Glycolytic Metabolites Research Articles

Physiological Adaptation of Fenneropenaeus chinensis in Response to Saline-Alkaline Stress Revealed by a Combined Proteomics and Metabolomics Method.

The rapid development of the mariculture industry has been hindered by limited coastal aquaculture space. To utilize the abundant inland saline-alkaline water, we studied the physiological effects of high carbonate alkalinity stress and high pH stress on Fenneropenaeus chinensis. The study employed quantitative proteomics by tandem mass tag (TMT) and non-targeted metabolomics analysis using a liquid chromatograph mass spectrometer (LC-MS) to understand the physiological and biochemical adaptive mechanisms of the hepatopancreas of F. chinensis in response to saline-alkaline stress at the molecular level. We designed two stress groups as follows: a high carbonate alkalinity (CA) group and a combined high carbonate alkalinity and high pH (CP) group. The study found that the protein and metabolic profiles of the two stress groups were changed, and the CP group, which was exposed to dual stresses, incurred more severe damage to the hepatopancreas compared to that of the CA group. After exposure to CA and CP, the hepatopancreas of F. chinensis showed significant alterations in 455 proteins and 50 metabolites, and 1988 proteins and 272 metabolites, respectively. In addition, F. chinensis upregulated the level of energy metabolism in the hepatopancreas to defend against osmotic imbalance caused by CA or CP stress, which was demonstrated by the significant upregulation of important proteins and metabolites in glycolysis, pyruvate metabolism, TCA cycle, and fatty acid oxidation. Additionally, pattern recognition receptors, the phenol oxidase system, and various immune-related metabolic enzymes and metabolites were also affected. The immune homeostasis of F. chinensis was affected by the alteration of the antioxidant system following exposure to CA or CP. These findings provide valuable information for F. chinensis saline-alkaline water cultivation practices.

Lactic acid: a narrative review of a promoter of the liver cancer microenvironment.

Lactic acid is a metabolite of glycolysis produced in the body, and its production is thought to be a mechanism by which cancer cells evade immune surveillance. Immune evasion and metabolic changes are well established as basic hallmarks of cancer. Although lactate has long been considered a waste product, it is now generally recognized to be a versatile small-molecule chemical that plays an important part in the tumor microenvironment (TME), with increased lactate production linked to the development of human malignancies. Metabolism in liver cancer is redirected toward glycolysis, which enhances the production of metabolic compounds used by tumor cells to produce proteins, lipids, and nucleotides, enabling them to maintain high proliferation rates and to establish the TME. Dysregulation of metabolic activity in liver cancer may impair antitumor responses owing to the immunosuppressive activity of the lactate produced by anaerobic glycolytic rates in tumor cells. This review primarily explores the link connection between lactic acid and the TME; evaluates the role of lactic acid in the occurrence, metastasis, prognosis, and treatment of liver cancer. Additionally, it investigates the associated pathways as potential targets for liver cancer treatment. Literature searches were conducted in PubMed, Web of Science, and Google Scholar, with the publication date of the most recent article included being January 2024. After eliminating duplicate articles and less relevant articles through titles and abstracts, we selected 113 articles for this review. We categorized references into two categories. One is to classify the content into lactate-related, liver cancer-related and tumor metabolism-related. The other is to classify the article types, which are divided into reviews, research articles and clinical trials. Additionally, we consulted the reference lists of the relevant articles to ensure coverage was comprehensive and unbiased. The connection between lactic acid and the TME has recently become an area of intense research interest, and many related articles have been published in this field. The main finding of this review is to summarize the proven link between lactate and the TME and its possible impact on the TME of liver cancer. And analyzed the potential of lactate in liver cancer treatment and prognosis prediction. Lactate may be key to developing novel approaches in the future treatment of liver cancer. Related research on the combination of classic therapies and molecular targeted drugs may provide innovative medicines that more selectively regulate immune cell activity.

Mycobacterium dormancy and antibiotic tolerance within the retinal pigment epithelium of ocular tuberculosis.

