine in hippocampal astrocytes rescues synaptic plasticity and memory loss in a mouse model of Alzheimer’s disease

AbstractBackgroundImpairment of glycolysis has often been observed early in the course of Alzheimer’s disease, although it is not known whether this deregulation contributes to the cognitive deficits observed in the disease. Aside from ATP production, a major function of glycolysis is to provide precursors, such as L‐serine, to support macromolecular synthesis. L‐serine is a metabolic hub and plays pivotal role in the brain. It is the precursor of D‐serine, the principal co‐agonist of synaptic N‐methyl D‐aspartate receptors (NMDARs) required for the induction of long‐term synaptic plasticity in the hippocampus. It also contributes to the homeostasis of synapses and of mitochondrial function by providing lipids.MethodWe used young female 3xTG‐AD mice to determine whether a shortage in the synthesis of L‐serine produced by hippocampal astrocytes from a glycolytic intermediate is responsible for the impairment of synaptic plasticity and spatial memory.ResultWe found that 3xTG‐AD mice in the early stage of the disease have reduced extracellular L‐serine levels in the hippocampus where in vivo glucose metabolism is reduced, but not in the frontal cortex that does not present metabolic alterations. Acute application of D‐serine or L‐serine rescues long‐term synaptic plasticity and chronic oral L‐serine supplementation restores memory deficits in AD mice. Such chronic treatment also restores the atrophy of hippocampal astrocytes in these AD‐mice.ConclusionThese results indicate that a metabolic deficit of L‐serine synthesis by astrocytes contributes to cognitive deficits in AD.

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.

Mechanical Force Modulates Alveolar Bone Marrow Mesenchymal Cells Characteristics for Bone Remodeling during Orthodontic Tooth Movement through Lactate Production

Orthodontic tooth movement (OTM) relies on mechanical force-induced bone remodeling. As a metabolic intermediate of glycolysis, lactate has recently been discovered to participate in bone remodeling by serving as a signaling molecule. However, whether lactate could respond to mechanical stimulus during OTM, as well as whether lactate has an impact on the alveolar bone remodeling during orthodontics, remain to be further elucidated. In the current study, we observed physiologically elevated production of lactate along with increased osteogenic differentiation, proliferation, and migration of alveolar bone marrow mesenchymal cells (ABMMCs) under mechanical force. Inhibition of lactate, induced by cyclic mechanical stretch by GNE-140, remarkably suppressed the osteogenic differentiation, proliferation, and migration, yet enhanced apoptosis of ABMMCs. Mechanistically, these regulatory effects of lactate were mediated by histone lactylation. Taken together, our results suggest that force-induced lactate is involved in controlling bone remodeling-related cellular activities in ABMMCs and plays a vital role in the alveolar bone remodeling during OTM. Our findings indicate that lactate might be a critical modulator for alveolar bone remodeling during OTM, providing a novel therapeutic target for the purpose of more effectively controlling tooth movement and improving the stability of orthodontic results.

Metabolic Adaptation to Tyrosine Kinase Inhibition in Chronic Myelogenous Leukemia Stem Cells

