Pentose Phosphate Pathway Activity Research Articles

Virtually identical does not mean exactly identical: Discrepancy in energy metabolism between glucose and fructose fermentation influences the reproductive potential of yeast cells

The physiological efficiency of cells largely depends on the possibility of metabolic adaptations to changing conditions, especially on the availability of nutrients. Central carbon metabolism has an essential role in cellular function. In most cells is based on glucose, which is the primary energy source, provides the carbon skeleton for the biosynthesis of important cell macromolecules, and acts as a signaling molecule. The metabolic flux between pathways of carbon metabolism such as glycolysis, pentose phosphate pathway, and mitochondrial oxidative phosphorylation is dynamically adjusted by specific cellular economics responding to extracellular conditions and intracellular demands. Using Saccharomyces cerevisiae yeast cells and potentially similar fermentable carbon sources i.e. glucose and fructose we analyzed the parameters concerning the metabolic status of the cells and connected with them alteration in cell reproductive potential. Those parameters were related to the specific metabolic network: the hexose uptake - glycolysis and activity of the cAMP/PKA pathway - pentose phosphate pathway and biosynthetic capacities - the oxidative respiration and energy generation. The results showed that yeast cells growing in a fructose medium slightly increased metabolism redirection toward respiratory activity, which decreased pentose phosphate pathway activity and cellular biosynthetic capabilities. These differences between the fermentative metabolism of glucose and fructose, lead to long-term effects, manifested by changes in the maximum reproductive potential of cells.

Insulin and glycolysis dependency of cardioprotection by nicotinamide riboside.

Decreased nicotinamide adenine dinucleotide (NAD+) levels contribute to various pathologies such as ageing, diabetes, heart failure and ischemia-reperfusion injury (IRI). Nicotinamide riboside (NR) has emerged as a promising therapeutic NAD+ precursor due to efficient NAD+ elevation and was recently shown to be the only agent able to reduce cardiac IRI in models employing clinically relevant anesthesia. However, through which metabolic pathway(s) NR mediates IRI protection remains unknown. Furthermore, the influence of insulin, a known modulator of cardioprotective efficacy, on the protective effects of NR has not been investigated. Here, we used the isolated mouse heart allowing cardiac metabolic control to investigate: (1) whether NR can protect the isolated heart against IRI, (2) the metabolic pathways underlying NR-mediated protection, and (3) whether insulin abrogates NR protection. NR protection against cardiac IRI and effects on metabolic pathways employing metabolomics for determination of changes in metabolic intermediates, and 13C-glucose fluxomics for determination of metabolic pathway activities (glycolysis, pentose phosphate pathway (PPP) and mitochondrial/tricarboxylic acid cycle (TCA cycle) activities), were examined in isolated C57BL/6N mouse hearts perfused with either (a) glucose + fatty acids (FA) ("mild glycolysis group"), (b) lactate + pyruvate + FA ("no glycolysis group"), or (c) glucose + FA + insulin ("high glycolysis group"). NR increased cardiac NAD+ in all three metabolic groups. In glucose + FA perfused hearts, NR reduced IR injury, increased glycolytic intermediate phosphoenolpyruvate (PEP), TCA intermediate succinate and PPP intermediates ribose-5P (R5P) / sedoheptulose-7P (S7P), and was associated with activated glycolysis, without changes in TCA cycle or PPP activities. In the "no glycolysis" hearts, NR protection was lost, whereas NR still increased S7P. In the insulin hearts, glycolysis was largely accelerated, and NR protection abrogated. NR still increased PPP intermediates, with now high 13C-labeling of S7P, but NR was unable to increase metabolic pathway activities, including glycolysis. Protection by NR against IRI is only present in hearts with low glycolysis, and is associated with activation of glycolysis. When activation of glycolysis was prevented, through either examining "no glycolysis" hearts or "high glycolysis" hearts, NR protection was abolished. The data suggest that NR's acute cardioprotective effects are mediated through glycolysis activation and are lost in the presence of insulin because of already elevated glycolysis.