Understanding the metabolic environment within the retinal pigment epithelium (RPE) cells altered by infection with Mycobacterium tuberculosis and mycobacterial dormancy is crucial to identify new therapeutic methods to cure ocular tuberculosis. The present study showed that RPE cellular metabolism is altered to foster intracellular M. tuberculosis to enter into the dormant and drug-tolerant state, thereby blunting the efficacy of anti-tuberculosis chemotherapy. RPE cells serve as an anatomical niche as the cells protect invading bacilli from antibiotic treatment. LC-MS metabolomics of RPE cells after co-treatment with H2O2 and M. tuberculosis infection showed that the intracellular environment within RPE cells is enriched with a greater level of oxidative stress. The antibiotic tolerance of intracellular M. tuberculosis within RPE cells can be restored by a metabolic manipulation strategy such as co-treatment of antibiotic with the most downstream glycolysis metabolite, phosphoenolpyruvate.

ATF4 and mTOR regulate metabolic reprogramming in TGF-β-treated lung fibroblasts.

Idiopathic pulmonary fibrosis is a fatal disease characterized by the TGF-β-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive replacement of healthy lung with scar tissue. We and others have shown that fibroblast activation is supported by metabolic reprogramming, including the upregulation of the de novo synthesis of glycine, the most abundant amino acid found in collagen protein. How fibroblast metabolic reprogramming is regulated downstream of TGF-β is incompletely understood. We and others have shown that TGF-β-mediated activation of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and downstream upregulation of Activating Transcription Factor 4 (ATF4) promote increased expression of the enzymes required for de novo glycine synthesis; however, whether mTOR and ATF4 regulate other metabolic pathways in lung fibroblasts has not been explored. Here, we used RNA sequencing to determine how both ATF4 and mTOR regulate gene expression in human lung fibroblasts following TGF-β. We found that ATF4 primarily regulates enzymes and transporters involved in amino acid homeostasis as well as aminoacyl-tRNA synthetases. mTOR inhibition resulted not only in the loss of ATF4 target gene expression, but also in the reduced expression of glycolytic enzymes and mitochondrial electron transport chain subunits. Analysis of TGF-β-induced changes in cellular metabolite levels confirmed that ATF4 regulates amino acid homeostasis in lung fibroblasts while mTOR also regulates glycolytic and TCA cycle metabolites. We further analyzed publicly available single cell RNAseq data sets and found increased expression of ATF4 and mTOR metabolic targets in pathologic fibroblast populations from the lungs of IPF patients. Our results provide insight into the mechanisms of metabolic reprogramming in lung fibroblasts and highlight novel ATF4 and mTOR-dependent pathways that may be targeted to inhibit fibrotic processes.

Loss of mitochondrial pyruvate transport initiates cardiac glycogen accumulation and heart failure.

