Increase In Glycolytic Flux Research Articles

Disrupting Na+ ion homeostasis and Na+/K+ ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo

BackgroundGlycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells.MethodsLive whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]d-glucose to [2H]-labelled l-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET).ResultsIntracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected.ConclusionsGlycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the ‘centrality of the [Na+]i-NKA nexus’ in the mechanistic basis of the Warburg effect.

A transcriptomic analysis of correlation between mitochondrial function and energy metabolism remodeling in mice with myocardial fibrosis following myocardial infarction

To investigate the changes of mitochondrial respiratory function during myocardial fibrosis in mice with myocardial infarction (MI) and its correlation with the increase of glycolytic flux. Forty C57BL/6N mice were randomized into two equal groups to receive sham operation or ligation of the left anterior descending coronary artery to induce acute MI. At 28 days after the operation, 5 mice from each group were euthanized and left ventricular tissue samples were collected for transcriptomic sequencing. FPKM method was used to calculate gene expression levels to identify the differentially expressed genes (DEGs) in MI mice, which were analyzed using GO and KEGG databases to determine the pathways affecting the disease process. Heat maps were drawn to show the differential expressions of the pathways and the related genes in the enrichment analysis. In primary cultures of neonatal mouse cardiac fibroblasts (CFs), the changes in mitochondrial respiration and glycolysis levels in response to treatment with the pro-fibrotic agonist TGF-β1 were analyzed using Seahorse experiment. The mouse models of MI showed significantly increased diastolic and systolic left ventricular diameter (P < 0.05) and decreased left ventricular ejection fraction (P < 0.0001). A total of 124 up-regulated and 106 down-regulated DEGs were identified in the myocardial tissues of MI mice, and GO and KEGG enrichment analysis showed that these DEGs were significantly enriched in fatty acid metabolism, organelles and other metabolic pathways and in the mitochondria. Heat maps revealed fatty acid beta oxidation, mitochondrial dysfunction and increased glycolysis levels in MI mice. In the primary culture of CFs, treatment with TGF-β1 significantly reduced the basal and maximum respiratory levels and increased the basal and maximum glycolysis levels (P < 0.0001). During myocardial fibrosis, energy metabolism remodeling occurs in the CFs, manifested by lowered mitochondrial function and increased energy generation through glycolysis.

Knockdown of RGMA improves ischemic stroke via Reprogramming of Neuronal Metabolism

Neuronal energy metabolism dysregulation is involved in various pathologies of Ischemia-reperfusion (I/R), yet the role of RGMA in neuronal metabolic reprogramming has not been reported. In this study, we found that RGMA expression significantly increased after I/R, and compared to control mice, mice with MCAO/R showed an increase in glycolytic metabolic products and the expression of glycolytic pathway proteins. Furthermore, RGMA levels are closely related to neuronal energy metabolism. We discovered that knockdown of RGMA can shift neuronal energy metabolism towards oxidative phosphorylation and the pentose phosphate pathway, thereby protecting mice from ischemic reperfusion injury. Mechanistically, knockdown of RGMA can downregulate PGK1 expression, reducing the increase in glycolytic flux following ischemia reperfusion. Moreover, we found that knockdown of RGMA can reduce the interaction between USP10 and PGK1, thus affecting the ubiquitination degradation of PGK1. In summary, our data suggest that RGMA may regulate neuronal energy metabolism by inhibiting the USP10-mediated deubiquitination of PGK1, thus protecting it from I/R injury. This study provides new ideas for clarifying the intrinsic mechanism of neuronal damage after I/R.