Abstract 4424: Differential metabolic response of triple-negative and estrogen receptor-positive breast cancer cells to abemaciclib, a CDK4/6 inhibitor

Abstract Background: Inhibition of the cell cycle kinases CDK4/6 is the standard-of-care for advanced hormone receptor-positive breast cancer. The clinical efficacy of CDK4/6 inhibitors in triple-negative breast cancer remains uncertain. Mechanisms of sensitivity and resistance are not fully understood. This study comprehensively investigated the metabolic response of triple-negative and estrogen receptor-positive breast cancer cells to abemaciclib (Abe), with the aim of providing metabolic insights for resistance mechanisms. Methods: MCF-7 (MCF) and MDA-MB-231 (MDA) cells were treated with Abe (0, 100, and 500 nM) for 1, 2, or 7 days. Cell cycle and cell senescence analyses were performed to assess drug effects. Cellular metabolism was investigated using the Seahorse assay, LC-MS/MS based targeted metabolomics, and stable isotope-assisted metabolic flux analyses with [U-13C]glutamine, [1,2-13C]glucose, and [U-13C]glucose as the tracers. Results: Abe induced cell cycle arrest at G0/G1 in both cell lines, more pronounced in MCF. Dose- and time-dependent cell senescence occurred in both treated cell lines, more pronounced in MDA. The Seahorse assay indicated that energy metabolism (assessed by oxygen consumption rate and extracellular acidification rate) was reduced in both treated cell lines. Targeted metabolomics revealed that post Abe treatment, intracellular lactate significantly decreased in both cell lines; intracellular TCA intermediates significantly decreased in MCF while being unchanged in MDA; intracellular deoxynucleotides decreased in both cell lines, to a lesser extent in MDA; cellular energy levels (assessed as the ratios of ATP/AMP, ATP/ADP, CTP/CMP, CTP/CDP) were significantly lower in MCF while remaining unchanged or increased in MDA; intracellular levels of many amino acids significantly increased in MDA while decreasing in MCF. [U-13C]glutamine flux revealed a significant decrease of intracellular glutamine-derived TCA intermediates in both treated cell lines. [1,2-13C]glucose flux indicated an enhanced oxidate pentose phosphate pathway (PPP) activity and increased de novo syntheses of purine nucleotides (ATP and GTP) in treated MDA (but not MCF). [U-13C]glucose flux suggested that while both cell lines responded to Abe with decreased anaerobic glycolysis (pyruvate converting to lactate), MDA (but not MCF) showed an enhanced capability of utilizing glucose-derived carbon to replenish TCA intermediates and increased amino acid synthesis. Conclusion: Adaptive cellular metabolism, characterized by enhanced utilization of glucose-derived carbon to replenish TCA intermediates and increased PPP activity, thus promoting biosynthesis of amino acids and purine nucleotides to maintain cellular energy and redox balance, may contribute to the intrinsic resistance of triple-negative breast cancer to CDK4/6 inhibitors. Citation Format: Jun Jiang, Xun Bao, Yuanyuan Jiang, Yang Yue, Maik Huettemann, Jing Li. Differential metabolic response of triple-negative and estrogen receptor-positive breast cancer cells to abemaciclib, a CDK4/6 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4424.

Metabolic reprogramming and macrophage polarization in granuloma formation.

This review article delves into the complexities of granuloma formation, focusing on the metabolic reprogramming within these immune structures, especially in tuberculosis and sarcoidosis. It underscores the role of the monocyte-macrophage lineage in granuloma formation and maintenance, emphasizing the adaptability of these cells to environmental cues and inflammatory stimuli. Key to the discussion is the macrophage polarization influenced by various cytokines, with a detailed exploration of the metabolic shifts towards glycolysis under hypoxic conditions and the utilization of the pentose phosphate pathway (PPP) for crucial biosynthetic processes. Significant attention is given to the metabolism of L-arginine in macrophages and its impact on immune response and granuloma function. The review also highlights the role of mechanistic target of rapamycin (mTOR) signaling in macrophage differentiation and its implications in granulomatous diseases. Discoveries such as elevated PPP activity in granuloma-associated macrophages and the protective role of NADPH against oxidative stress offer novel insights into granuloma biology. The review concludes by suggesting potential therapeutic targets within these metabolic pathways to modulate granuloma formation and function, proposing new treatment avenues for conditions characterized by chronic inflammation and granuloma formation. This work contributes significantly to the understanding of immune regulation and chronic inflammation, presenting avenues for future research and therapy in granulomatous diseases.