Heart failure involves metabolic alterations including increased glycolysis despite unchanged or decreased glucose oxidation. The mitochondrial pyruvate carrier (MPC) regulates pyruvate entry into the mitochondrial matrix, and cardiac deletion of the MPC in mice causes heart failure. How MPC deletion results in heart failure is unknown. We performed targeted metabolomics and isotope tracing in wildtype (fl/fl) and cardiac-specific Mpc2-/- (CS-Mpc2-/-) hearts after in vivo injection of U-13C-glucose. Cardiac glycogen was assessed biochemically and by transmission electron microscopy. Cardiac uptake of 2-deoxyglucose was measured and western blotting performed to analyze insulin signaling and enzymatic regulators of glycogen synthesis and degradation. Isotope tracing and glycogen analysis was also performed in hearts from mice fed either low-fat diet or a ketogenic diet previously shown to reverse the CS-Mpc2-/- heart failure. Cardiac glycogen was also assessed in mice infused with angiotensin-II that were fed low-fat or ketogenic diet. Failing CS-Mpc2-/- hearts contained normal levels of ATP and phosphocreatine, yet these hearts displayed increased enrichment from U-13C-glucose and increased glycolytic metabolite pool sizes. 13C enrichment and pool size was also increased for the glycogen intermediate UDP-glucose, as well as increased enrichment of the glycogen pool. Glycogen levels were increased ~6-fold in the failing CS-Mpc2-/- hearts, and glycogen granules were easily detected by electron microscopy. This increased glycogen synthesis occurred despite enhanced inhibitory phosphorylation of glycogen synthase and reduced expression of glycogenin-1. In young, non-failing CS-Mpc2-/- hearts, increased glycolytic 13C enrichment occurred, but glycogen levels remained low and unchanged compared to fl/fl hearts. Feeding a ketogenic diet to CS-Mpc2-/- mice reversed the heart failure and normalized the cardiac glycogen and glycolytic metabolite accumulation. Cardiac glycogen levels were also elevated in mice infused with angiotensin-II, and both the cardiac hypertrophy and glycogen levels were improved by ketogenic diet. Our results indicate that loss of MPC in the heart causes glycogen accumulation and heart failure, while a ketogenic diet can reverse both the glycogen accumulation and heart failure. We conclude that maintaining mitochondrial pyruvate import and metabolism is critical for the heart, unless cardiac pyruvate metabolism is reduced by consumption of a ketogenic diet.

Effect of transcription factor WT1 on triple-negative breast cancer metastasis through PFKFB4-mediated glycolysis.

e13145 Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high probability of metastasis as well as a lack of specific targets and targeted therapeutics. Preliminary study suggested that Wilms' tumor gene 1 (WT1) is highly expressed in breast cancer patients with poor prognosis and may promote TNBC metastasis, but the underlying mechanism remains poorly defined. Methods: WT1 expression was evaluated by immunohistochemistry (IHC) in breast cancer patients. Kaplan–Meier survival analysis was performed to assess the prognostic significance of WT1 expression. WT1 was silenced in MDA-MB-231 and BT549 cells or overexpressed in HCC1806 cells. qRT-PCR and Western blot were used to detect the WT1 expression in tissues and cells. Wound healing assays, transwell assays and 3D spheroid assays were used to examine the migration and invasion abilities in TNBC cells. The lung metastasis model of mice was used to evaluate metastasis of TNBC in vivo. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome sequencing (RNA-seq) were performed to find PFKFB4, a downstream target gene for WT1. ChIP-PCR and dual-luciferase reporter assays were used to explore the transcriptional regulation of PFKFB4 by WT1. Seahorse XF glycolysis stress assay, glucose uptake assay, and lactate production assay were used to investigate the role of WT1 in regulating glycolysis metabolites. Results: WT1 was highly expressed in TNBC and correlated to poor prognosis in TNBC patients. Functional assays revealed that WT1 promoted TNBC cell metastasis in vitro and in vivo. Our mechanistic investigations demonstrated that WT1 promoted TNBC cells migration and invasion by transcriptionally activating the expression of PFKFB4.This action leaded to increased glycolytic capacity, glucose uptake, and lactate production in cancer cells, therefore promoting metastasis of TNBC. Clinically, the combined expression of WT1 and PFKFB4 provides a reliable predictive biomarker for the prognosis of TNBC patients. Conclusions: Our findings reveal a molecular mechanism of WT1 promoting TNBC metastasis, which provides new targets for the precision treatment of TNBC and new perspectives for the development of targeted metabolic anticancer drugs.