Cell-free ascites from ovarian cancer patients induces Warburg metabolism and cell proliferation through TGFβ-ERK signaling

Ascites plays a key role in supporting the metastatic potential of ovarian cancer cells. Shear stress and carry-over of cancer cells by ascites flow support carcinogenesis and metastasis formation. In addition, soluble factors may participate in the procarcinogenic effects of ascites in ovarian cancer. This study aimed to determine the biological effects of cell-free ascites on carcinogenesis in ovarian cancer cells. Cell-free ascites from ovarian cancer patients (ASC) non-selectively induced cell proliferation in multiple models of ovarian cancer and untransformed primary human dermal fibroblasts. Furthermore, ASC induced a Warburg-type rearrangement of cellular metabolism in A2780 ovarian cancer cells characterized by increases in cellular oxygen consumption and glycolytic flux; increases in glycolytic flux were dominant. ASC induced mitochondrial uncoupling and fundamentally reduced fatty acid oxidation. Ascites-elicited effects were uniform among ascites specimens. ASC-elicited transcriptomic changes in A2780 ovarian cancer cells included induction of the TGFβ-ERK/MEK pathway, which plays a key role in inducing cell proliferation and oncometabolism. ASC-induced gene expression changes, as well as the overexpression of members of the TGFβ signaling system, were associated with poor survival in ovarian cancer patients. We provided evidence that the activation of the autocrine/paracrine of TGFβ signaling system may be present in bladder urothelial carcinoma and stomach adenocarcinoma. Database analysis suggests that the TGFβ system may feed forward bladder urothelial carcinoma and stomach adenocarcinoma. Soluble components of ASC support the progression of ovarian cancer. These results suggest that reducing ascites production may play an essential role in the treatment of ovarian cancer by inhibiting the progression and reducing the severity of the disease.

Radiation-induced changes in energy metabolism result in mitochondrial dysfunction in salivary glands

Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.

Glycometabolic reprogramming in cementoblasts: A vital target for enhancing cell mineralization.

Cementum, a constituent part of periodontal tissues, has important adaptive and reparative functions. It serves to attach the tooth to alveolar bone and acts as a barrier delimit epithelial growth and bacteria evasion. A dynamic and highly responsive cementum is essential for maintaining occlusal relationships and the integrity of the root surface. It is a thin layer of mineralized tissue mainly produced by cementoblasts. Cementoblasts are osteoblast-like cells essential for the restoration of periodontal tissues. In recent years, glucose metabolism has been found to be critical in bone remodeling and osteoblast differentiation. However, the glucose metabolism of cementoblasts remains incompletely understood. First, immunohistochemistry staining and in vivo tracing with 18 F-fluorodeoxyglucose (18 F-FDG) revealed significantly higher glucose metabolism in cementum formation. To test the bioenergetic pathways of cementoblast differentiation, we compared the bioenergetic profiles of mineralized and unmineralized cementoblasts. As a result, we observed a significant increase in the consumption of glucose and production of lactate, coupled with the higher expression of glycolysis-related genes. However, the expression of oxidative phosphorylation-related genes was downregulated. The verified results were consistent with the RNA sequencing results. Likewise, targeted energy metabolomics shows that the levels of glycolytic metabolites were significantly higher in the mineralized cementoblasts. Seahorse assays identified an increase in glycolytic flux and reduced oxygen consumption during cementoblast mineralization. Apart from that, we also found that lactate dehydrogenase A (LDHA), a key glycolysis enzyme, positively regulates the mineralization of cementoblasts. In summary, cementoblasts mainly utilized glycolysis rather than oxidative phosphorylation during the mineralization process.

Abstract P3-07-21: Therapeutic efficacy of NGY-091 against triple negative breast cancer is mediated by direct cell killing and activation of antitumor immunity