Objectives: BCR-ABL tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myelogenous leukemia (CML) but fail to eliminate leukemia stem cells, which persist as a source of disease recurrence. Previous studies have suggested that primitive CML cells rely on upregulated oxidative metabolism for survival. However, there is limited knowledge about the effects of TKI treatment on CML stem cell metabolism and the role of metabolic adaptations in stem cell persistence after TKI treatment. Here we evaluated the metabolic response to TKI treatment in CML stem and progenitor cells using a representative SCL-tTA/BCR-ABL mouse model of CML. Methods: CML mice were treated with the TKI Nilotinib or vehicle for 2 days, to evaluate initial effects of treatment, and for 2 weeks, to evaluate response to continued treatment. Bioenergetics, metabolite profiling, and stable isotope metabolic labeling was performed on BM c-Kit+ progenitor cells obtained from vehicle and TKI treated mice. The effects of TKI treatment on metabolism-related gene expression in CML hematopoietic stem cells (HSC) was studied using single cell RNA-Seq (scRNA) analysis of BM Lin- c-Kit+ Sca-1+ (LSK) cells from CML mice treated with vehicle or TKI for 2 days or 2 weeks. Results: Extracellular flux analysis revealed that TKI treatment initially inhibited oxygen consumption rate (OCR), representing mitochondrial respiration, and extracellular acidification rate (ECAR), representing glycolysis, in CML progenitor cells, but that these were restored with continued TKI treatment. Metabolite profiling using high-resolution mass spectrometry (HRMS) identified significant initial reduction in glycolytic, glutaminolytic and tricarboxylic acid cycle (TCA) intermediates after 2 days of TKI treatment. However, metabolite levels were restored to levels similar to controls with continued TKI treatment. Steady-state metabolic labeling with [U-13C6]-glucose or [U-13C5]-Glutamine showed reduced glycolytic flux, glutaminolysis and TCA intermediates after treatment with TKI for 2 days, but restoration of glycolytic activity and increased glucose-mediated lactate generation, and increased glutaminolytic and TCA cycle activity with continued treatment. The above studies indicate initial inhibition of energy metabolism in CML progenitors with TKI treatment, but metabolic adaptation to continued TKI exposure. scRNA analysis of CML HSC populations showed that TKI exposure led to selective enrichment of primitive HSC subsets characterized by reduced OXPHOS, glycolysis, nucleotide metabolism, and MYC gene signatures. Treatment with TKI for 2 days further reduced metabolic gene signatures, but continued treatment led to restoration of MYC and OXPHOS gene signature expression coupled with enrichment of HSC and quiescence signatures in persistent HSC. TKI-treated stem cells showed increased levels of Hif1a protein and enhanced activity of HIF-1, a key transcriptional regulator of energy metabolism. Treatment of CML mice with a HIF-1 inhibitor, Echinomycin, in combination with TKI treatment, resulted in significant depletion of CML stem cells compared to TKI alone. The combination of HIF-1 inhibitor with TKI treatment also depleted human CML stem cells in xenografted mice. Conclusions: Our results demonstrate metabolic adaptation in CML and progenitor stem cells following TKI treatment, and identify HIF-1 activation as a targetable mechanism to enhance elimination of CML stem cell that persist after TKI treatment.

AG-946 Normalizes Glycolysis and Improves Red Cell Indices in a Humanized Sickle Cell Mouse Model

