Glycolysis For Energy Production Research Articles

Metabolic dysfunction and the development of physical frailty: an aging war of attrition.

The World Health Organization recently declared 2021-2030 the decade of healthy aging. Such emphasis on healthy aging requires an understanding of the biologic challenges aging populations face. Physical frailty is a syndrome of vulnerability that puts a subset of older adults at high risk for adverse health outcomes including functional and cognitive decline, falls, hospitalization, and mortality. The physiology driving physical frailty is complex with age-related biological changes, dysregulated stress response systems, chronic inflammatory pathway activation, and altered energy metabolism all likely contributing. Indeed, a series of recent studies suggests circulating metabolomic distinctions can be made between frail and non-frail older adults. For example, marked restrictions on glycolytic and mitochondrial energy production have been independently observed in frail older adults and collectively appear to yield a reliance on the highly fatigable ATP-phosphocreatine (PCr) energy system. Further, there is evidence that age-associated impairments in the primary ATP generating systems (glycolysis, TCA cycle, electron transport) yield cumulative deficits and fail to adequately support the ATP-PCr system. This in turn may acutely contribute to several major components of the physical frailty phenotype including muscular fatigue, weakness, slow walking speed and, over time, result in low physical activity and accelerate reductions in lean body mass. This review describes specific age-associated metabolic declines and how they can collectively lead to metabolic inflexibility, ATP-PCr reliance, and the development of physical frailty. Further investigation remains necessary to understand the etiology of age-associated metabolic deficits and develop targeted preventive strategies that maintain robust metabolic health in older adults.

Advances in metabolic reprogramming of renal tubular epithelial cells in sepsis-associated acute kidney injury.

Sepsis-associated acute kidney injury presents as a critical condition characterized by prolonged hospital stays, elevated mortality rates, and an increased likelihood of transition to chronic kidney disease. Sepsis-associated acute kidney injury suppresses fatty acid oxidation and oxidative phosphorylation in the mitochondria of renal tubular epithelial cells, thus favoring a metabolic shift towards glycolysis for energy production. This shift acts as a protective mechanism for the kidneys. However, an extended reliance on glycolysis may contribute to tubular atrophy, fibrosis, and subsequent chronic kidney disease progression. Metabolic reprogramming interventions have emerged as prospective strategies to counteract sepsis-associated acute kidney injury by restoring normal metabolic function, offering potential therapeutic and preventive modalities. This review delves into the metabolic alterations of tubular epithelial cells associated with sepsis-associated acute kidney injury, stressing the importance of metabolic reprogramming for the immune response and the urgency of metabolic normalization. We present various intervention targets that could facilitate the recovery of oxidative phosphorylation-centric metabolism. These novel insights and strategies aim to transform the clinical prevention and treatment landscape of sepsis-associated acute kidney injury, with a focus on metabolic mechanisms. This investigation could provide valuable insights for clinicians aiming to enhance patient outcomes in the context of sepsis-associated acute kidney injury.

Initial Phase of Anthracycline Cardiotoxicity Involves Cardiac Fibroblasts Activation and Metabolic Switch.

The application of doxorubicin (DOX) is hampered by cardiotoxicity, with diastolic dysfunction as the earliest manifestation. Fibrosis leads to impaired relaxation, but the mechanisms that operate shortly after DOX exposure are not clear. We asked whether the activation of cardiac fibroblasts (CFs) anticipates myocardial dysfunction and evaluated the effects of DOX on CF metabolism. CFs were isolated from the hearts of rats after the first injection of DOX. In another experiment, CFs were exposed to DOX in vitro. Cell phenotype and metabolism were determined. Early effects of DOX consisted of diastolic dysfunction and unchanged ejection fraction. Markers of pro-fibrotic remodeling and evidence of CF transformation were present immediately after treatment completion. Oxygen consumption rate and extracellular acidification revealed an increased metabolic activity of CFs and a switch to glycolytic energy production. These effects were consistent in CFs isolated from the hearts of DOX-treated animals and in naïve CFs exposed to DOX in vitro. The metabolic switch was paralleled with the phenotype change of CFs that upregulated markers of myofibroblast differentiation and the activation of pro-fibrotic signaling. In conclusion, the metabolic switch and activation of CFs anticipate DOX-induced damage and represent a novel target in the early phase of anthracycline cardiomyopathy.

Biological Impact of Organic Extracts from Urban-Air Particulate Matter: An In Vitro Study of Cytotoxic and Metabolic Effects in Lung Cells

Atmospheric particulate matter (PM) with diameters below 10 µm (PM10) may enter the lungs through inhalation and are linked to various negative health consequences. Emergent evidence emphasizes the significance of cell metabolism as a sensitive target of PM exposure. However, the current understanding of the relationship between PM composition, conventional toxicity measures, and the rewiring of intracellular metabolic processes remains limited. In this work, PM10 sampled at a residential area (urban background, UB) and a traffic-impacted location (roadside, RS) of a Portuguese city was comprehensively characterized in terms of polycyclic aromatic hydrocarbons and plasticizers. Epithelial lung cells (A549) were then exposed for 72 h to PM10 organic extracts and different biological outcomes were assessed. UB and RS PM10 extracts dose-dependently decreased cell viability, induced reactive oxygen species (ROS), decreased mitochondrial membrane potential, caused cell cycle arrest at the G0/G1 phase, and modulated the intracellular metabolic profile. Interestingly, the RS sample, richer in particularly toxic PAHs and plasticizers, had a greater metabolic impact than the UB extract. Changes comprised significant increases in glutathione, reflecting activation of antioxidant defences to counterbalance ROS production, together with increases in lactate, NAD+, and ATP, which suggest stimulation of glycolytic energy production, possibly to compensate for reduced mitochondrial activity. Furthermore, a number of other metabolic variations hinted at changes in membrane turnover and TCA cycle dynamics, which represent novel clues on potential PM10 biological effects.