Abstract Aberrant metabolic reprogramming is known to drive triple negative breast cancer progression and metastasis. The increase in glycolytic flux in cancer cells creates a lactate-rich tumor microenvironment (TME) that is exploited by tumors to their advantage by activating immunosuppressive cell populations, such as Tregs and MDSCs, that thrive on lactate as a fuel source. Therefore, blockade of lactate export from glycolytic cancer cells while inhibiting lactate entry into suppressive immune cells is a novel therapeutic strategy to treat cancer. Lactate transport in cells is predominantly mediated by MCT1 and MCT4 transporters. We have developed a compound, NGY-091, that is a first-in-class small molecule dual inhibitor of the MCT1 and MCT4 lactate transporters. NGY-091 treatment exhibited a potent in vitro cytotoxicity against breast cancer cells with various levels of MCT1 and MCT4 expressions. The on-target activity of NGY-091 was validated by measuring intracellular and extracellular lactate levels. NGY-091 strongly blocked MCT1 mediated lactate import and lactate export through MCT4 in vitro. Furthermore, a direct in vivo tumor cell killing was evident in human TNBC CDX (MDA MB 231) and PDX models with the treatment of NGY-091. In a syngeneic model of 4T1, we observed a significant reduction in tumor growth and synergistic tumor regression when combined with immune checkpoint blockade therapy. Profiling of lymphoid and myeloid cells in NGY-091 treated tumors by flow cytometry revealed a significant alteration in immune architecture suggesting activation of antitumor immunity. NGY-091 treated tumors induced a profound increase in effector T cell populations, CD8/Treg ratio and tumor-suppressive M1 macrophages while significantly downregulating M2 macrophages. To further investigate if NGY-091 directly alters immune cell activation and functionality in vitro, we treated CD4 T, CD8 T, Tregs and MDSCs in a lactate-rich culture condition mimicking lactate level in the TME. NGY-091 treatment strongly increased effector CD4 and CD8 T cells while significantly reducing suppressive function of Treg and MDSCs in vitro. These findings indicate the direct effect of NGY-091 on immune cell and validate the in vivo observations in 4T1 tumors. Therefore, NGY-091 intervenes two key hallmarks of cancer – metabolism and immunity and provides a novel avenue to therapeutically target aggressive breast cancer. Citation Format: Sambad Sharma, Sanath Wijerathna, Kerui Wu, Abhishek Tyagi, Shih-Ying Wu, Kounosuke Watabe, Nelly Kuklin, Jaime Escobedo, Vincent Sandanayaka. Therapeutic efficacy of NGY-091 against triple negative breast cancer is mediated by direct cell killing and activation of antitumor immunity [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 P3-07-21.

Abstract TMP74: HIF-1a And MTORC2 Co-regulate Macrophage Inflammation And Metabolism In Intracerebral Hemorrhage

Introduction: Recruited macrophages are known to spur secondary inflammation-induced damage in intracerebral hemorrhage (ICH), and targeted metabolic modulation of these macrophages could limit inflammation. Recent macrophage transcriptomic work has associated increased expression of HIF-1a, master regulator of the hypoxia response, and mTORC2, the more recently-discovered mTOR kinase complex, with good patient outcomes. The goal of this work was to identify the inflammatory and metabolic effects of these factors, providing insight into their putatively beneficial role in acute brain injury. Methods: Human monocyte-derived macrophages were stimulated either with ICH-associated danger signals (DAMP) (S100A9 (2 ug/mL, R&amp;D) + IL-1β (10 ng/mL, Biolegend)), or 1 ng/mL LPS (Invivogen). Echinomycin (Sigma Aldrich), NR1 (Aobious), JR-AB2-011 (Aobious), and Torin-1 (LC Labs) were used to inhibit HIF-1, mTORC1, mTORC2, and mTOR signaling respectively. Cytokine levels were quantified using cytometric bead array (BD) and prostaglandin-E2 (PGE 2 ) levels were quantified using ELISA (Cayman). Metabolic flux was measured using the Seahorse platform (Agilent). Results/Conclusions: HIF-1a was necessary for the production of inflammatory and reparative cytokines, consistent with previous literature. Inhibition of mTORC1 or mTORC2 had little effect on cytokine levels, while broad inhibition of mTOR resulted in a step-wise decrease in chemokine secretion. mTORC2 was required for the production of PGE 2 (p&lt;0.01; Figure 1A), a lipid mediator highly expressed in ICH patients with good outcomes. This effect was dependent on HIF-1a signaling (p&lt;0.01; Figure 1A) and resulted in an increase in glycolytic flux (p&lt;0.001; Figure 1B), suggesting a neuroprotective mTORC2-HIF-PGE 2 axis. Future research will explore the role of HIF-1a and mTORC2 in murine ICH models using pharmacological inhibition and LysM-Cre HIF-1a-floxed mixed bone marrow chimeras.

