Glycolytic Intermediates Research Articles

Abstract 272: Autophagy and MEK inhibition promotes ferroptosis in liver kinase B1 (Lkb1)-deficient Kras-driven lung tumors

Abstract Tumor suppressor Liver Kinase B1 (LKB1) activates 5’-adenosine monophosphate protein kinase (AMPK) and maintains energy homeostasis in response to energy crises. LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC), causing aggressive tumor growth and metastases. Unfortunately, standard treatment with RAS-RAF-MEK-ERK signaling pathway inhibitors has minimal therapeutic efficacy in LKB1-mutant KRAS-driven NSCLC. Thus, identifying a novel treatment for patients harboring co-mutations in LKB1 and KRAS is urgently needed. Autophagy degrades and recycles the building blocks for cancer cells to survive metabolic challenges. Using genetically engineered mouse models (GEMMs), we have previously demonstrated that autophagy compensates for Lkb1 loss for Kras-driven lung tumorigenesis; loss of an autophagy-essential gene Atg7 dramatically impaired tumor initiation and tumor growth in KrasG12D/+;Lkb1−/− (KL) lung tumors. This is in sharp contrast to Lkb1 wild-type (WT) (KrasG12D/+;p53−/− (KP)) tumors that are less sensitive to autophagy gene ablation. To further value our discoveries in clinical translational ability, we treated mouse lung tumor derived cell lines (TDCLs) with FDA-approved autophagy inhibitor hydroxychloroquine (HCQ) and MEK inhibitor Trametinib and found that the combination treatment displayed synergistic anti-proliferative effects in KL TDCLs compared to KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomic profiling of KL TDCLs with Trametinib, HCQ, or combination treatment and found that several glycolytic and TCA cycle intermediates, amino acids, and ATP levels were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combination treatment. In addition, the combination treatment significantly reduced mitochondrial membrane potential, basal respiration, and ATP production in KL TDCLs. In vivo studies using tumor allografts, genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) showed anti-tumor activity of the combination treatment on KL tumors, but not in KP tumors. Moreover, we found increased lipid peroxidation indicative of ferroptosis in KL TDCLs and KL PDX tumors with the combination treatment compared to the single agent treatments. Finally, treatment with a ferroptosis inhibitor rescued the reduced KL allograft tumor growth caused by the combination treatment. Taken together, our observations indicate that autophagy upregulation in KL tumors causes resistance to Trametinib treatment by maintaining energy homeostasis for cell survival and inhibits ferroptosis. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient KRAS-driven NSCLC. Citation Format: Vrushank Bhatt, Taijin Lan, Wenping Wang, Jerry Kong, Eduardo Cararo Lopes, Khoosheh Khayati, Jianming Wang, Akash Raju, Michael Rangel, Enrique Lopez, Zhixian Sherrie Hu, Xuefei Luo, Xiaoyang Su, Jyoti Malhotra, Wenwei Hu, Sharon R. Pine, Eileen White, Jessie Yanxiang Guo. Autophagy and MEK inhibition promotes ferroptosis in liver kinase B1 (Lkb1)-deficient Kras-driven lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 272.

Abstract 4254: The ratio of key metabolic transcripts is a predictive biomarker of breast cancer metastasis to the lung

Abstract Understanding the rewired metabolism of organ-specific metastasis in breast cancer is an under-appreciated problem, with implications for the treatment and prevention of metastatic disease. Here, we used a systems biology approach and the MDA-MB 231 model to compare metabolic fluxes used by parental breast cancer cells and their brain- and lung-homing derivatives. We combined metabolomic profiling, flux measurements, spatial tissue mimetic systems, and mathematical modeling to dissect the cells’ metabolic rewiring, and identified changes specific for lung metastatic selection. We validated our results in other cell lines as well as patient data from project GENIE and the MBCP. Our results produced five biological insights: First, levels of mRNA and metabolic intermediates may anticorrelate with flux. Second, different lineages evolved from the same line can have distinct heritable metabolic fluxes. Metastatic lineages in our model display higher glycolytic flux and bioenergetics than parental cells, with lung-homing cells exhibiting by far the greatest glucose uptake and lactate production. Importantly, this occurs despite low levels of glycolytic intermediates in lung-homing cells. Third, this apparent paradox can be reconciled if feedback inhibition of the glycolysis pathway is prevented, a finding we modeled with flux-balance analysis and confirmed by 13C glucose tracing and measuring glycolytic enzymatic activities directly. Fourth, this distinct metabolic behavior in lung-homing cells is maintained by a high ratio of lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) gene expression, which also correlates with lung metastases in patients with breast cancer. Feature classification models trained on clinical characteristics alone were unable to predict tropism; however, the LDH/PDH ratio was a significant predictor for lung but not brain metastases, independent of other transcriptomic signatures, suggesting that this feature is a potential biomarker for lung metastasis. Fifth, this metabolic effect did not increase cellular growth rate, suggesting that lactate secretion may itself be a trait under selection in breast cancer lung metastasis. Follow up experiments and game theory-based models in a spatially structured tissue-mimetic system showed that lung-homing cells grow more favorably than brain-homing cells in nutrient-deprived gradients, demonstrating how divergent metabolisms of the lineages could lead to selection in different environments. Notably, lung metastases in a mouse model of pancreatic cancer had higher lactate production than other metastases, suggesting that this metabolic trait could be important for other cancer types. Together, our in vitro, in silico, and clinical data analyses highlight that metabolism—currency of all physiological processes—plays an essential role in the connection from gene to phenotype in metastatic disease. Citation Format: Deepti Mathur, Chen Liao, Wendy Lin, Alessandro La Ferlita, Salvatore Alaimo, Alfredo Ferro, Yi Zhong, Christine Iacobuzio-Donahue, Joao Xavier. The ratio of key metabolic transcripts is a predictive biomarker of breast cancer metastasis to the lung. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4254.