We have previously demonstrated that enzymes of the glycolysis pathway and deoxy hemoglobin (deoxyHb) reversibly bind to the cytoplasmic domain of Band 3 (CdB3). For these studies we generated 3 transgenic mice in which the endogenous mouse Band 3 was replaced with: i) human wild type CdB3 (WT); ii) human CdB3 lacking the deoxyHb binding site (Hb-); iii) human CdB3 with high-affinity binding site for deoxyHb (Hb+). In the humanized WT strain, glycolytic enzymes (including fructose-1,6-biphosphate [FBP], dihydroxyacetone phosphate/ glyceraldehyde-3-phosphate [G3P] and 3-phosphoglycerate/2-phosphoglycerate [PG]) bind to CdB3 in oxygenated conditions and are displaced by the binding of deoxyHb in deoxygenated conditions. Erythrocytes from the mutant lines were insensitive to oxygen concentration (Chu et al. Blood 128, 2016, Zheng et al. JBC 294, 2019, Zhou et al. Sci. Adv. 5, 2019), showing that CdB3 constitutes a molecular switch regulating assembly of glycolytic enzymes on the erythrocyte membrane based on the oxygenation state. To study if this mechanism plays a role in Sickle Cell Disease (SCD), we crossed the humanized Band 3 mouse strains to the Townes SCD mouse model. -Hb_SS mice had a significantly higher percentage of sickled cells and a higher rate of sickling compared to WT_SS animals. ++Hb_SS mice showed a decrease in the percentage of sickled cells and the rate of sickling. We hypothesized that the inability of the glycolytic enzymes to reversibly bind to CdB3 in the -Hb mice inhibited glycolysis. To test this hypothesis, we analyzed a panel of 28 cellular metabolites in each genotype (WT_AA, _AS, and _SS; -Hb_AA, _AS, _SS; and ++Hb_AA, _AS, _SS) using an API 4500 triple quadrupole mass spectrometer (AB Sciex), with a polymeric amino column (apHera by Supelco) with stable isotope spike in controls allowing the absolute quantification of each metabolite. Consistent with the constitutive binding of the terminal glycolytic enzymes to CdB3 in -Hb erythrocytes, glycolysis was inhibited, as evidenced by significant accumulation of the intermediates at the top of the glycolysis pathway, including FBP, G3P and PG (p values all <0.01) compared to WT cells. In the ++Hb mutant where the terminal glycolytic enzymes are constitutively displaced from CdB3, significantly lower levels of FBP, G3P and PG compared to WT cells were observed (p values all <0.01). AG-946 is a novel small-molecule activator of erythrocyte pyruvate kinase that mediates the final step of glycolysis yielding 2 ATP and 2 pyruvate molecules per glucose molecule. We hypothesized that treatment of the -Hb-SS animals treated with AG-946 would reduce the levels of the glycolytic intermediates by accelerating the terminal stage of glycolysis. We designed a pilot study in which -Hb-SS mice were fed control chow for 4 weeks and then randomly separated into a group receiving control chow and a group receiving AG-946 at an approximate dose of 10 mg/kg/day. After 8 weeks on study, blood was collected from treated and untreated animals for analysis of complete blood counts, plasma levels of AG-946, the levels of 28 metabolites, intracellular 2,3-DPG, and the rate and degree of sickling. AG946 was well tolerated, all mice in both arms of the study gained weight over the 8 weeks. At the end of the study, the plasma levels of AG946 in the treated animals ranged from 736 to 2281 nM. In the treated animals we observed a complete normalization of the levels of the glycolytic intermediates and 2,3-DPG while the levels in the untreated group were unchanged. Treated animals showed significant increases in the red blood cell counts (from 4.6 + 0.52 in the control group to 7.07 + 0.21 1012/L in the treated group), hematocrit (from 24.7 + 1.8% to 33 + 1.8%) and hemoglobin (6.9 + 0.8 to 8.9 + 0.3 g/dL; p values all <0.016). We observed significant decreases in the MCV (53.5 + 2.3 to 46.6 + 1.1) and MCH (14.9 + 0.05 to 12.6 + 0.25; p values all <0.01). These differences did not correlate with the plasma levels of AG-946 indicating that levels of 736 nM are sufficient to correct the block in glycolysis and improve the red cell indices. No differences were observed in MCHC or WBC, or in the rate or degree of sickling. We conclude that AG-946 treatment effectively enhances glycolysis in humanized Band 3/SS mice. The normalization of glycolytic intermediates is accompanied by increases in critical red cell indices (RBC, HCT, Hb, and MCV). AG946 may be an effective treatment for sickle cell disease.

Loss of the Nuclear Receptor Pparγ Primes T Effector (TEFF) Differentiation of Antigen-Specific CD8+ T Cells

Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to the nuclear hormone superfamily, with three distinct types identified: PPARalpha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). PPARs possess a critical role in the regulation of lipid metabolism, and thus dictate the differentiation and fate of immune cells. PPARγ is a lipid-activated transcription factor mainly known to mediate anti-inflammatory effects on both innate and adaptive immune system. Synthetic ligands for activating PPARγ ameliorate disease outcomes in various models of inflammation and autoimmunity. The role of PPARγ in the immune system has been mainly studied in macrophages, where it is known to mediate M2 polarization. In the present study we investigated how PPARγ affects the differentiation and function of antigen-specific effector (TEFF) and memory (TM) CD8+ T cells using OTI TCR transgenic mice and OTI mice with T cell-specific PPARγ deletion (OTI/PPARγf/f-CD4-cre; thereafter named OTI/PPARγ-KO). Antigen-specific TEFF were generated in vitro by stimulation of OTI and OTI/PPARγ-KO T cells with Ova257-264 followed by culture with IL-2, whereas TM were generated by stimulation with Ova257-264 followed by culture with IL-15. Under both TEFF and TM conditions in vitro, antigen-specific stimulation of OTI/PPARγ-KO cells resulted in significantly greater proliferation and expansion relative to OTI control. Additionally, OTI/PPARγ-KO TM cells cultured with IL-15 co-expressed significantly higher CD44+/CD62L+ fractions indicating a greater expansion of central memory cells. Consistent with these, metabolomics analysis showed that under both culture conditions, PPARγ-deficient OTI cells had increased levels of glycolysis intermediates but also TCA metabolites consistent with a program of metabolically active cells with high levels of glycolysis and OXPHOS. RNA-sequencing studies showed that upon TEFF differentiation in vitro, OTI/PPARγ-KO T cells displayed a differential gene expression program compared to OTI controls. Pathway enrichment analysis revealed that PPARγ-deficient OTI cells were significantly enriched for genes related to T cell activation and proliferation, integrin activation, cytokine production, ribosomal function and biogenesis, RNA processing and protein translation. During antigen-specific stimulation in vivo performed after adoptive transfer of OTI/PPARγ-KO and OTI cells into syngeneic recipients followed by immunization with Ova-expressing Listeria monocytogenes (Lm-OVA), PPARγ deletion resulted in a rapid and enhanced TEFF cell activation and expansion as determined by the higher mitotic index, higher enrichment of Dextramer-positive antigen-specific T cells, and the higher expression of activation markers including KLRG1, CD44 and CD69, but also CTLA-4, PD-1, Lag-3 and Tim-3. These cells also developed higher numbers of TNF-α and IFN-γ-producing T cells, as well as ROR-γt expressing T cells consistent with Tc17 differentiation. In vivo stimulated antigen-specific PPARγ-deficient and control OTI T cells showed no numerical differences in TM differentiation or expansion. However, upon rechallenge in vitro, PPARγ-deficient TM cells exhibited improved capacity for TEFF differentiation with higher release of inflammatory cytokines and expression of CD69, PD-1 and CTLA-4. Our findings uncover a new role of PPARγ ablation in programming CD8+ T responses by regulating metabolism-mediated cues, which imprint T cell differentiation and long-term effector capacity.

EXTH-10. TARGETING MITOCHONDRIAL ENERGETICS REVERSES THERAPEUTIC RESISTANCE IN PEDIATRIC DMG AND HGG

Abstract Diffuse midline gliomas (DMGs) and other high-grade gliomas (HGG) are the most commonly lethal intrinsic brain tumors of childhood. Accordingly, novel treatment approaches are needed that consider the heterogeneity of these tumors. Using an extensive pharmacological screen, we identified several two-drug combinations that were particularly effective against a range of DMG and HGG cell lines, the most active being the combination of the histone deacetylase inhibitor, panobinostat, and a proteasome inhibitor, marizomib, both in clinical trials, which synergistically induced tumor-specific apoptosis. However, at clinically achievable concentrations, a small population of resistant tumor cells survived and dominated, mimicking the clinical scenario, which prompted our development of drug resistance models as mechanistic tools. Resistance mechanisms were examined using RNA sequencing of drug-resistant versus -naïve cells. NMNAT2, a critical mediator of NAD synthesis, was overexpressed in DIPGs in the context of high baseline QPRT expression, suggesting a critical role of NAD synthesis and utilization pathways in resistance. Gene-set enrichment analysis suggested a key role for glycolytic pathways in the resistance phenotype. In support of this hypothesis, we observed increased levels of mitochondrial mass and NAD and ATP in resistant cells compared to drug-naïve cells, as well as upregulation of glycolytic intermediates by LC/MS. Conversely, inhibition of OXPHOS and glycolysis pathways resensitized resistant glioma cells to apoptosis in vitro. To test this hypothesis in vivo, mice were injected with drug-naïve or resistant DIPG-013 glioma cells and were exposed to an OXPHOS and glycolysis inhibitor, lonidamine. Lonidamine alone extended survival of naïve (18.6 + 1.4 d vs. 25 + 1.6 d, p=0.0003) and resistant (18.7 + 1 d vs. 62 + 1 d, p=0.0002) DIPG13p models (Log-Rank). This supports the application of metabolic therapy as a novel strategy for treating resistant MGs that has activity in a clinically relevant DIPG model. Supported by NIHS10OD023402, the DIPG/DMG Collaborative, the CNI, and Connor’s Cure.