Sec14l4 SNP Mice Have Increased Susceptibility to Oxidative Hemolysis through Modulation of G6PD and the Pentose Phosphate Pathway

Red blood cell function and recovery following transfusion is influenced by a variety of factors including sex, race, sickle cell trait and single nucleotide polymorphisms (SNPs). In collaboration with the REDS III program, we performed a genome wide association study identifying SNPs that are associated with in vitro osmotic, oxidative and spontaneous storage hemolysis stress. Sec14L4, was one of 27 genes identified with enhanced hemolysis, specifically following oxidative insult and later confirmed by an independent study to be associated with decreased hemoglobin increments following transfusion. SEC14L4 is an understudied, highly conserved phosphatidylinositol transfer protein that is predicted to reside with the Golgi Apparatus. While the function of SEC14L4 is not understood, it is clear that polymorphisms in this gene have clinical impact and understanding its role in red cell biology and transfusion is critical. We generated a novel humanized Sec14L4 SNP and knockout mouse model to assess RBC metabolism, function and transfusion outcomes following storage. While Sec14l4 KO mice have complete loss of SEC14L4 protein expression, the SNP has an approximate 50% decrease in expression, suggesting the mutation decreases protein and/or mRNA stability. Interestingly, knockout mice experience anemia with decreased hemoglobin levels and lower hematocrit at baseline. Following 12 days of storage (equivalent to 42 days of human blood storage) Sec14l4 knockout RBCs have increased hemolysis and are more susceptible to lysis following treatment with the oxidant diamide. Tracing experiments with 1,2,313C3-glucose from Sec14l4 SNP mouse RBCs reveals decreased levels of glucose-6-phosaphate dehydrogenase (G6PD) and depression of metabolic fluxes through the pentose phosphate pathway with lower levels of 13C3-6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP+), and reduced ADP and ATP. Compensatory increases in glycolytic flux was also observed. G6PD is the rate liming enzyme of the pentose phosphate pathway which is the sole producer of NADPH in the red blood cell. NADPH is an essential cofactor for glutathione reductase and peroxidase, the primary erythrocyte antioxidant enzymes. Red blood cells deficient for G6PD undergo acute hemolysis if exposed to oxidative stress due to decreased antioxidant capacity. These data suggest that red blood cells deficient in SEC14L4 phenocopy G6PD deficient red cells, possibly explaining increased susceptibility to oxidative insult. Lipidomic evaluation also reveled increased accumulation of oxidized lipids during storage (12-HETE and 15-HETE). Oxilipins are markers of poor post-transfusion recovery in stored mouse and human red blood cells. Together these data indicate that SEC14L4 is an important player in red blood cell stability during storage, oxidative stress, and following transfusion. How a Golgi resident protein influences the adult red cell is not yet understood but suggests a role for SEC14L4 in early erythrocyte development which has an effect over the lifetime of the cell, with the result of increased stability and antioxidant capacity. In support of this hypothesis, Sec14l4 is highly expressed in the bone marrow with decreasing expression as the red cell terminally differentiates. Future work will confirm proteomic, metabolomic and lipidomic findings and focus on how SEC14L4 may be regulating the G6PD pathway in mature red cells.