In vivo evaluation of the effect of sickle cell hemoglobin S, C and therapeutic transfusion on erythrocyte metabolism and cardiorenal dysfunction.

Despite a wealth of exploratory plasma metabolomics studies in sickle cell disease (SCD), no study to date has evaluate a large and well phenotyped cohort to compare the primary erythrocyte metabolome of hemoglobin SS, SC and transfused AA red blood cells (RBCs) in vivo. The current study evaluates the RBC metabolome of 587 subjects with sickle cell sickle cell disease (SCD) from the WALK-PHaSST clinical cohort. The set includes hemoglobin SS, hemoglobin SC SCD patients, with variable levels of HbA related to RBC transfusion events. Here we explore the modulating effects of genotype, age, sex, severity of hemolysis, and transfusion therapy on sickle RBC metabolism. Results show that RBCs from patients with Hb SS genotypes-compared to AA RBCs from recent transfusion events or SC RBCs-are characterized by significant alterations of RBC acylcarnitines, pyruvate, sphingosine 1-phosphate, creatinine, kynurenine and urate metabolism. Surprisingly, the RBC metabolism of SC RBCs is dramatically different from SS, with all glycolytic intermediates significantly elevated in SS RBCs, with the exception of pyruvate. This result suggests a metabolic blockade at the ATP-generating phosphoenolpyruvate to pyruvate step of glycolysis, which is catalyzed by redox-sensitive pyruvate kinase. Metabolomics, clinical and hematological data were collated in a novel online portal. In conclusion, we identified metabolic signatures of HbS RBCs that correlate with the degree of steady state hemolytic anemia, cardiovascular and renal dysfunction and mortality.

Basic Fibroblast Growth Factor Promotes Mesenchymal Stem Cell Migration by Regulating Glycolysis-Dependent β-Catenin Signaling.

Migration of mesenchymal stem cells (MSCs) to the site of injury is crucial in transplantation therapy. Studies have shown that cell migration is regulated by the cellular microenvironment and accompanied by changes in cellular metabolism. However, limited information is available about the relationship between MSC migration and cellular metabolism. Here, we show that basic fibroblast growth factor (bFGF) promotes the migration of MSCs with high levels of glycolysis and high expression of hexokinase 2 (HK2), a rate-limiting enzyme in glycolysis. The enhancement of glycolysis via the activation of HK2 expression promoted the migration of MSCs, whereas the inhibition of glycolysis, but not of oxidative phosphorylation, inhibited the bFGF-induced migration of these cells. Furthermore, bFGF enhanced glycolysis by increasing HK2 expression, which consequently promoted β-catenin accumulation, and the inhibition of glycolysis inhibited the bFGF-induced accumulation of β-catenin. When the accumulation of glycolytic intermediates was altered, phosphoenolpyruvate was found to be directly involved in the regulation of β-catenin expression and activation, suggesting that bFGF regulates β-catenin signaling through glycolytic intermediates. Moreover, transplantation with HK2-overexpressing MSCs significantly improved the effect of cell therapy on skull injury in rats. In conclusion, we propose a novel glycolysis-dependent β-catenin signaling regulatory mechanism and provide an experimental and theoretical basis for the clinical application of MSCs.

Crabtree effect in kidney proximal tubule cells via late-stage glycolytic intermediates

The Crabtree effect is defined as a rapid glucose-induced repression of mitochondrial oxidative metabolism and has been described in yeasts and tumor cells. Using plate-based respirometry, we identified the Crabtree effect in normal (non-tumor) kidney proximal tubule epithelial cells (PTEC) but not in other kidney cells (podocytes or mesangial cells) or mammalian cells (C2C12 myoblasts). Glucose-induced repression of respiration was prevented by reducing glycolysis at the proximal step with 2-deoxyglucose and partially reversed by pyruvate. The late-stage glycolytic intermediates glyceraldehyde 3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate, but not the early-stage glycolytic intermediates or lactate, inhibited respiration in permeabilized PTEC and kidney cortex mitochondria, mimicking the Crabtree effect. Studies in diabetic mice indicated a pattern of increased late-stage glycolytic intermediates consistent with a similar pattern occurring invivo. Our results show the unique presence of the Crabtree effect in kidney PTEC and identify the major mediators of this effect.