Immunometabolism in Sepsis

Immunometabolism is a fascinating field of research that investigates the interactions between metabolic processes and the immune response. This intricate connection plays a pivotal role in regulating inflammatory reactions and consequently exerts a significant impact on the course of sepsis. The proinflammatory response during an immune reaction is closely tied to a high energy demand in immune cells. As a result, proinflammatory immune cells rapidly require substantial amounts of energy in the form of ATP, necessitating a fundamental and swift shift in their metabolism, i.e., their means of generating energy. This entails a marked increase in glycolysis within the proinflammatory response, thereby promptly meeting the energy requirements and providing essential metabolic building blocks for the biosynthesis of macromolecules. Alongside glycolysis, there is heightened activity in the pentose phosphate pathway (PPP). The PPP significantly contributes to NADPH production within the cell, thus maintaining redox equilibrium. Elevated PPP activity consequently leads to an increased NADPH level, resulting in enhanced production of reactive oxygen species (ROS) and nitric oxide (NO). While these molecules are crucial for pathogen elimination, an excess can also induce tissue damage. Simultaneously, there are dual interruptions in the citric acid cycle. In the cellular resting state, the citric acid cycle acts as a sort of "universal processor", where metabolic byproducts of glycolysis, fatty acid breakdown, and amino acid degradation are initially transformed into NADH and FADH2, subsequently yielding ATP. While the citric acid cycle and its connected oxidative phosphorylation predominantly generate energy at rest, it becomes downregulated in the proinflammatory phase of sepsis. The two interruptions lead to an accumulation of citrate and succinate within cells, reflecting mitochondrial dysfunction. Additionally, the significantly heightened glycolysis through fermentation yields lactate, a pivotal metabolite for sepsis diagnosis and prognosis. Conversely, cells in an anti-inflammatory state revert to a metabolic profile akin to the resting state: Glycolysis is attenuated, PPP is suppressed, and the citric acid cycle is reactivated. Of particular interest is that not only does the immune reaction influence metabolic pathways, but this connection also operates in reverse. Thus, modulation of metabolic pathways also modulates the immunity of the corresponding cell and thereby the state of the immune system itself. This could potentially serve as an intriguing avenue in sepsis therapy.

Deciphering the metabolic heterogeneity of hematopoietic stem cells with single-cell resolution

Metabolic status is crucial for stem cell functions; however, the metabolic heterogeneity of endogenous stem cells has never been directly assessed. Here, we develop a platform for high-throughput single-cell metabolomics (hi-scMet) of hematopoietic stem cells (HSCs). By combining flow cytometric isolation and nanoparticle-enhanced laser desorption/ionization mass spectrometry, we routinely detected >100 features from single cells. We mapped the single-cell metabolomes of all hematopoietic cell populations and HSC subpopulations with different division times, detecting 33 features whose levels exhibited trending changes during HSC proliferation. We found progressive activation of the oxidative pentose phosphate pathway (OxiPPP) from dormant to active HSCs. Genetic or pharmacological interference with OxiPPP increased reactive oxygen species level in HSCs, reducing HSC self-renewal upon oxidative stress. Together, our work uncovers the metabolic dynamics during HSC proliferation, reveals a role of OxiPPP for HSC activation, and illustrates the utility of hi-scMet in dissecting metabolic heterogeneity of immunophenotypically defined cell populations.

Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit (CLPP) As a Novel Mediator of, and Target in High Risk Multiple Myeloma