355 (PB135) - Cellular transporters as novel metabolic immune checkpoints: NGY-091, a small molecule dual MCT1/4 inhibitor for immuno oncology

Background: Aberrant metabolic reprogramming of malignant cells is known to drive cancer progression and metastasis. The increase in glycolytic flux in cancer cells creates a lactate-rich tumor microenvironment (TME), which tumors exploit by expanding immunosuppressive cell populations such as Tregs and MDSCs that thrive on lactate as a fuel source. Blockade of lactate export from glycolytic cancer cells, while inhibiting lactate uptake by suppressive immune cells, is a novel therapeutic strategy to treat cancer. Lactate transport in cells is predominantly mediated by MCT1 and MCT4 transporters. We have developed a compound, NGY-091, a first-in-class small molecule dual inhibitor of the MCT1 and MCT4 lactate transporters. Materials and Methods: The direct cytotoxic effect of NGY-091 was examined in multiple cancer cell lines. The on-target activity of NGY-091 was validated by measuring intracellular and extracellular lactate levels. in vivo efficacy of NGY-091 was studied using CDX, PDX and syngeneic tumor models. Immune profiling of tumor was performed 2 weeks after treating animals with NGY-091 by flow cytometry. Results: NGY-091 treatment exhibited a potent in vitro cytotoxicity against cancer cells with various levels of MCT1 and MCT4 expressions. NGY-091 strongly blocked lactate import through MCT1 under high lactate conditions and lactate export through MCT4 in high glucose conditions. NGY-091 treatment also inhibited pyruvate entry into the mitochondria through MPC1. Furthermore, direct in vivo tumor cell killing was evident in multiple human CDX and PDX models with the treatment of NGY-091. In syngeneic models of 4T1 and MC38 murine tumors, we observed significant tumor growth inhibition with NGY-091 treatment, and strong synergistic tumor reduction when treatment was combined with various immune checkpoint inhibitors. Profiling of lymphoid and myeloid cells in NGY-091 treated tumors by flow cytometry revealed a significant alteration in immune architecture suggesting activation of antitumor immunity. NGY-091 treated tumors induced a profound increase in effector T cell populations, CD8/Treg ratio, and tumor-suppressive M1 macrophages, while significantly downregulating M2 macrophages. To further investigate if NGY-091 directly alters immune cell activation and functionality in vitro, we treated CD4 T, CD8T, Tregs, and MDSCs in a lactate-rich culture condition, which mimicked the lactate level in the TME. NGY-091 treatment strongly increased effector CD4+ and CD8+ T cells, meanwhile it significantly reduced suppressive function of Treg and MDSCs in vitro. Conclusions: These findings indicate the direct effect of NGY-091 on immune cells and validate the in vivo observations in 4T1 tumors. Therefore, NGY-091 intervenes with two key hallmarks of cancer, metabolism and immunity and provides a novel modality to therapeutically target cancer. Conflict of interest: Ownership: Nirogy Therapeutics

Inflammation and apoptosis, two key events induced by hyperglycemia mediated reactive nitrogen species in RGC-5 cells

Nitrosative stress plays a critical role in retinal injury in high glucose (HG) environment of eye, but the mechanisms remain poorly understood. Here we tested the hypothesis that HG induced reactive nitrogen species (RNS) production acts as a key functional mediator of antioxidant depletion, mitochondrial dysfunction, biomolecule damage, inflammation and apoptosis. Our findings illustrated that exposure of cultured RGC-5 cells to HG significantly disrupts the antioxidant defense mechanism and mitochondrial machineries by increasing the loss of mitochondrial membrane potential (ΔѰM) and elevating mitochondrial mass. Furthermore, we used biochemical tools to analyze the changes in metabolites, sulfur amino acids (SAAs) such as L-glutathione (GSH) and L-cysteine (Cys), in the presence of HG environment. These metabolic changes were followed by an increase in glycolytic flux that is phosphofructokinase-2 (PFK-2) activity. Moreover, HG exposure results in a significant disruption of protein carbonylation (PC) and lipid peroxidation (LPO), downregulation of OGG1 and increase in 8-OHdG accumulations in RGC-5 cells. In addition, our results demonstrated that HG environment coinciding with increased expression of inflammatory mediators, cell cycle deregulation, decreased in cell viability and expression of FoxOs, increased lysosomal content leading to apoptosis. Pre-treatment of selective inhibitors of RNS significantly reduced the HG-induced cell cycle deregulation and apoptosis in RGC-5 cells. Collectively, these results illustrated that accumulated RNS exacerbates the antioxidant depletion, mitochondrial dysfunction, biomolecule damage, inflammation and apoptosis induced by HG exposure in RGC-5 cells. Treatment of pharmacological inhibitors attenuated the HG induced in retinal cells.