Development of pseudo-targeted profiling of isotopic metabolomics using combined platform of high resolution mass spectrometry and triple quadrupole mass spectrometry with application of 13C6-glucose tracing in HepG2 cells

Isotope tracing assisted metabolic analysis is becoming a unique tool to understand metabolic regulation in cell biology and biomedical research. Targeted mass spectrometry analysis based on selected reaction monitoring (SRM) has been widely applied in isotope tracing experiment with the advantages of high sensitivity and broad linearity. However, its application for new pathway discovery is largely restrained by molecular coverage. To overcome this limitation, we describe a strategy called pseudo-targeted profiling of isotopic metabolomics (PtPIM) to expand the analysis of isotope labeled metabolites beyond the limit of known pathways and chemical standards. Pseudo-targeted metabolomics was first established with ion transitions and retention times transformed from high resolution (orbitrap) mass spectrometry. Isotope labeled MRM transitions were then generated according to chemical formulas of fragments, which were derived from accurate ion masses acquired by HRMS. An in-house software “PseudoIsoMRM” was developed to simulate isotope labeled ion transitions in batch mode and correct the interference of natural isotopologues. This PtPIM strategy was successfully applied to study 13C6-glucose traced HepG2 cells. As 313 molecules determined as analysis targets, a total of 4104 ion transitions were simulated to monitor 13C labeled metabolites in positive-negative switching mode of QQQ mass spectrometer with minimum dwell time of 0.3 ms achieved. A total of 68 metabolites covering glycolysis, TCA cycle, nucleotide biosynthesis, one-carbon metabolism and related derivatives were found to be labeled (> 2%) in HepG2 cells. Active pentose phosphate pathway was observed with diverse labeling status of glycolysis intermediates. Meanwhile, our PtPIM strategy revealed that rotenone severely suppressed mitochondrial function e.g. oxidative phosphorylation and fatty acid beta-oxidation. In this case, anaerobic respiration became the major source of energy metabolism by producing abundant lactate. Conclusively, the simulation based PtPIM method demonstrates a strategy to broaden metabolite coverage in isotope tracing analysis independent of standard chemicals.

Consequences of reprogramming acetyl-CoA metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the mouse liver

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces the progression of steatosis to steatohepatitis with fibrosis in mice. Furthermore, TCDD reprograms hepatic metabolism by redirecting glycolytic intermediates while inhibiting lipid metabolism. Here, we examined the effect of TCDD on hepatic acetyl-coenzyme A (acetyl-CoA) and β-hydroxybutyrate levels as well as protein acetylation and β-hydroxybutyrylation. Acetyl-CoA is not only a central metabolite in multiple anabolic and catabolic pathways, but also a substrate used for posttranslational modification of proteins and a surrogate indicator of cellular energy status. Targeted metabolomic analysis revealed a dose-dependent decrease in hepatic acetyl-CoA levels coincident with the phosphorylation of pyruvate dehydrogenase (E1), and the induction of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase phosphatase, while repressing ATP citrate lyase and short-chain acyl-CoA synthetase gene expression. In addition, TCDD dose-dependently reduced the levels of hepatic β-hydroxybutyrate and repressed ketone body biosynthesis gene expression. Moreover, levels of total hepatic protein acetylation and β-hydroxybutyrylation were reduced. AMPK phosphorylation was induced consistent with acetyl-CoA serving as a cellular energy status surrogate, yet subsequent targets associated with re-establishing energy homeostasis were not activated. Collectively, TCDD reduced hepatic acetyl-CoA and β-hydroxybutyrate levels eliciting starvation-like conditions despite normal levels of food intake.

Metabolic adjustments in response to ATP spilling by the small DX protein in a Streptomyces strain.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms. However, such strategy has been rarely implemented in Streptomyces species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of Streptomyces, the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong ErmE promoter. This construct and the empty vector were introduced into the species Streptomyces albogriseolus/viridodiastaticus yielding A37 and A36, respectively. A37 yielded higher biomass than A36 indicating that the DX-mediated ATP degradation resulted into a stimulation of A37 metabolism, consistently with what was reported in other microorganisms. The comparative analysis of the metabolomes of A36 and A37 revealed that A37 had a lower content in glycolytic and Tricarboxylic Acid Cycle intermediates as well as in amino acids than A36, these metabolites being consumed for biomass generation in A37. In contrast, the abundance of other molecules indicative either of energetic stress (ADP, AMP, UMP, ornithine and thymine), of activation (NAD and threonic acid) or inhibition (citramalic acid, fatty acids, TAG and L-alanine) of the oxidative metabolism, was higher in A37 than in A36. Furthermore, hydroxyl-pyrimidine derivatives and polycyclic aromatic polyketide antibiotics belonging to the angucycline class and thought to have a negative impact on respiration were also more abundantly produced by A37 than by A36. This comparative analysis thus revealed the occurrence in A37 of antagonistic metabolic strategies, namely, activation or slowing down of oxidative metabolism and respiration, to maintain the cellular energetic balance. This study thus demonstrated that DX constitutes an efficient biotechnological tool to enhance the expression of the specialized metabolic pathways present in the Streptomyces genomes that may include cryptic pathways. Its use thus might lead to the discovery of novel bioactive molecules potentially useful to human health.