The sterol element binding protein 1c (SREBP1c) preserves cellular metabolism and immunosuppressive function of regulatory t cells

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Cariplo Foundation PRIN (Italian Ministry of University and Research) Aim Balance of cellular metabolism is increasingly recognized as a hallmark of physiologic cellular functions; this also extends to cells of the immune system. Here, we aimed at investigating the role of SREBP1c, a key protein regulating intracellular fatty acid (FA) metabolism, on the metabolism and function of Treg, that play a key role in immune tolerance maintenance. Material and Methods A detailed immunophenotyping through flow cytometry and metabolic profiling (metabolomics and seahorse analysis) of isolated Tregulatory (CD4+CD25+, nTreg) and in vitro induced Treg (iTreg) cells were performed together with in vitro and in vivo assays of Treg function from SREBP1c KO and WT littermates. RNAseq and lipidomics was performed on iTreg. Results Srebp1c deficiency reduced suppressive (-21%,p<0,01) and increased migratory function (+40%,p<0,05) of nTreg and iTreg, tested by in vitro assays. In vivo, Srebp1c KO mice presented reduced circulating and tissues’ level of Treg compared to WT mice (-66%,p<0,01) and nTreg from Srebp1c KO mice worsened Experimental Autoimmune Encephalomyelitis progression compared to WT nTreg. We addressed impaired Treg function to metabolic alteration due to Srebp1c deficiency: KO iTreg showed an increased glycolytic potential with preserved mitochondrial function coupled to accumulation of glycolytic metabolites and lactate (+20%,p<0,01) and reduced energy charge (-40%,p<0.01). We associated the switch to anaerobic glycolysis in KO Treg to an impaired lipid metabolism, as suggested by impaired abundance of lipid species (TG, LPC and PC); this was confirmed by RNAseq showing a downregulation of lipid metabolism’s pathways and upregulation of glycolysis. In parallel, tolerogenic response was impaired, a phenotype associated to a reduced transcription and activation of Foxp3, the master regulator of Treg function. Conclusion By restrain glycolysis and preserving lipid metabolism, our data have identified SREBP1c as a checkpoint crucial for the immunometabolic suppressive response of Tregs.

#2756 Metabolomic prediction of end stage renal disease in large national biobanks

Abstract Background and Aims Identifying at-risk individuals likely to progress to end-stage renal disease as early as possible is important to effectively concentrate preventative interventions. Existing predictive tools, like the kidney failure risk score, are effective for individuals who already have moderate to severe kidney disease. Here we examine whether comprehensive metabolomics can improve performance in individuals with mildly impaired kidney function. Method Using an NMR metabolomics platform we measured >200 biomarkers including creatinine, albumin, lipids, fatty acids, amino acids, glycolysis metabolites and inflammation markers in blood samples from all 500,000 participants of the UK Biobank, who have >10 years of linked prospective follow-up data. We used machine learning to develop a score to predict kidney disease progression based on biomarkers, age and sex. We compared its performance to eGFR in a held out set of individuals in UK Biobank, and replicated in 200,000 participants of the Estonian Biobank. Results Among individuals with prevalent diabetes, hypertension or eGFR < 90 the biomarker score performs as well as or better than eGFR for all individuals. In particular it outperforms among individuals with only moderate (eGFR 60-75) or mild (eGFR 75-90) impairment of kidney function with an AUC increase from 0.63 to 0.72 and 0.48 to 0.70, respectively (Figure). This improvement is driven by contributions from numerous biomarkers, including lipids and inflammation markers. Furthermore, because these biomarkers capture diverse metabolic risk, the biomarker score also strongly predicts major adverse cardiovascular outcomes. Conclusion This large dataset allows detailed investigation of the value of metabolomics in identifying future risk of severe kidney disease early. In particular these biomarkers can highlight individuals who do not already have very low eGFR.

GDE5/Gpcpd1 activity determines phosphatidylcholine composition in skeletal muscle and regulates contractile force in mice

Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline and glycerol 3-phosphate; this study aimed to elucidate its physiological function in vivo. Heterozygous whole-body GDE5-deficient mice reveal a significant GPC accumulation across tissues, while homozygous whole-body knockout results in embryonic lethality. Skeletal muscle-specific GDE5 deletion (Gde5 skKO) exhibits reduced passive force and improved fatigue resistance in electrically stimulated gastrocnemius muscles in vivo. GDE5 deficiency also results in higher glycolytic metabolites and glycogen levels, and glycerophospholipids alteration, including reduced levels of phospholipids that bind polyunsaturated fatty acids (PUFAs), such as DHA. Interestingly, this PC fatty acid compositional change is similar to that observed in skeletal muscles of denervated and Duchenne muscular dystrophy mouse models. These are accompanied by decrease of GDE5 expression, suggesting a regulatory role of GDE5 activity for glycerophospholipid profiles. Furthermore, a DHA-rich diet enhances contractile force and lowers fatigue resistance, suggesting a functional relationship between PC fatty acid composition and muscle function. Finally, skinned fiber experiments show that GDE5 loss increases the probability of the ryanodine receptor opening and lowers the maximum Ca2+-activated force. Collectively, GDE5 activity plays roles in PC and glucose/glycogen metabolism in skeletal muscle.

Postmortem mitochondria function in longissimus lumborum of Angus and Brahman steers

Mitochondria function and integrity may impact postmortem metabolism and meat quality development. Adaptations in heat tolerant Brahman may persist to limit cellular stress postmortem. Our objective was to evaluate glycolysis, pH decline, and mitochondria function in longissimus lumborum (LL) from Angus and Brahman steers (N = 28) early postmortem (1 to 6 h) and after rigor (24 h). We evaluated metabolites of anaerobic glycolysis, ATP, pH, and temperature, and determined mitochondria oxygen consumption rate (OCR) in permeabilized fibers. The main effects of breed (b) and time (t) and the interaction were tested. Brahman LL contained greater ATP during the first 6 h postmortem; Brahman also tended to exhibit a slower pH decline (b × t, P = 0.07) and more rapid temperature decline (b × t, P < 0.001), but metabolites of anaerobic glycolysis were not different. Mitochondria in Brahman and Angus LL were well-coupled and respired at 1 h postmortem. However, outer membrane integrity became increasingly compromised postmortem (t, P < 0.001). Brahman tended to exhibit greater electron transport system capacity (b, P < 0.1) and had greater capacity for oxidative phosphorylation (complex I and II substrates) at 6 h compared with Angus (P < 0.001). In totality, greater ATP, slower pH decline, and enhanced mitochondria capacity indicate that Brahman possess mitochondrial properties or cellular adaptations that help protect the cell during energy stress postmortem. Slower pH and more rapid temperature decline in LL from Brahman may also help preserve mitochondria function postmortem.

Glucagon-like peptide-1 analogs activate AMP kinase leading to reversal of the Warburg metabolic switch in breast cancer cells.

Breast cancer (BC) is associated with type 2 diabetes mellitus (T2DM) and obesity. Glucagon-like peptide (GLP)-1 regulates post-prandial insulin secretion, satiety, and gastric emptying. Several GLP-1 analogs have been FDA-approved for the treatment of T2DM and obesity. Moreover, GLP-1 regulates various metabolic activities across different tissues by activating metabolic signaling pathways like adenosine monophosphate (AMP) activated protein kinase (AMPK), and AKT. Rewiring metabolic pathways is a recognized hallmark of cancer, regulated by several cancer-related pathways, including AKT and AMPK. As GLP-1 regulates AKT and AMPK, we hypothesized that it alters BC cells' metabolism, thus inhibiting proliferation. The effect of the GLP-1 analogs exendin-4 (Ex4) and liraglutide on viability, AMPK signaling and metabolism of BC cell lines were assessed. Viability of BC cells was evaluated using colony formation and MTT/XTT assays. Activation of AMPK and related signaling effects were evaluated using western blot. Metabolism effects were measured for glucose, lactate and ATP. Exendin-4 and liraglutide activated AMPK in a cAMP-dependent manner. Blocking Ex4-induced activation of AMPK by inhibition of AMPK restored cell viability. Interestingly, Ex4 and liraglutide reduced the levels of glycolytic metabolites and decreased ATP production, suggesting that GLP-1 analogs impair glycolysis. Notably, inhibiting AMPK reversed the decline in ATP levels, highlighting the role of AMPK in this process. These results establish a novel signaling pathway for GLP-1 in BC cells through cAMP and AMPK modulation affecting proliferation and metabolism. This study suggests that GLP-1 analogs should be considered for diabetic patients with BC.