The dual role of the hexosamine biosynthetic pathway in cardiac physiology and pathophysiology

The heart is a highly metabolic organ with extensive energy demands and hence relies on numerous fuel substrates including fatty acids and glucose. However, oxidative stress is a natural by-product of metabolism that, in excess, can contribute towards DNA damage and poly-ADP-ribose polymerase activation. This activation inhibits key glycolytic enzymes, subsequently shunting glycolytic intermediates into non-oxidative glucose pathways such as the hexosamine biosynthetic pathway (HBP). In this review we provide evidence supporting the dual role of the HBP, i.e. playing a unique role in cardiac physiology and pathophysiology where acute upregulation confers cardioprotection while chronic activation contributes to the onset and progression of cardio-metabolic diseases such as diabetes, hypertrophy, ischemic heart disease, and heart failure. Thus although the HBP has emerged as a novel therapeutic target for such conditions, proposed interventions need to be applied in a context- and pathology-specific manner to avoid any potential drawbacks of relatively low cardiac HBP activity.

Microbial diversity and functional genes of red vinasse acid based on metagenome analysis.

Red vinasse acid has a distinct flavor and a vivid red color that are directly tied to the intricate metabolic activities of microorganisms that produce it. In this study, metagenomic technology was used to mine its functional genes and examine the microbial diversity of red vinasse acid. The findings revealed the identification of 2,609 species, 782 genera, and 63 phyla of microorganisms, and the dominant genus was Lactobacillus. Amino acid metabolism and carbohydrate metabolism were significant activities among the 16,093 and 49,652 genes that were annotated in the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. In gluconeogenesis, red vinasse acid encodes 194 genes controlling the transporter protein systems of different sugars and has key enzyme genes that catalyze the conversion of intracellular sugars into glycolytic intermediates. In amino acid flavor formation, red vinasse acid contains 32 control genes for branched-chain aminotransferase (BCAT), 27 control genes for aromatic-amino-acid transaminase (ArAT), 60 control genes for keto acid invertase, 123 control genes for alcohol/aldehyde dehydrogenase, and 27 control genes for acetyl esterase, which have the basis for the formation of strong flavor substances from amino acids.

Glyoxalase 1 as a Therapeutic Target in Cancer and Cancer Stem Cells.

Methylglyoxal (MG) is a dicarbonyl compound formed in cells mainly by the spontaneous degradation of the triose phosphate intermediates of glycolysis. MG is a powerful precursor of advanced glycation end products, which lead to strong dicarbonyl and oxidative stress. Although divergent functions of MG have been observed depending on its concentration, MG is considered to be a potential anti-tumor factor due to its cytotoxic effects within the oncologic domain. MG detoxification is carried out by the glyoxalase system. Glyoxalase 1 (Glo1), the ubiquitous glutathione-dependent enzyme responsible for MG degradation, is considered to be a tumor promoting factor due to it catalyzing the removal of cytotoxic MG. Indeed, various cancer types exhibit increased expression and activity of Glo1 that closely correlate with tumor cell growth and metastasis. Furthermore, mounting evidence suggests that Glo1 contributes to cancer stem cell survival. In this review, we discuss the role of Glo1 in the malignant progression of cancer and its possible use as a promising therapeutic target for tumor therapy. We also summarize therapeutic outcomes of Glo1 inhibitors as prospective treatments for the prevention of cancer.

Nutraceutical Prevention of Diabetic Complications-Focus on Dicarbonyl and Oxidative Stress.

Oxidative and dicarbonyl stress, driven by excess accumulation of glycolytic intermediates in cells that are highly permeable to glucose in the absence of effective insulin activity, appear to be the chief mediators of the complications of diabetes. The most pathogenically significant dicarbonyl stress reflects spontaneous dephosphorylation of glycolytic triose phosphates, giving rise to highly reactive methylglyoxal. This compound can be converted to harmless lactate by the sequential activity of glyoxalase I and II, employing glutathione as a catalyst. The transcription of glyoxalase I, rate-limiting for this process, is promoted by Nrf2, which can be activated by nutraceutical phase 2 inducers such as lipoic acid and sulforaphane. In cells exposed to hyperglycemia, glycine somehow up-regulates Nrf2 activity. Zinc can likewise promote glyoxalase I transcription, via activation of the metal-responsive transcription factor (MTF) that binds to the glyoxalase promoter. Induction of glyoxalase I and metallothionein may explain the protective impact of zinc in rodent models of diabetic complications. With respect to the contribution of oxidative stress to diabetic complications, promoters of mitophagy and mitochondrial biogenesis, UCP2 inducers, inhibitors of NAPDH oxidase, recouplers of eNOS, glutathione precursors, membrane oxidant scavengers, Nrf2 activators, and correction of diabetic thiamine deficiency should help to quell this.