Background: Up to 30% of newly diagnosed myeloma (NDMM) patients have high-risk disease characterized by increased proliferation, resistance to apoptosis, focal and bone marrow-independent growth, and intraclonal heterogeneity. High-risk can be defined by fluorescence in situ hybridization (FISH) features including 17p deletion, translocation (t)(4;14), t(14;16), or t(14;20), and gain of 1q. However, some standard risk patients also exhibit high-risk characteristics, indicating that identifying novel targetable mediators of this phenotype is of interest. Methods: We conducted single cell RNA-Sequencing (scRNA-Seq) of CD138 + monoclonal/neoplastic and polyclonal/normal plasma cells (PCs) from 72 individuals across the plasma cell dyscrasia spectrum and 3 healthy controls to identify differentially expressed genes associated with myeloma pathobiology. Also, we leveraged publicly available gene expression profiling (GEP) databases, and performed pre-clinical studies both in vitro and in vivo. Results: CLPP was expressed with increased frequency in NDMM and relapsed/refractory myeloma patients compared to the precursors monoclonal gammopathy of undetermined significance and smoldering myeloma (GSE6477; p=4.03x10 -14). Notably, CLPP is the proteolytic component, along with CLPX, of the CLP complex which cleaves peptides and proteins in an ATP-dependent manner and is associated with the inner mitochondrial membrane. The GEP data were confirmed in our scRNA-Seq database, which showed CLPP over-expression occurred preferentially in each patient's monoclonal versus their own polyclonal PCs. Moreover, analysis of the Broad Institute Cancer Cell Line Encyclopedia revealed MM cells had the highest mean CLPP expression of any tumor cell type. In the NDMM CoMMpass database, high CLPP levels were associated with markers of increased disease burden (beta-2-microglobulin (p<0.001), calcium (p=0.0027), circulating plasma cells (p<0.001), marrow plasmacytosis (p<0.001), and lactate dehydrogenase (p=0.0025)). Moreover, these patients showed a significantly shorter progression-free and overall survival (both p<0.0001), and did not cluster in any of the Arkansas GEP70-defined myeloma subtypes. In MM.1S and H929 myeloma cells, specific CLPP knockdown with an inducible shRNA decreased cell proliferation and activated apoptosis as shown by increased Annexin V positivity. Similarly, pharmacologic CLPP inhibition produced a time- and concentration-dependent induction of apoptosis in a panel of cell lines representing myeloma molecular subtypes and drug resistant phenotypes. Ectopic CLPP overexpression promoted myeloma organoid growth, while its suppression had the opposite effect. Functional analysis revealed that CLPP knockdown or inhibition impaired cell proteolytic activity, mitochondrial transmembrane potential, and reduced adhesion to stromal cells while inducing reactive oxygen species and autophagy. To further pursue the underlying molecular mechanisms, we first examined the net downstream effects of CLPP knockdown and inhibition by mass spectrometry followed by mapping to the KEGG database. Recurrently up-regulated pathways included the mitochondrial unfolded protein response (UPR mt), lysosome transport, and autophagy utilizing and metabolic processes. In contrast, down-regulated pathways included oxidative phosphorylation, c-Myc signaling, RNA polymerase I-directed transcription, DNA replication and elongation, and the G2M checkpoint. Next, our metabolomics analysis revealed a significant attenuation of the Warburg effect in myeloma cells upon CLPP inhibition consistent with reduced glycolysis and pentose phosphate pathway activity. Notably, mitochondrial ATP production was significantly reduced, as confirmed by a seahorse XF Real-time ATP Rate assay. Finally, in vivo experiments demonstrated that pharmacologic CLPP inhibition impaired myeloma growth in an MM.1S-based xenograft model ( Figure). Conclusions: These pre-clinical data support the hypothesis that CLPP represents a novel mediator of high-risk behavior in multiple myeloma, demonstrate that its targeting results in mitochondrial dysfunction that attenuates myeloma growth and survival, and support development of pharmacologic CLPP inhibitors to target myeloma.

Nicotinamide riboside cardioprotection is mediated through glycolysis activation

Abstract Purpose Low NAD+ levels are observed in pathologies such as ageing, diabetes, heart failure and ischemia-reperfusion (IR) injury. Nicotinamide riboside (NR) has emerged as promising therapeutic NAD+ precursor [1,2]. NR was recently reported to protect against in vivo cardiac IR injury [3], but underlying mechanisms of protection were unexplored. Here we used the isolated mouse heart allowing metabolic control to examine NR’s metabolic mechanisms of cardiac protection. Methods Isolated C57BL/6N hearts were Langendorff-perfused with 5.5 mM glucose and 0.2 mM fatty acids (FA), and subjected to 35 min global ischemia and 90 min reperfusion. NR (50 mg/L) or vehicle were administered for 25 min before ischemia, and during the first 25 min of reperfusion. Cardiac functional recovery and cell death parameters (lactate dehydrogenase (LDH) release, % infarct size) were determined. To examine NR’s effects on cardiac metabolism, hearts were perfused with 5.5 mM 13C-glucose and 0.2 mM FA and analyzed by metabolomics and fluxomics using LC-MS techniques before ischemia. The dependency of NR’s protection on glycolysis was tested in hearts perfused with glucose replaced by pyruvate and lactate ("no glycolysis" hearts). Finally, NR’s protection and effects on cardiac metabolism were also examined in "high glycolysis" hearts, by adding insulin to the perfusate. Results NR reduced infarct size, LDH release and improved cardiac functional recovery for glucose/FA perfused hearts, demonstrating NR’s cardioprotection. In these glucose/FA hearts, metabolomics analysis showed NR increased NAD+, glycolytic intermediate phosphoenolpyruvate (PEP), Krebs cycle intermediate succinate and pentose phosphate pathway (PPP) intermediates ribose-5P (R5P) and sedoheptulose-7P (S7P) (Fig. 1). Fluxomics analysis demonstrated that NR’s protection was associated with activated glycolysis, without changes in tricarboxylic acid (TCA) cycle or PPP activities. No 13C-labeling of S7P was observed (Fig. 1). NR protection was lost in the "no glycolysis" hearts. In the "high glycolysis" hearts, NR was unable to protect the heart and increase glycolysis and reduced anaerobic glycolysis and ATP with increases in AMP, indicative of NR-induced energetic stress (Fig. 2). Remarkably, in these insulin perfused hearts, NR still increased PPP intermediates, with high 13C-labeling of S7P (Fig. 2). Conclusions NR’s protection against IR injury is only present in hearts with low glycolysis, associated with activation of pre-ischemic glycolysis. When activation of glycolysis was prevented, through either examining "no glycolysis" hearts or "high glycolysis" hearts, NR protection was abolished. NR treatment increased pre-ischemic PPP intermediates in either low or high glycolysis hearts, whereby insulin was necessary to incorporate 13C-glucose into S7P. The data suggest that NR’s acute cardioprotective effects are mediated through the activation of glycolysis.NR effects in low-glycolysis heartsNR effects in high-glycolysis hearts