On the interdependence of ketone body oxidation, glycogen content, glycolysis and energy metabolism in the heart.

In heart, glucose and glycolysis are important for anaplerosis and potentially therefore for d-β-hydroxybutyrate (βHB) oxidation. As a glucose store, glycogen may also furnish anaplerosis. We determined the effects of glycogen content on βHB oxidation and glycolytic rates, and their downstream effects on energetics, in the isolated rat heart. High glycogen (HG) and low glycogen (LG) containing hearts were perfused with 11mM [5-3 H]glucose and/or 4mM [14 C]βHB to measure glycolytic rates or βHB oxidation, respectively, then freeze-clamped for glycogen and metabolomic analyses. Free cytosolic [NAD+ ]/[NADH] and mitochondrial [Q+ ]/[QH2 ] ratios were estimated using the lactate dehydrogenase and succinate dehydrogenase reaction, respectively. Phosphocreatine (PCr) and inorganic phosphate (Pi ) concentrations were measured using 31 P-nuclear magnetic resonance spectroscopy. Rates of βHB oxidation in LG hearts were half that in HG hearts, with βHB oxidation directly proportional to glycogen content. βHB oxidation decreased glycolysis in all hearts. Glycogenolysis in glycogen-replete hearts perfused with βHB alone was twice that of hearts perfused with βHB and glucose, which had significantly higher levels of the glycolytic intermediates fructose 1,6-bisphosphate and 3-phosphoglycerate, and higher free cytosolic [NAD+ ]/[NADH]. βHB oxidation increased the Krebs cycle intermediates citrate, 2-oxoglutarate and succinate, the total NADP/H pool, reduced mitochondrial [Q+ ]/[QH2 ], and increased the calculated free energy of ATP hydrolysis (∆GATP ). Although βHB oxidation inhibited glycolysis, glycolytic intermediates were not depleted, and cytosolic free NAD remained oxidised. βHB oxidation alone increased Krebs cycle intermediates, reduced mitochondrial Q and increased ∆GATP . We conclude that glycogen facilitates cardiac βHB oxidation by anaplerosis. KEY POINTS: Ketone bodies (d-β-hydroxybutyrate, acetoacetate) are increasingly recognised as important cardiac energetic substrates, in both healthy and diseased hearts. As 2-carbon equivalents they are cataplerotic, causing depletion of Krebs cycle intermediates; therefore their utilisation requires anaplerotic supplementation, and intra-myocardial glycogen has been suggested as a potential anaplerotic source during ketone oxidation. It is demonstrated here that cardiac glycogen does indeed provide anaplerotic substrate to facilitate β-hydroxybutyrate oxidation in isolated perfused rat heart, and this contribution was quantified using a novel pulse-chase metabolic approach. Further, using metabolomics and 31 P-MR, it was shown that glycolytic flux from myocardial glycogen increased the heart's ability to oxidise βHB, and βHB oxidation increased the mitochondrial redox potential, ultimately increasing the free energy of ATP hydrolysis.

Abstract P6-11-16: Withaferin A induces metabolic crisis in breast cancer cell lines via decreasing c-myc expression: potential therapeutic implication