Increased glycolysis is an early consequence of palmitate lipotoxicity mediated by redox signaling

Exposure to toxic levels of fatty acids (lipotoxicity) leads to cell damage and death and is involved in the pathogenesis of the metabolic syndrome. Since the metabolic consequences of lipotoxicity are still poorly understood, we studied the bioenergetic effects of the saturated fatty acid palmitate, quantifying changes in mitochondrial morphology, real-time oxygen consumption, ATP production sources, and extracellular acidification in hepatoma cells. Surprisingly, glycolysis was enhanced by the presence of palmitate as soon as 1 h after stimulus, while oxygen consumption and oxidative phosphorylation were unchanged, despite overt mitochondrial fragmentation. Palmitate only induced mitochondrial fragmentation if glucose and glutamine were available, while glycolytic enhancement did not require glutamine, showing it is independent of mitochondrial morphological changes. Redox state was altered by palmitate, as indicated by NAD(P)H quantification. Furthermore, the mitochondrial antioxidant mitoquinone, or a selective inhibitor of complex I electron leakage (S1QEL) further enhanced palmitate-induced glycolysis. Our results demonstrate that palmitate overload and lipotoxicity involves an unexpected and early increase in glycolytic flux, while, surprisingly, no changes in oxidative phosphorylation are observed. Interestingly, enhanced glycolysis involves signaling by mitochondrially-generated oxidants, uncovering a novel regulatory mechanism for this pathway.

The Warburg Effect Occurs Rapidly in Stimulated Human Adult but Not Umbilical Cord Blood Derived Macrophages.

The Warburg effect, defined as increased glycolysis and decreased oxidative phosphorylation, occurs in murine macrophages following LPS stimulation and is required for activation. There are differences between human and murine macrophage metabolic responses to stimulation, with peak metabolite concentrations occurring earlier in humans than mice. Complex changes occur in the human immune system with age, resulting in the very young and the very old being more susceptible to infections. Anti-bacterial immune responses in umbilical cord immune cells are considered deficient but there is a paucity of data on the role that metabolism plays. We hypothesized that metabolic responses in human macrophages occur early during activation. In addition, we hypothesized that umbilical cord derived macrophages have an altered immunometabolic response compared with adult macrophages. We demonstrate that adult and cord blood monocyte derived macrophages (MDM) immediately increase glycolysis in response to stimulation with LPS or Mycobacterium tuberculosis (Mtb), however only adult MDM decrease oxidative phosphorylation. At 24 hours post stimulation, glycolysis remains elevated in both adult and cord blood MDM, oxidative phosphorylation remains unchanged in the cord blood MDM and has normalized in the adult MDM stimulated with Mtb. However, LPS stimulated adult MDM have increased oxidative phosphorylation at 24 hours, illustrating differences in metabolic responses to different stimuli, time-dependent variation in responses and differences in macrophage metabolism in adults compared with umbilical cord blood. We compared the phenotype and function of macrophages derived from adult or cord blood. Cord blood MDM secreted less TNF following Mtb stimulation and more IL-6 following LPS stimulation compared with adult MDM. Our findings demonstrate that whilst cord blood MDM exhibit an immediate increase in glycolytic flux in response to stimulation, similar to adult MDM, cord blood MDM do not concomitantly decrease oxygen consumption. This indicates that adult macrophages shift to Warburg metabolism immediately after stimulation, but cord blood macrophages do not. Understanding the differences in the metabolic profiles of macrophages over a human lifetime will enable the translation of immunometabolism into effective immuno-supportive therapies that could potentially be targeted at vulnerable populations, such as the very old and the very young.