IL-6/JAK2-dependent G6PD phosphorylation promotes nucleotide synthesis and supports tumor growth

ObjectiveTumor cells hijack inflammatory mechanisms to promote their own growth. IL-6 is one of the major cytokines, and is frequently upregulated in tumors. The pentose phosphate pathway (PPP) generates the indispensable building blocks to produce various nucleotides. Here we aimed to determine whether and how PPP is timely tuned in response to IL-6 to support tumor growth. MethodsProtein expression was examined by immunoblot. Protein interaction was examined by immunoprecipitation. Tumor cell proliferation in in vitro culture was examined by BrdU assay and colony formation assay. Tumor cell proliferation in mouse xenograft model was examined by Ki-67 staining. ResultsHere we show that the metabolic flux of PPP and enzymatic activity of glucose-6-phosphate dehydrogenase (G6PD) is rapidly induced under IL-6 treatment, without obvious changes in G6PD expression level. Mechanistically, Janus kinase 2 (JAK2) phosphorylates G6PD Y437 under IL-6 treatment, which accentuates G6PD enzymatic activity by promoting G6PD binding with its substrate G6P. Further, JAK2-dependent G6PD Y437 phosphorylation is required for IL-6-induced nucleotide biosynthesis and tumor cell proliferation, and is associated with the progression of oral squamous cell carcinoma. ConclusionsOur findings report a new mechanism implicated in the crosstalk between tumor cells and inflammatory microenvironment, by which JAK2-dependent activation of G6PD governs nucleotide synthesis to support tumor cell proliferation, thereby highlighting its value as a potential anti-tumor target.

Insulin-induced gene 2 protects against hepatic ischemia–reperfusion injury via metabolic remodeling

BackgroundHepatic ischemia–reperfusion (IR) injury is the primary reason for complications following hepatectomy and liver transplantation (LT). Insulin-induced gene 2 (Insig2) is one of several proteins that anchor the reticulum in the cytoplasm and is essential for metabolism and inflammatory responses. However, its function in IR injury remains ambiguous.MethodsInsig2 global knock-out (KO) mice and mice with adeno-associated-virus8 (AAV8)-delivered Insig2 hepatocyte-specific overexpression were subjected to a 70% hepatic IR model. Liver injury was assessed by monitoring hepatic histology, inflammatory responses, and apoptosis. Hypoxia/reoxygenation stimulation (H/R) of primary hepatocytes and hypoxia model induced by cobalt chloride (CoCl2) were used for in vitro experiments. Multi-omics analysis of transcriptomics, proteomics, and metabolomics was used to investigate the molecular mechanisms underlying Insig2.ResultsHepatic Insig2 expression was significantly reduced in clinical samples undergoing LT and the mouse IR model. Our findings showed that Insig2 depletion significantly aggravated IR-induced hepatic inflammation, cell death and injury, whereas Insig2 overexpression caused the opposite phenotypes. The results of in vitro H/R experiments were consistent with those in vivo. Mechanistically, multi-omics analysis revealed that Insig2 is associated with increased antioxidant pentose phosphate pathway (PPP) activity. The inhibition of glucose-6-phosphate-dehydrogenase (G6PD), a rate-limiting enzyme of PPP, rescued the protective effect of Insig2 overexpression, exacerbating liver injury. Finally, our findings indicated that mouse IR injury could be attenuated by developing a nanoparticle delivery system that enables liver-targeted delivery of substrate of PPP (glucose 6-phosphate).ConclusionsInsig2 has a protective function in liver IR by upregulating the PPP activity and remodeling glucose metabolism. The supplementary glucose 6-phosphate (G6P) salt may serve as a viable therapeutic target for alleviating hepatic IR.