Abstract Background: Breast cancer is the most diagnosed cancer and the leading cause of mortality among women in India and worldwide. Reprogrammed glucose metabolism is considered as the hallmark of the cancer with immense therapeutic relevance. Withaferin A (WA), a phytocompound isolated from the plant Withania somnifera, commonly known as Ashwagandha has the remarkable anticancer role. However, the mechanism of action of WA in breast cancer metabolism is still unelucidated. Breast cancer cells have metabolic vulnerability and high glucose dependency. Thus targeting glycolysis could be the better therapeutic approach. Chemotherapy and radiotherapy have the side effects, to overcome this natural products can be used to treat cancer. The potential of natural compounds in targeting metabolic vulnerabilities of cancer and highlighting prospective therapeutic benefits that can be determined by improving our understanding of this field. Aims and Objective: To explore the therapeutic effect of WA in breast cancer cell lines and to identify the role of WA in targeting Warburg effect via c-myc. Cancer cells exhibit upregulated Warburg effect, to fulfill the bioenergetics and biosynthetic demands of the rapidly growing cancer cells. Thus targeting the Warburg effect could be the better therapeutic approach. Materials and methods: Breast cancer cell lines (MBA-MB-231, MBA-MB-468, MBA-MB-453 and MCF-7) were procured from NCCS Pune and maintained in DMEM media supplemented with 10% FBS. SRB dye was used for cell viability as it binds to the protein of the cells. Further Colony formation assay (using crystal violet dye) was done to evaluate the effect of WA on colony formation. Metabolic assays \ (lactate production, glucose uptake and ATP generation) were performed using kits (Eton Bioscience). Transient silencing using si-RNA of c-myc was done with lipofectamine 3000. Silencing of c-myc was done for Warburg effect and protein expression. RNA was isolated from breast cancer cells using Qaigen kit and RT-PCR was performed to evaluate the glycolytic gene expression before and after the treatment of the WA in breast cancer cell lines. To check the expression of glycolytic genes at protein level we had done the western blot. Protein isolation was done in RIPA lysis buffer and western blot was performed using primary antibodies of GLUT1, HK2, PKM2, c-myc. Statistical analysis was analyzed in graph pad prism. Breast cancer patient METABRIC data was analyzed and pathway deregulation score was calculated from Pathifier algorithm. Results: Withaferin A decreased the glucose uptake, lactate production and ATP production in different breast cancer cell lines. Further, WA induced suppression of key glycolytic enzymes via c-myc, decreased cell proliferation, biomass and colony formation ability of the breast cancer cells. Silencing of c-myc gene also showed the similar results to WA such as decrease in Warburg effect and reduction in glycolytic proteins expression. Through the LC-MS analysis WA decreases the key glycolytic metabolites and other pathway metabolites, induce metabolic catastrophe in breast cancer cells. Clinical relevance of our experiments was validated in dataset of ~2000 breast tumors (METABRIC) using Pathifier algorithm wherein we calculated deregulation score of glycolysis pathways in each of the tumor and normal sample. Importantly, higher deregulation of glycolysis was observed in breast tumor compared to normal tissues and found to be associated with poor prognosis. Conclusion: Our results highlight the anti- carcinogenic effect of Withaferin A in modulating breast cancer metabolism and the clinical significance of glycolysis in general. WA decreases the glucose metabolism and its flux through key metabolic pathways associated with the glycolytic intermediates. Therefore it could be argued that therapeutic targeting of breast cancer metabolism by WA may improve clinical outcome. Citation Format: Asifa Khan, Shumaila Siddiqui, Sheersh Massey, Daman Saluja, Syed Akhtar Husain, Mohammad Askandar Iqbal. Withaferin A induces metabolic crisis in breast cancer cell lines via decreasing c-myc expression: potential therapeutic implication [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-16.

Coenzyme Q0 defeats NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects by inhibiting HIF-1α expression in human triple-negative breast cancer cells.

Coenzyme Q0 (CoQ0) is a derivative quinone from Antrodia camphorata (AC) that exerts anticancer activities. This study examined the anticancer attributes of CoQ0 (0-4µM) on inhibited anti-EMT/metastasis and NLRP3 inflammasome, and altered Warburg effects via HIF-1α inhibition in triple-negative breast cancer (MDA-MB-231 and 468) cells. MTT assay, cell migration/invasion assays, Western blotting, immunofluorescence, metabolic reprogramming, and LC-ESI-MS were carried out to assess the therapy potential of CoQ0. CoQ0 inhibited HIF-1α expression and suppressed the NLRP3 inflammasome and ASC/caspase-1 expression, followed by downregulation of IL-1β and IL-18 expression in MDA-MB-231 and 468 cells. CoQ0 ameliorated cancer stem-like markers by decreasing CD44 and increasing CD24 expression. Notably, CoQ0 modulated EMT by upregulating the epithelial marker E-cadherin and downregulating the mesenchymal marker N-cadherin. CoQ0 inhibited glucose uptake and lactate accumulation. CoQ0 also inhibited HIF-1α downstream genes involved in glycolysis, such as HK-2, LDH-A, PDK-1, and PKM-2 enzymes. CoQ0 decreased extracellular acidification rate (ECAR), glycolysis, glycolytic capacity, and glycolytic reserve in MDA-MB-231 and 468 cells under normoxic and hypoxic (CoCl2) conditions. CoQ0 inhibited the glycolytic intermediates lactate, FBP, and 2/3-PG, and PEP levels. CoQ0 increased oxygen consumption rate (OCR), basal respiration, ATP production, maximal respiration, and spare capacity under normoxic and hypoxic (CoCl2) conditions. CoQ0 increased TCA cycle metabolites, such as citrate, isocitrate, and succinate. CoQ0 inhibited aerobic glycolysis and enhanced mitochondrial oxidative phosphorylation in TNBC cells. Under hypoxic conditions, CoQ0 also mitigated HIF-1α, GLUT1, glycolytic-related (HK-2, LDH-A, and PFK-1), and metastasis-related (E-cadherin, N-cadherin, and MMP-9) protein or mRNA expression in MDA-MB-231 and/or 468 cells. Under LPS/ATP stimulation, CoQ0 inhibited NLRP3 inflammasome/procaspase-1/IL-18 activation and NFκB/iNOS expression. CoQ0 also hindered LPS/ATP-stimulated tumor migration and downregulated LPS/ATP-stimulated N-cadherin and MMP-2/-9 expression. The present study revealed that suppression of HIF-1α expression caused by CoQ0 may contribute to inhibition of NLRP3-mediated inflammation, EMT/metastasis, and Warburg effects of triple-negative breast cancers.