Positive feedback regulation between glycolysis and histone lactylation drives oncogenesis in pancreatic ductal adenocarcinoma

BackgroundMetabolic reprogramming and epigenetic alterations contribute to the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). Lactate-dependent histone modification is a new type of histone mark, which links glycolysis metabolite to the epigenetic process of lactylation. However, the role of histone lactylation in PDAC remains unclear.MethodsThe level of histone lactylation in PDAC was identified by western blot and immunohistochemistry, and its relationship with the overall survival was evaluated using a Kaplan-Meier survival plot. The participation of histone lactylation in the growth and progression of PDAC was confirmed through inhibition of histone lactylation by glycolysis inhibitors or lactate dehydrogenase A (LDHA) knockdown both in vitro and in vivo. The potential writers and erasers of histone lactylation in PDAC were identified by western blot and functional experiments. The potential target genes of H3K18 lactylation (H3K18la) were screened by CUT&Tag and RNA-seq analyses. The candidate target genes TTK protein kinase (TTK) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) were validated through ChIP-qPCR, RT-qPCR and western blot analyses. Next, the effects of these two genes in PDAC were confirmed by knockdown or overexpression. The interaction between TTK and LDHA was identified by Co-IP assay.ResultsHistone lactylation, especially H3K18la level was elevated in PDAC, and the high level of H3K18la was associated with poor prognosis. The suppression of glycolytic activity by different kinds of inhibitors or LDHA knockdown contributed to the anti-tumor effects of PDAC in vitro and in vivo. E1A binding protein p300 (P300) and histone deacetylase 2 were the potential writer and eraser of histone lactylation in PDAC cells, respectively. H3K18la was enriched at the promoters and activated the transcription of mitotic checkpoint regulators TTK and BUB1B. Interestingly, TTK and BUB1B could elevate the expression of P300 which in turn increased glycolysis. Moreover, TTK phosphorylated LDHA at tyrosine 239 (Y239) and activated LDHA, and subsequently upregulated lactate and H3K18la levels.ConclusionsThe glycolysis-H3K18la-TTK/BUB1B positive feedback loop exacerbates dysfunction in PDAC. These findings delivered a new exploration and significant inter-relationship between lactate metabolic reprogramming and epigenetic regulation, which might pave the way toward novel lactylation treatment strategies in PDAC therapy.

A versatile delivery vehicle for cellular oxygen and fuels or metabolic sensor? A review and perspective on the functions of myoglobin.

A canonical view of the primary physiological function of myoglobin (Mb) is that it is an oxygen (O2) storage protein supporting mitochondrial oxidative phosphorylation, especially as the tissue O2 partial pressure (Po2) drops and Mb off-loads O2. Besides O2 storage/transport, recent findings support functions for Mb in lipid trafficking and sequestration, interacting with cellular glycolytic metabolites such as lactate (LAC) and pyruvate (PYR), and "ectopic" expression in some types of cancer cells and in brown adipose tissue (BAT). Data from Mb knockout (Mb-/-) mice and biochemical models suggest additional metabolic roles for Mb, especially regulation of nitric oxide (NO) pools, modulation of BAT bioenergetics, thermogenesis, and lipid storage phenotypes. From these and other findings in the literature over many decades, Mb's function is not confined to delivering O2 in support of oxidative phosphorylation but may serve as an O2 sensor that modulates intracellular Po2- and NO-responsive molecular signaling pathways. This paradigm reflects a fundamental change in how oxidative metabolism and cell regulation are viewed in Mb-expressing cells such as skeletal muscle, heart, brown adipocytes, and select cancer cells. Here, we review historic and emerging views related to the physiological roles for Mb and present working models illustrating the possible importance of interactions between Mb, gases, and small-molecule metabolites in regulation of cell signaling and bioenergetics.