Alternative splicing signature of alveolar type II epithelial cells of Tibetan pigs under hypoxia-induced.

Alternative splicing (AS) allows the generation of multiple transcript variants from a single gene and affects biological processes by generating protein diversity in organisms. In total, 41,642 AS events corresponding to 9,924 genes were identified, and SE is the most abundant alternatively spliced type. The analysis of functional categories demonstrates that alternatively spliced differentially expressed genes (DEGs) were enriched in the MAPK signaling pathway and hypoxia-inducible factor 1 (HIF-1) signaling pathway. Proteoglycans in cancer between the normoxic (21% O2, TN and LN) and hypoxic (2% O2, TL and LL) groups, such as SLC2A1, HK1, HK2, ENO3, and PFKFB3, have the potential to rapidly proliferate alveolar type II epithelial (ATII) cells by increasing the intracellular levels of glucose and quickly divert to anabolic pathways by glycolysis intermediates under hypoxia. ACADL, EHHADH, and CPT1A undergo one or two AS types with different frequencies in ATII cells between TN and TL groups (excluding alternatively spliced DEGs shared between normoxic and hypoxic groups), and a constant supply of lipids might be obtained either from the circulation or de novo synthesis for better growth of ATII cells under hypoxia condition. MCM7 and MCM3 undergo different AS types between LN and LL groups (excluding alternatively spliced DEGs shared between normoxic and hypoxic groups), which may bind to the amino-terminal PER-SIM-ARNT domain and the carboxyl terminus of HIF-1α to maintain their stability. Overall, AS and expression levels of candidate mRNAs between Tibetan pigs and Landrace pigs revealed by RNA-seq suggest their potential involvement in the ATII cells grown under hypoxia conditions.

Carbon and nitrogen metabolic regulation in freshwater plant Ottelia alismoides in response to carbon limitation: A metabolite perspective.

Carbon and nitrogen metabolism are basic, but pivotal metabolic pathways in plants and are tightly coupled. Maintaining the balance of carbon and nitrogen metabolism is critical for plant survival. Comprehensively revealing the metabolic balance of carbon–nitrogen interactions is important and helpful for understanding the adaptation of freshwater plants to CO2 limited aqueous environment. A comprehensive metabolomics analysis combined with physiological measurement was performed in the freshwater plant Ottelia alismoides acclimated to high and low CO2, respectively, for a better understanding of how the carbon and nitrogen metabolic adjustment in freshwater plants respond to carbon limitation. The present results showed that low CO2 acclimated O. alismoides exhibited significant diurnal titratable acidity and malate fluctuations, as well as an opposite diel pattern of starch change and high enzymatic activities required for crassulacean acid metabolism (CAM) photosynthesis, which indicates that CAM was induced under low CO2. Moreover, the metabolomic analysis showed that most intermediates of glycolysis, pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle, were increased under low CO2, indicative of active respiration in low-CO2-treated O. alismoides. Meanwhile, the majority of amino acids involved in pathways of glutamate and arginine metabolism, aspartate metabolism, and the branched-chain amino acids (BCAAs) metabolism were significantly increased under low CO2. Notably, γ-aminobutyric acid (GABA) level was significantly higher in low CO2 conditions, indicating a typical response with GABA shunt compensated for energy deprivation at low CO2. Taken together, we conclude that in low-CO2-stressed O. alismoides, CAM photosynthesis was induced, leading to higher carbon and nitrogen as well as energy requirements. Correspondingly, the respiration was greatly fueled via numerous starch degradation to ensure CO2 fixation in dark, while accompanied by linked promoted N metabolism, presumably to produce energy and alternative carbon sources and nitrogenous substances for supporting the operation of CAM and enhancing tolerance for carbon limitation. This study not only helps to elucidate the regulating interaction between C and N metabolism to adapt to different CO2 but also provides novel insights into the effects of CO2 variation on the metabolic profiling of O. alismoides.