Peptide deregulated in hypertrophic scar-1 alleviates hypertrophic scar fibrosis by targeting focal adhesion kinase and pyruvate kinase M2 and remodeling the metabolic landscape

Hypertrophic scarring is a fibrotic skin disease characterized by excessive deposition of collagens. Emerging evidence has suggested important roles for peptides in fibrosis-related diseases. Here, we demonstrate that a skin-derived endogenous peptide, peptide deregulated in hypertrophic scar-1 (PDHS1), with the sequence IATTTASAATAAAIGATPRAK, inhibits cell proliferation, promotes apoptosis, decreases the proportion of cells in S phase, and decreases collagen synthesis in hypertrophic scar fibroblasts. Additionally, treatment with PDHPS1 alleviates hypertrophic scarring in a rabbit ear model. PDHPS1 was found to bind to focal adhesion kinase (FAK) and to decrease its activity. PDHPS1 was also shown to bind to pyruvate kinase M2 (PKM2) and to decreased its expression. Smad2 phosphorylation is also inhibited by treatment with PDHPS1. Overexpression of FAK rescues the decreased expression of COL3A1 induced by PDHPS1 treatment. Targeted metabolomics revealed that PDHPS1 reprogramed metabolism that related to amino acid synthesis, leading to decreases of the key glycolysis intermediates glucose-6-phosphate and fructose-6-phosphate. These results demonstrated that the endogenous peptide PDHPS1 alleviates hypertrophic scar fibrosis in vitro and in vivo by targeting FAK and PKM2 and remodeling the metabolic landscape. Overall, treatment with PDHPS1 is a potential therapeutic strategy for hypertrophic scarring.

Increased fatty acid metabolism and decreased glycolysis are hallmarks of metabolic reprogramming within microglia in degenerating white matter during recovery from experimental stroke

The goal of this study was to evaluate changes in metabolic homeostasis during the first 12 weeks of recovery in a distal middle cerebral artery occlusion mouse model of stroke. To achieve this goal, we compared the brain metabolomes of ipsilateral and contralateral hemispheres from aged male mice up to 12 weeks after stroke to that of age-matched naïve and sham mice. There were 707 biochemicals detected in each sample by liquid chromatography-mass spectroscopy (LC-MS). Mitochondrial fatty acid β-oxidation, indicated by acyl carnitine levels, was increased in stroked tissue at 1 day and 4 weeks following stroke. Glucose and several glycolytic intermediates were elevated in the ipsilateral hemisphere for 12 weeks compared to the aged naïve controls, but pyruvate was decreased. Additionally, itaconate, a glycolysis inhibitor associated with activation of anti-inflammatory mechanisms in myeloid cells, was higher in the same comparisons. Spatial transcriptomics and RNA in situ hybridization localized these alterations to microglia within the area of axonal degeneration. These results indicate that chronic metabolic differences exist between stroked and control brains, including alterations in fatty acid metabolism and glycolysis within microglia in areas of degenerating white matter for at least 12 weeks after stroke.

Determination of the intermediates in glycolysis and tricarboxylic acid cycle with an improved derivatization strategy using gas chromatography-mass spectrometry in complex samples

Traditional Chinese medicine (TCM) is recognized as a complex matrix, and improved analytical methods are crucial to extract the key indicators and depict the interaction and alteration of the complex matrix. Shenqi Fuzheng Injection (SQ), a water extract of Radix Codonopsis and Radix Astragali, has demonstrated preventative effects on myotube atrophy induced by chemotherapeutic agents. To achieve the improved analytical capability of complex biological samples, we established a highly reproducible, sensitive, specific, and robust gas chromatography-tandem mass spectrometry (GC–MS) method to detect glycolysis and tricarboxylic acid (TCA) cycle intermediates with optimized factors in the extraction and derivatization process. Our method detected fifteen metabolites and covered most intermediate metabolites in glycolysis and TCA cycles, including glucose, glucose-6-phosphate, fructose-6-phosphate, dihydroxyacetone phosphate, 3-diphosphoglycerate, phosphoenolpyruvate, pyruvate, lactate, citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate, and malate. Through methodological verification of the method, it was found that the linear correlation coefficients of each compound in the method were greater than 0.98, all of which had lower limits of quantification, the recovery rate was 84.94–104.45%, and the accuracy was 77.72–104.92%. The intraday precision was 3.72–15.37%, the interday precision was 5.00–18.02%, and the stability was 7.85–15.51%. Therefore, the method has good linearity, accuracy, precision, and stability. The method was further applied to study the attenuating effects of the SQ in a chemotherapeutic agents-induced C2C12 myotube atrophy model to evaluate the changes in the tricarboxylic acid cycle and glycolytic products under the action by the complex systems of TCM and disease model. Our study provided an improved method to explore TCM's pharmacodynamic constituents and action mechanisms.

Plasma central carbon metabolite changes associated with KRAS mutation and circulating tumor DNA (ctDNA) status in colorectal cancer (CRC).