Lack of Sestrins Is Associated with Increased Glycolytic Flux

Introduction: Sestrin1, 2, and 3 are a family of stress-response proteins implicated in many diseases. How Sestrins affect cell survival through AMP-activated kinase (AMPK) and protein synthesis through mechanistic target of rapamycin in complex 1 (mTORC1) is most studied; however, how Sestrins affect metabolism is poorly understood. Hypothesis: Independent of AMPK/mTORC1 activation, Sestrins negatively regulate glycolytic flux by inhibiting glycolytic enzyme activity through binding inhibition. Results: Wildtype (WT) HEK293T cells were transfected with plasmids expressing individual FLAG-tagged Sestrins and then deprived of glucose for 180 min or deprived of glucose for 150 min followed by glucose addback for 30 min. FLAG-tagged Sestrins were immunoprecipitated and found to directly interact with Hexokinase2 and Phosphofructokinase such that binding inversely correlates with glucose concentration. Additionally, WT and Sestrin-knockout (TKO) HEK293T cells were subjected to the glycolytic and mitochondrial stress tests and analyzed for metabolic flux via Seahorse analysis. TKO cells demonstrated higher glycolytic flux, but lower mitochondrial flux, compared to WT cells. Cells were then treated with inhibitors against AMPK and mTORC1 and subjected to the glycolytic stress test. Surprisingly, glycolytic flux in TKO cells was unaffected by the inhibitors while WT cells reduced glycolytic flux suggesting Sestrins can regulate glycolytic flux independent of AMPK/mTORC1 activation. Thereafter, WT and TKO cells were glucose-deprived for 120 min or glucose-deprived for 110 min followed by glucose addback for 10 min and then characterized by steady-state metabolomics. As expected, TKO cells possessed elevations in glycolytic metabolites with glucose addback compared to WT cells. Similarly, WT and TKO cells were deprived of glucose for 110 min followed by stable isotope tracing with [13C6]glucose for 10- and 30-minutes and metabolites were profiled by mass spectrometry. TKO cells possessed higher abundances of glycolytic metabolites as well as elevated labeled extracellular lactate. Conclusions: These data support a model wherein Sestrins directly act as negative regulators for glycolytic enzyme activity, thus regulating glycolytic flux. This work is funded by F32DK126312 (P.A.R.), P30DK048520 Pilot and Feasibility Award (P.A.R.), T32DK120521 (A.E.L.), P30CA046934 Cancer Center Support Grant, R01AR080051 (A.B.), R01DK015658 (S.R.K.), P30DK048520 (P.S.M.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BCG Vaccination-Associated Lower HbA1c and Increased CD25 Expression on CD8+ T Cells in Patients with Type 1 Diabetes in Ghana.

BCG vaccination affects other diseases beyond tuberculosis by unknown-potentially immunomodulatory-mechanisms. Recent studies have shown that BCG vaccination administered during overt type 1 diabetes (T1D) improved glycemic control and affected immune and metabolic parameters. Here, we comprehensively characterized Ghanaian T1D patients with or without routine neonatal BCG vaccination to identify vaccine-associated alterations. Ghanaian long-term T1D patients (n = 108) and matched healthy controls (n = 214) were evaluated for disease-related clinical, metabolic, and immunophenotypic parameters and compared based on their neonatal BCG vaccination status. The majority of study participants were BCG-vaccinated at birth and no differences in vaccination rates were detected between the study groups. Notably, glycemic control metrics, i.e., HbA1c and IDAA1c, showed significantly lower levels in BCG-vaccinated as compared to unvaccinated patients. Immunophenotype comparisons identified higher expression of the T cell activation marker CD25 on CD8+ T cells from BCG-vaccinated T1D patients. Correlation analysis identified a negative correlation between HbA1c levels and CD25 expression on CD8+ T cells. In addition, we observed fractional increases in glycolysis metabolites (phosphoenolpyruvate and 2/3-phosphoglycerate) in BCG-vaccinated T1D patients. These results suggest that neonatal BCG vaccination is associated with better glycemic control and increased activation of CD8+ T cells in T1D patients.