Model Parameterization with Quantitative Proteomics: Case Study with Trehalose Metabolism in Saccharomyces cerevisiae

When Saccharomyces cerevisiae undergoes heat stress it stimulates several changes that are necessary for its survival, notably in carbon metabolism. Notable changes include increase in trehalose production and glycolytic flux. The increase in glycolytic flux has been postulated to be due to the regulatory effects in upper glycolysis, but this has not been confirmed. Additionally, trehalose is a useful industrial compound for its protective properties. A model of trehalose metabolism in S. cerevisiae was constructed using Convenient Modeller, a software that uses a combination of convenience kinetics and a genetic algorithm. The model was parameterized with quantitative omics under standard conditions and validated using data collected under heat stress conditions. The completed model was used to show that feedforward activation of pyruvate kinase by fructose 1,6-bisphosphate during heat stress contributes to the increase in metabolic flux. We were also able to demonstrate in silico that overexpression of enzymes involved in production and degradation of trehalose can lead to higher trehalose yield in the cell. By integrating quantitative proteomics with metabolic modelling, we were able to confirm that the flux increase in trehalose metabolic pathways during heat stress is due to regulatory effects and not purely changes in enzyme expression. The overexpression of enzymes involved in trehalose metabolism is a potential approach to be exploited for trehalose production without need for increasing temperature.

Metabolic Dysregulation and Neurovascular Dysfunction in Diabetic Retinopathy.

Diabetic retinopathy is a major cause of ocular complications in patients with type 1 and type 2 diabetes in developed countries. Due to the continued increase in the number of people with obesity and diabetes in the United States of America and globally, the incidence of diabetic retinopathy is expected to increase significantly in the coming years. Diabetic retinopathy is widely accepted as a combination of neurodegenerative and microvascular changes; however, which change occurs first is not yet understood. Although the pathogenesis of diabetic retinopathy is very complex, regulated by numerous signaling pathways and cellular processes, maintaining glucose homeostasis is still an essential component for normal physiological functioning of retinal cells. The maintenance of glucose homeostasis is finely regulated by coordinated interplay between glycolysis, Krebs cycle, and oxidative phosphorylation. Glycolysis is the most conserved metabolic pathway in biology and is tightly regulated to maintain a steady-state concentration of glycolytic intermediates; this regulation is called scheduled or regulated glycolysis. However, an abnormal increase in glycolytic flux generates large amounts of intermediate metabolites that can be shunted into different damaging pathways including the polyol pathway, hexosamine pathway, diacylglycerol-dependent activation of the protein kinase C pathway, and Amadori/advanced glycation end products (AGEs) pathway. In addition, disrupting the balance between glycolysis and oxidative phosphorylation leads to other biochemical and molecular changes observed in diabetic retinopathy including endoplasmic reticulum-mitochondria miscommunication and mitophagy dysregulation. This review will focus on how dysregulation of glycolysis contributes to diabetic retinopathy.

Regulation of glycolysis by the hypoxia-inducible factor (HIF): implications for cellular physiology.

Under conditions of hypoxia, most eukaryotic cells can shift their primary metabolic strategy from predominantly mitochondrial respiration towards increased glycolysis to maintain ATP levels. This hypoxia-induced reprogramming of metabolism is key to satisfying cellular energetic requirements during acute hypoxic stress. At a transcriptional level, this metabolic switch can be regulated by several pathways including the hypoxia inducible factor-1α (HIF-1α) which induces an increased expression of glycolytic enzymes. While this increase in glycolytic flux is beneficial for maintaining bioenergetic homeostasis during hypoxia, the pathways mediating this increase can also be exploited by cancer cells to promote tumour survival and growth, an area which has been extensively studied. It has recently become appreciated that increased glycolytic metabolism in hypoxia may also have profound effects on cellular physiology in hypoxic immune and endothelial cells. Therefore, understanding the mechanisms central to mediating this reprogramming are of importance from both physiological and pathophysiological standpoints. In this review, we highlight the role of HIF-1α in the regulation of hypoxic glycolysis and its implications for physiological processes such as angiogenesis and immune cell effector function.