181 Background: KRAS mutations have been widely characterized as markers of poor prognosis in CRC. In stage IV CRC, KRAS mutations are predictive of benefit to anti-EGFR therapy. ctDNA has increasingly been recognized as a prognostic biomarker in CRC as well. We evaluated the association between plasma metabolites and KRAS mutation or ctDNA status in a longitudinal, observational cohort of patients with stage I-IV CRC. Methods: This was a retrospective analysis of prospectively collected blood samples from a single-institute cohort of patients with stage I-IV CRC. All blood samples were collected at pre-chemotherapy baseline. A modified Epi proColon 2.0 CE (Epigenomics AG) assay was used for plasma ctDNA testing on the methylated SEPTIN9 gene (mSEPT9). ctDNA positivity was defined as a mSEPT9 percentage of methylation reference (PMR) value greater than zero. Up to 150 metabolites of central carbon metabolism were analyzed by mass spectrometry and high-performance liquid chromatography. Analytes were compared by relative area under the curve (AUC) and differences evaluated by ANOVA. The mean AUC was used in patients with metabolites measured from > 1 timepoint of collection. Patients were stratified by ctDNA status (positive or negative) and KRAS mutant (MT) or wildtype (WT) status. Results: A total of 32 patients were included with median age 65 years (range 20-90). The majority were female (53%) and had stage IV disease (78%). Of 25 patients with stage IV CRC, 88% had pre-chemotherapy blood samples collected in the first-line setting. Most patients were KRAS MT (44%) compared to KRAS WT (37%) or unknown KRAS status (19%). The most common KRAS MT subtypes were G12D (29%), G12V (29%), G13D (21%), and G12C (14%). The mean overall survival in this cohort was 27.4 months while the mean mSEPT9 PMR value was 2553.6. When stratified by ctDNA status, ctDNA positivity was associated with decreased levels of essential amino acids (lysine, methionine, threonine) and the non-essential amino acid arginine (all p < 0.05). Compared to KRAS WT tumors, KRAS MT tumors were associated with increased levels of proline, phenylalanine, and intermediates of glycolysis (lactate), MTA cycle (SAM, 5-Methioadenosine), and O-GlcNAcylation (GlcNac, all p < 0.05). Conclusions: We are the first to demonstrate the feasibility of associating central carbon metabolites with ctDNA and KRAS mutation status. As ctDNA positivity and KRAS MT status have evolving prognostic potential in CRC, associated metabolic signatures may identify metabolic pathways for novel biomarker development. Our findings also show that KRAS MT CRC appears to be enriched in intermediates of glycolytic, methyl donor, and O-GlcNAcylation pathways.

Altered energy metabolism in Fatal Familial Insomnia cerebral organoids is associated with astrogliosis and neuronal dysfunction.

Fatal familial insomnia (FFI) is a rare neurodegenerative disease caused by a dominantly inherited single amino acid substitution (D178N) within the prion protein (PrP). No in vitro human brain tissue model for this disease has previously been available. Consequently, how this mutation exerts its damaging effect on brain cells is still unknown. Using CRISPR-Cas9 engineered induced pluripotent stem cells, we made D178N cerebral organoids and compared these with isotype control organoids. We found that, in the absence of other hallmarks of FFI, the D178N organoids exhibited astrogliosis with cellular oxidative stress. Abnormal post-translational processing of PrP was evident but no tissue deposition or propagation of mis-folded PrP isoforms were observed. Neuronal electrophysiological function was compromised and levels of neurotransmitters, particularly acetylcholine and GABA, altered. Underlying these dysfunctions were changes in cellular energy homeostasis, with substantially increased glycolytic and Krebs cycle intermediates, and greater mitochondrial activity. This increased energy demand in D178N organoids was associated with increased mitophagy and depletion of lipid droplets, in turn resulting in shifts of cellular lipid composition. Using a double mutation (178NN) we could confirm that most changes were caused by the presence of the mutation rather than interaction with PrP molecules lacking the mutation. Our data strongly suggests that shifting biosynthetic intermediates and oxidative stress, caused by an imbalance of energy supply and demand, results in astrogliosis with compromised neuronal activity in FFI organoids. They further support that many of the disease associated changes are due to a corruption of PrP function and do not require propagation of PrP mis-folding.

Pyruvate Dehydrogenase Inhibition Leads to Decreased Glycolysis, Increased Reliance on Gluconeogenesis and Alternative Sources of Acetyl-CoA in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease characterized by poor outcomes and therapy resistance. Devimistat is a novel agent that inhibits pyruvate dehydrogenase complex (PDH). A phase III clinical trial in AML patients combining devimistat and chemotherapy was terminated for futility, suggesting AML cells were able to circumvent the metabolic inhibition of devimistat. The means by which AML cells resist PDH inhibition is unknown. AML cell lines treated with devimistat or deleted for the essential PDH subunit, PDHA, showed a decrease in glycolysis and decreased glucose uptake due to a reduction of the glucose transporter GLUT1 and hexokinase II. Both devimistat-treated and PDHA knockout cells displayed increased sensitivity to 2-deoxyglucose, demonstrating reliance on residual glycolysis. The rate limiting gluconeogenic enzyme phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly upregulated in devimistat-treated cells, and its inhibition increased sensitivity to devimistat. The gluconeogenic amino acids glutamine and asparagine protected AML cells from devimistat. Non-glycolytic sources of acetyl-CoA were also important with fatty acid oxidation, ATP citrate lyase (ACLY) and acyl-CoA synthetase short chain family member 2 (ACSS2) contributing to resistance. Finally, devimistat reduced fatty acid synthase (FASN) activity. Taken together, this suggests that AML cells compensate for PDH and glycolysis inhibition by gluconeogenesis for maintenance of essential glycolytic intermediates and fatty acid oxidation, ACLY and ACSS2 for non-glycolytic production of acetyl-CoA. Strategies to target these escape pathways should be explored in AML.