High pyroptosis activity in pancreatic adenocarcinoma: poor prognosis and oxaliplatin resistance.

Pyroptosis, as a type of inflammatory programmed cell death, has been studied in inflammatory diseases and numerous cancers but its role in pancreatic ductal adenocarcinoma (PDAC) remains further exploration. A TCGA-PDAC cohort was enrolled for bioinformatics analysis to investigate the effect of pyroptosis on the prognosis and drug sensitivity of patients. PA-TU-8988T and CFPAC-1 cells were selected for investigating the role of GSDMC in PDAC. A distinct classification pattern of PDAC mediated by 21 pyroptosis-related genes (PRGs) was identified. It was suggested that higher pyroptosis activity was associated with poor prognosis of patients and higher tumor proliferation rates. We further established a prognostic model based on three PRGs (GSDMC, CASP4 and NLRP1) and the TCGA-PDAC cohort was classified into low and high-risk subgroups. It is noteworthy that the high-risk group showed significantly higher tumor proliferation rates and was proved to be highly correlated with oxaliplatin resistance. Further experiments suggested that overexpression of GSDMC promoted the proliferation and oxaliplatin resistance of PA-TU-8988T cells in vitro and vivo, while downregulation of GSDMC showed opposite effects in CFPAC-1 cells. Finally, we found that the activation of pentose phosphate pathway (PPP) was the mechanism by which GSDMC overexpression promoted the proliferation and oxaliplatin resistance of pancreatic cancer cells. In this study, we found that higher pyroptosis activity is associated with worse prognosis and oxaliplatin resistance of PDAC patients. In addition, as a core effector of pyroptosis, GSDMC promoted proliferation and oxaliplatin resistance of pancreatic cancer cells, which will provide new therapeutic target for PDAC patients.

Glucose and redox metabolism in meiotically blocked in vitro grown mouse antral follicles.

Glucose and redox metabolism characterization in mouse antral follicles with meiotically blocked oocytes, after in vitro follicle culture (IFC) from the early secondary stage. Following IFC (10 days), oocytes, corresponding cumulus (CC), and granulosa cells (GC) were collected from antral follicles: (i) on day 9-immature, germinal vesicle (GV) stage; (ii) on day 10, after hCG/EGF stimulation-mature, metaphase II (MII) stage and meiotically blocked (MB) immature GV stage. The metabolic profiles of all samples (GV, MII, and MB) were compared by measuring changes in metabolites involved in glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), and redox activity via enzymatic spectrophotometric assays in each cell type. Within MB follicles, GCs drive higher levels of glycolysis and lactic acid fermentation (LAF) while oocytes exert more PPP activity. MB-oocytes had significantly larger diameters compared to day 9 GVs. MB follicles revealed limited metabolic changes in the somatic compartment compared to their GV counterparts (before stimulation). MB-CCs showed increased aconitase and glucose-6-phosphate dehydrogenase activities with lower malate levels comparted to GV-CCs. MB and MII in vitro grown follicles displayed comparable metabolic profiles, suggesting culture induces metabolic exhaustion regardless of the maturation stage. Current results suggest that in addition to impaired nuclear maturation, metabolic disruption is present in MB follicles. MB follicles either compensate with high levels of TCA cycle and PPP activities in CCs, or are unable to drive proper levels of aerobic metabolism, which might be due to the current culture conditions.

Integrated transcriptomics and metabolomics reveal protective effects on heart of hibernating Daurian ground squirrels.

Hibernating mammals are natural models of resistance to ischemia, hypoxia-reperfusion injury, and hypothermia. Daurian ground squirrels (spermophilus dauricus) can adapt to endure multiple torpor-arousal cycles without sustaining cardiac damage. However, the molecular regulatory mechanisms that underlie this adaptive response are not yet fully understood. This study investigates morphological, functional, genetic, and metabolic changes that occur in the heart of ground squirrels in three groups: summer active (SA),late torpor (LT),and interbout arousal (IBA).Morphological and functional changes in the heart were measured using hematoxylin-eosin (HE)staining, Masson staining, echocardiography, and enzyme-linked immunosorbent assay (ELISA).Results showed significant changes in cardiac function in the LT group as compared with SA or IBA groups, but no irreversible damage occurred. To understand the molecular mechanisms underlying these phenotypic changes, transcriptomic and metabolomic analyses were conducted to assess differential changes in gene expression and metabolite levels in the three groups of ground squirrels, with a focus on GOand KEGG pathway analysis. Transcriptomic analysis showed that differentially expressed genes were involved in the remodeling of cytoskeletal proteins, reduction in protein synthesis, and downregulation of the ubiquitin-proteasome pathway during hibernation (including LT and IBA groups), as compared with the SA group. Metabolomic analysis revealed increased free amino acids, activation of the glutathione antioxidant system, altered cardiac fatty acid metabolic preferences, and enhanced pentose phosphate pathway activity during hibernation as compared with the SA group. Combining the transcriptomic and metabolomic data, active mitochondrial oxidative phosphorylation and creatine-phosphocreatine energy shuttle systems were observed, as well as inhibition of ferroptosis signaling pathways during hibernation as compared with the SA group. In conclusion, these results provide new insights into cardio-protection in hibernators from the perspective of gene and metabolite changes and deepen our understanding of adaptive cardio-protection mechanisms in mammalian hibernators.