Inhibition of pyruvate dehydrogenase accelerates anaerobic glycolysis under postmortem simulating conditions

This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 μM CPI-613, 1.5 U/ml Avidin, 400 μM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 μM CPI-613 or control, were employed to track the fates of pyruvate using [13C6]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 μM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism.

Coculture with Colon-26 cancer cells decreases the protein synthesis rate and shifts energy metabolism toward glycolysis dominance in C2C12 myotubes.

Cancer cachexia is the result of complex interorgan interactions initiated by cancer cells and changes in patient behavior such as decreased physical activity and energy intake. Therefore, it is crucial to distinguish between the direct and indirect effects of cancer cells on muscle mass regulation and bioenergetics to identify novel therapeutic targets. In this study, we investigated the direct effects of Colon-26 cancer cells on the molecular regulating machinery of muscle mass and its bioenergetics using a coculture system with C2C12 myotubes. Our results demonstrated that coculture with Colon-26 cells induced myotube atrophy and reduced skeletal muscle protein synthesis and its regulating mechanistic target of rapamycin complex 1 signal transduction. However, we did not observe any activating effects on protein degradation pathways including ubiquitin-proteasome and autophagy-lysosome systems. From a bioenergetic perspective, coculture with Colon-26 cells decreased the complex I-driven, but not complex II-driven, mitochondrial ATP production capacity, while increasing glycolytic enzyme activity and glycolytic metabolites, suggesting a shift in energy metabolism toward glycolysis dominance. Gene expression profiling by RNA sequencing showed that the increased activity of glycolytic enzymes was consistent with changes in gene expression. However, the decreased ATP production capacity of mitochondria was not in line with the gene expression. The potential direct interaction between cancer cells and skeletal muscle cells revealed in this study may contribute to a better fundamental understanding of the complex pathophysiology of cancer cachexia.NEW & NOTEWORTHY We explored the potential direct interplay between colon cancer cells (Colon-26) and skeletal muscle cells (C2C12 myotubes) employing a noncontact coculture experimental model. Our findings reveal that coculturing with Colon-26 cells substantially impairs the protein synthesis rate, concurrently instigating a metabolic shift toward glycolytic dominance in C2C12 myotubes. This research unveils critical insights into the intricate cellular cross talk underpinning the complex pathophysiology of cancer cachexia.

A glycolytic metabolite bypasses “two-hit” tumor suppression by BRCA2

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

A glycolytic metabolite that drives BRCA2 haploinsufficiency

Many types of tumor cells alter metabolic pathways to meet their energy and biosynthetic demands for proliferation or stress adaptation. In this issue of Cell, Kong etal. find that the glycolytic metabolite methylglyoxal causes cancer-associated mutant single-base substitution features by inducing BRCA2 proteolysis, leading to functional haploinsufficiency of BRCA2.

A Targeted Approach for the Metabolome Analysis of E. coli Biofilms

AbstractBiofilms of pathogenic bacteria are responsible for persistent infections in humans, therefore investigations of biofilm formation and treatment strategies are required. The gram‐negative enterobacterium Escherichia (E.) coli is the most common pathogen causing chronic or recurring urinary tract infections. Metabolomics approaches targeted the bacterium to investigate specific metabolic patterns of biofilms and regulatory influences on biofilm formation. In this study, we aimed to investigate the metabolome of biofilms formed by the multidrug‐resistant extended‐spectrum beta‐lactamase‐producing (ESBL) E. coli PBIO729. For this purpose, a protocol for fast sampling of the macrocolony biofilms and efficient extraction of metabolites was optimized. Validation of an LC‐MS/MS method confirmed its usability for the analysis of nucleotides and other phosphorylated metabolites. A GC‐MS approach was used to monitor nutrient uptake from the medium in addition to the analysis of amino acid content and metabolites of glycolysis and TCA cycle in E. coli biofilms.