Characterization of metabolic reprogramming by metabolomics in the oncocytic thyroid cancer cell line XTC.UC1

Oncocytic thyroid cancer is characterized by the aberrant accumulation of abnormal mitochondria in the cytoplasm and a defect in oxidative phosphorylation. We performed metabolomics analysis to compare metabolic reprogramming among the oncocytic and non-oncocytic thyroid cancer cell lines XTC.UC1 and TPC1, respectively, and a normal thyroid cell line Nthy-ori 3-1. We found that although XTC.UC1 cells exhibit higher glucose uptake than TPC1 cells, the glycolytic intermediates are not only utilized to generate end-products of glycolysis, but also diverted to branching pathways such as lipid metabolism and the serine synthesis pathway. Glutamine is preferentially used to produce glutathione to reduce oxidative stress in XTC.UC1 cells, rather than to generate α-ketoglutarate for anaplerotic flux into the TCA cycle. Thus, growth, survival and redox homeostasis of XTC.UC1 cells rely more on both glucose and glutamine than do TPC1 cells. Furthermore, XTC.UC1 cells contained higher amounts of intracellular amino acids which is due to higher expression of the amino acid transporter ASCT2 and enhanced autophagy, thus providing the building blocks for macromolecules and energy production. These metabolic alterations are required for oncocytic cancer cells to compensate their defective mitochondrial function and to alleviate excess oxidative stress.

Metabolomic Alterations in Mammary Glands from Pubertal Mice Fed a High-Fat Diet.

Dietary malpractice is a risk factor for obesity. This study tested the hypothesis that consumption of a high-fat diet alters mammary metabolome in pubertal mice. We performed untargeted metabolomic analysis of primary metabolism on mammary glands from pubertal mice fed the AIN93G standard diet or a high-fat diet (HFD) for 3 weeks. We identified 97 metabolites for statistical comparisons. The HFD altered the amino acid metabolism considerably. This included elevated expression of branched-chain amino acids, non-essential amino acids (aspartic acid and glutamic acid), and methionine sulfoxide (oxidized methionine) and an alteration in the aminoacyl-tRNA biosynthesis pathway. Furthermore, elevations of fumaric acid and malic acid (both are citrate cycle intermediates) and glyceric acid (its phosphate derivatives are intermediates of glycolysis) in HFD-fed mice suggest an acceleration of both citrate cycle and glycolysis. Lower expression of glycerol, oleic acid, and palmitoleic acid, as well as decreased mammary expression of genes encoding lipid metabolism (Acaca, Fads1, Fasn, Scd1, and Srebf1) in HFD-fed mice indicate an attenuated lipid metabolism in the presence of adequate dietary fat. In conclusion, consumption of the HFD for 3 weeks alters metabolic profile of pubertal mammary glands. This alteration may affect mammary development and growth in pubertal mice.

Rapid probing of glucose influx into cancer cell metabolism: using adjuvant and a pH-dependent collection of central metabolites to improve in-cell D-DNP NMR.

Changes to metabolism are a hallmark of many diseases. Disease metabolism under physiological conditions can be probed in real time with in-cell NMR assays. Here, we pursued a systematic approach towards improved in-cell NMR assays. Unambiguous identifications of metabolites and of intracellular pH are afforded by a comprehensive, downloadable collection of spectral data for central carbon metabolites in the physiological pH range (4.0-8.0). Chemical shifts of glycolytic intermediates provide unique pH dependent patterns akin to a barcode. Using hyperpolarized 13C1 enriched glucose as the probe molecule of central metabolism in cancer, we find that early glycolytic intermediates are detectable in PC-3 prostate cancer cell lines, concurrently yielding intracellular pH. Using non-enriched and non-enhanced pyruvate as an adjuvant, reactions of the pentose phosphate pathway become additionally detectable, without significant changes to the barriers in upper glycolysis and to intracellular pH. The scope of tracers for in-cell observations can thus be improved by the presence of adjuvants, showing that a recently proposed effect of pyruvate in the tumor environment is paralleled by a rerouting of cancer cell metabolism towards producing building blocks for proliferation. Overall, the combined use of reference data for compound identification, site specific labelling for reducing overlap, and use of adjuvant afford increasingly detailed insight into disease metabolism.