Effect of fatty acids on intracellular pneumocandin B0 storage in the fermentation of Glarea lozoyensis

The natural product pneumocandin B0 is the precursor of the antifungal drug caspofungin. To explore the relationship between pneumocandin B0 and oil. We found that the addition of 1 g/L of oil to the fermentation medium is more conducive to the production of pneumocandin B0. The metabolic reaction mechanism was explored using different fatty acids and the results showed that stearic acid and acetic acid increased the total production of pneumocandin B0 by 22.98% and 9.08%, respectively, as well as increasing the content of intracellular lipid droplets. We also analyzed gene expression and pathway differences between the two different fatty acids using transcriptome analyses. The addition of both acetic acid and stearic acid promoted an active pentose phosphate pathway, providing cells with higher intracellular reducing power. We found that the addition of fatty acids can lead to lipid accumulation, and lipid droplets can sequester lipophilic secondary metabolites such as pneumocandin B0 to reduce cell damage. These results provide novel insights into the relationship between pneumocandin B0 biosynthesis and fatty acids in G. lozoyensis. In addition, this study provides important genetic information for improving the yield of pneumocandin B0 through a strategy of metabolic engineering in the future.Graphical

Metabolic reprogramming in Nrf2-driven proliferation of normal rat hepatocytes.

Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.

Neutrophil metabolomics in severe COVID-19 reveal GAPDH as a suppressor of neutrophil extracellular trap formation

Severe COVID-19 is characterized by an increase in the number and changes in the function of innate immune cells including neutrophils. However, it is not known how the metabolome of immune cells changes in patients with COVID-19. To address these questions, we analyzed the metabolome of neutrophils from patients with severe or mild COVID-19 and healthy controls. We identified widespread dysregulation of neutrophil metabolism with disease progression including in amino acid, redox, and central carbon metabolism. Metabolic changes in neutrophils from patients with severe COVID-19 were consistent with reduced activity of the glycolytic enzyme GAPDH. Inhibition of GAPDH blocked glycolysis and promoted pentose phosphate pathway activity but blunted the neutrophil respiratory burst. Inhibition of GAPDH was sufficient to cause neutrophil extracellular trap (NET) formation which required neutrophil elastase activity. GAPDH inhibition increased neutrophil pH, and blocking this increase prevented cell death and NET formation. These findings indicate that neutrophils in severe COVID-19 have an aberrant metabolism which can contribute to their dysfunction. Our work also shows that NET formation, a pathogenic feature of many inflammatory diseases, is actively suppressed in neutrophils by a cell-intrinsic mechanism controlled by GAPDH.

Temporal changes in glucose metabolism reflect polarization in resident and monocyte-derived macrophages after myocardial infarction.