Therapeutic Targeting of Cancer Metabolism with Triosephosphate Isomerase.

The increase in glycolytic flux in cancer, known as aerobic glycolysis, is one of the most important hallmarks of cancer. Therefore, glycolytic enzymes have importance in understanding the molecular mechanism of cancer progression. Triosephosphate isomerase (TPI) is one of the key glycolytic enzymes. Furthermore, it takes a part in gluconeogenesis, pentose phosphate pathway and fatty acid biosynthesis. To date, it has been shown altered levels of TPI in various cancer types, especially in metastatic phenotype. According to other studies, TPI might be considered as a potential therapeutic target and a cancer-related biomarker in different types of cancer. However, its function in tumor formation and development has not been fully understood. Here, we reviewed the relationship between TPI and cancer for the first time.

Gene-Diet Interactions: Dietary Rescue of Metabolic Effects in spen-Depleted Drosophila melanogaster.

Obesity and its comorbidities are a growing health epidemic. Interactions between genetic background, the environment, and behavior (i.e., diet) greatly influence organismal energy balance. Previously, we described obesogenic mutations in the gene Split ends (Spen) in Drosophila melanogaster, and roles for Spen in fat storage and metabolic state. Lipid catabolism is impaired in Spen-deficient fat storage cells, accompanied by a compensatory increase in glycolytic flux and protein catabolism. Here, we investigate gene-diet interactions to determine if diets supplemented with specific macronutrients can rescue metabolic dysfunction in Spen-depleted animals. We show that a high-yeast diet partially rescues adiposity and developmental defects. High sugar partially improves developmental timing as well as longevity of mated females. Gene-diet interactions were heavily influenced by developmental-stage-specific organismal needs: extra yeast provides benefits early in development (larval stages) but becomes detrimental in adulthood. High sugar confers benefits to Spen-depleted animals at both larval and adult stages, with the caveat of increased adiposity. A high-fat diet is detrimental according to all tested criteria, regardless of genotype. Whereas Spen depletion influenced phenotypic responses to supplemented diets, diet was the dominant factor in directing the whole-organism steady-state metabolome. Obesity is a complex disease of genetic, environmental, and behavioral inputs. Our results show that diet customization can ameliorate metabolic dysfunction underpinned by a genetic factor.

Metabolic Reprogramming Promotes Neural Crest Migration via Yap/Tead Signaling.

The Warburg effect is one of the metabolic hallmarks of cancer cells, characterized by enhanced glycolysis even under aerobic conditions. This physiological adaptation is associated with metastasis , but we still have a superficial understanding of how it affects cellular processes during embryonic development. Here we report that the neural crest,a migratory stem cell population in vertebrate embryos, undergoes an extensive metabolic remodeling to engage in aerobic glycolysis prior to delamination. This increase in glycolytic flux promotes Yap/Tead signaling, which activates the expression of a set of transcription factors to drive epithelial-to-mesenchymal transition. Our results demonstrate how shifts in carbon metabolism can trigger the gene regulatory circuits that control complex cell behaviors. These findings support the hypothesis that the Warburg effect is a precisely regulated developmental mechanism that is anomalously reactivated during tumorigenesis and metastasis.

Metabolic adaptations in spontaneously immortalized PGC-1α knock-out mouse embryonic fibroblasts increase their oncogenic potential

PGC-1α controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1α in cancer. To investigate how loss of PGC-1α activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear β-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1α, restraining metabolic adaptations in cancer.