Metabolic reprogramming from glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation may mediate macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We hypothesized that changes in cardiac macrophage glucose metabolism would reflect polarization status after myocardial infarction (MI), ranging from the early inflammatory phase to the later wound healing phase. MI was induced by permanent ligation of the left coronary artery in adult male C57BL/6J mice for 1 (D1), 3 (D3), or 7 (D7) days. Infarct macrophages were subjected to metabolic flux analysis or gene expression analysis. Monocyte versus resident cardiac macrophage metabolism was assessed using mice lacking the Ccr2 gene (CCR2 KO). By flow cytometry and RT-PCR, D1 macrophages exhibited an M1 phenotype while D7 macrophages exhibited an M2 phenotype. Macrophage glycolysis (extracellular acidification rate) was increased at D1 and D3, returning to basal levels at D7. Glucose oxidation (oxygen consumption rate) was decreased at D3, returning to basal levels at D7. At D1, glycolytic genes were elevated (Gapdh, Ldha, Pkm2), while TCA cycle genes were elevated at D3 (Idh1 and Idh2) and D7 (Pdha1, Idh1/2, Sdha/b). Surprisingly, Slc2a1 and Hk1/2 were increased at D7, as well as pentose phosphate pathway (PPP) genes (G6pdx, G6pd2, Pgd, Rpia, Taldo1), indicating increased PPP activity. Macrophages from CCR2 KO mice showed decreased glycolysis and increased glucose oxidation at D3, and decreases in Ldha and Pkm2 expression. Administration of dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, robustly decreased pyruvate dehydrogenase phosphorylation in the non-infarcted remote zone, but did not affect macrophage phenotype or metabolism in the infarct zone. Our results indicate that changes in glucose metabolism and the PPP underlie macrophage polarization following MI, and that metabolic reprogramming is a key feature of monocyte-derived but not resident macrophages.

ATP13A2 activates the pentose phosphate pathway to promote colorectal cancer growth though TFEB-PGD axis.

The pentose phosphate pathway (PPP) is an important mechanism by which tumour cells resist stressful environments and maintain malignant proliferation. However, the mechanism by which the PPP regulates these processes in colorectal cancer (CRC) remains elusive. Closely related PPP genes were obtained from the TCGA and GEO databases. The effect of ATP13A2 on CRC cell proliferation was evaluated by performing in vitro assays. The connection between the PPP and ATP13A2 was explored by assessing proliferation and antioxidative stress. The molecular mechanism by which ATP13A2 regulates the PPP was investigated using chromatin immunoprecipitation and dual luciferase experiments. The clinical therapeutic potential of ATP13A2 was explored using patient-derived xenograft (PDX), patient-derived organoid (PDO) and AOM/DSS models. We identified ATP13A2 as a novel PPP-related gene. ATP13A2 deficiency inhibited CRC growth and PPP activity, as manifested by a decrease in the levels of PPP products and an increase in reactive oxygen species levels, whereas ATP13A2 overexpression induced the opposite effect. Mechanistically, ATP13A2 regulated the PPP mainly by affecting phosphogluconate dehydrogenase (PGD) mRNA expression. Subsequent studies showed that ATP13A2 overexpression promoted TFEB nuclear localization by inhibiting the phosphorylation of TFEB, thereby enhancing the transcription of PGD and ultimately affecting the activity of the PPP. Finally, ATP13A2 knockdown inhibited CRC growth in PDO and PDX models. ATP13A2- /- mice had a lower CRC growth capacity than ATP13A2+/+ in the AOM/DSS model.Our findings revealed that ATP13A2 overexpression-driven dephosphorylation of TFEB promotes PPP activation by increasing PGD transcription, suggesting that ATP13A2 may serve as a potential target for CRC therapy.

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.

A High-Throughput Sequencing Data-Based Classifier Reveals the Metabolic Heterogeneity of Hepatocellular Carcinoma.

Metabolic heterogeneity plays a key role in poor outcomes in malignant tumors, but its role in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we aim to disentangle the metabolic heterogeneity features of HCC by developing a classification system based on metabolism pathway activities in high-throughput sequencing datasets. As a result, HCC samples were classified into two distinct clusters: cluster 1 showed high levels of glycolysis and pentose phosphate pathway activity, while cluster 2 exhibited high fatty acid oxidation and glutaminolysis status. This metabolic reprogramming-based classifier was found to be highly correlated with several clinical variables, including overall survival, prognosis, TNM stage, and 𝛼-fetoprotein (AFP) expression. Of note, activated oncogenic pathways, a higher TP53 mutation rate, and increased stemness were also observed in cluster 1, indicating a causal relationship between metabolic reprogramming and carcinogenesis. Subsequently, distinct metabolism-targeted therapeutic strategies were proven in human HCC cell lines, which exhibit the same metabolic properties as corresponding patient samples based on this classification system. Furthermore, the metabolic patterns and effects of different types of cells in the tumor immune microenvironment were explored by referring to both bulk and single-cell data. It was found that malignant cells had the highest overall metabolic activities, which may impair the anti-tumor capacity of CD8+ T cells through metabolic competition, and this provided a potential explanation for why immunosuppressive cells had higher overall metabolic activities than those with anti-tumor functions. Collectively, this study established an HCC classification system based on the gene expression of energy metabolism pathways. Its prognostic and therapeutic value may provide novel insights into personalized clinical practice in patients with metabolic heterogeneity.