Initial phase of anthracycline cardiotoxicity involves metabolic switch and activation of cardiac fibroblasts

Abstract Background Doxorubicin (DOX) is an effective anticancer drug, but its application is hampered by cardiotoxicity with asymptomatic diastolic dysfunction as the earliest manifestation. Interstitial fibrosis leads to impaired relaxation, but the timing of fibrotic process and the mechanisms that operate shortly after DOX exposure are not clear. While changes in energy metabolism by DOX is recognized in cardiomyocytes, the effects of DOX on cardiac fibroblasts (CFs) metabolism are unknown.Thus, in a model of anthracycline cardiomyopathy, we asked whether the activation of CFs anticipates the onset of DOX-induced early diastolic dysfunction and evaluated the effects of DOX on the activation and metabolism of CFs. Methods Fischer 344 rats were exposed to 6 injections of 2.5 mg/kg of DOX over 2 weeks. Heart function was assessed by echocardiography and left ventricular catheterization. To test the very early in vivo effects of DOX on CFs, primary CFs were isolated from hearts of DOX-treated rats after the first injection of DOX. In another set of experiments, CFs were exposed to DOX in vitro. Cell metabolism was measured by a Seahorse-XF Real-Time ATP assay and cell phenotype was determined by immunocytochemistry and molecular biology. Results Early effects of DOX consisted of unchanged ejection fraction and the evidence of diastolic dysfunction. Markers of pro-fibrotic remodeling and histological evidence of CFs transformation were present in the heart immediately after treatment completion. Importantly, at the very beginning of the DOXO exposure, when cardiac function was normal and there was no histological evidence of increased fibrosis, the conversion of CFs to myofibroblasts, evidenced by SMA, was detected. Oxygen consumption rate and extracellular acidification, revealed the increased metabolic activity of CFs after DOX exposure and the switch to the glycolytic energy production. These effects were consistent in CFs isolated from the hearts of DOX-treated animals and in naive CFs exposed to DOX in vitro. The metabolic switch was paralleled with the phenotype change of CFs towards extracellular matrix-producing cells. Upon DOX, CFs upregulated markers of myofibroblast differentiation and the activation of pro-fibrotic signaling (aSMA, TGFb and phospho-SMAD). CFs maintained their functional properties in scratch assay. CFs upregulated also NOX2, indicating these cells as an intramyocardial source of reactive oxygen in the initial phase of the disease. Conclusion The metabolic switch and activation of CFs anticipate the onset of DOX-induced early diastolic dysfunction. The effect of DOX on the metabolic profile in CFs represents a novel and potentially targetable component of the very early phase of the pathogenesis of anthracycline cardiomyopathy.

LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity

BackgroundNeutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells.MethodsLrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2.ResultsfMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP.ConclusionsLRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells.-J661noP-dBkFUp6aS7aWrVideo

The fate of mitochondria during platelet activation

AbstractBlood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam–scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

Splicing regulation of GFPT1 muscle-specific isoform and its roles in glucose metabolisms and neuromuscular junction

Glutamine:fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP). A 54-bp exon 9 of GFPT1 is specifically included in skeletal and cardiac muscles to generate a long isoform of GFPT1 (GFPT1-L). We showed that SRSF1 and Rbfox1/2 cooperatively enhance, and hnRNP H/F suppresses, the inclusion of human GFPT1 exon 9 by modulating recruitment of U1 snRNP. Knockout (KO) of GFPT1-L in skeletal muscle markedly increased the amounts of GFPT1 and UDP-HexNAc, which subsequently suppressed the glycolytic pathway. Aged KO mice showed impaired insulin-mediated glucose uptake, as well as muscle weakness and fatigue likely due to abnormal formation and maintenance of the neuromuscular junction. Taken together, GFPT1-L is likely to be acquired in evolution in mammalian striated muscles to attenuate the HBP for efficient glycolytic energy production, insulin-mediated glucose uptake, and the formation and maintenance of the neuromuscular junction.

3-Bromopyruvate Inhibits the Growth and Glucose Metabolism of TNBC Xenografts in Nude Mice by Targeting c-Myc.

Due to the lack of effective drug treatment, triple-negative breast cancer (TNBC) is prone to recurrence and metastasis after an operation. As a glycolytic inhibitor, 3-bromopyruvic acid (3-BrPA) can inhibit the proliferation and induce apoptosis of TNBC cells. However, whether it has similar effects in animal models remains unclear. To observe the effect of 3-BrPA on the growth and glucose metabolism of human TNBC transplanted tumors in nude mice and to investigate the mechanism. We constructed subcutaneous xenografts of human TNBC in nude mice and treated them with low, medium and high concentrations of 3-BrPA. After 15 days, nude mice were sacrificed to detect hexokinase (HK) activity and adenosine triphosphate (ATP) content in tumor tissues. Hematoxylin-eosin (HE) staining was used to detect the damage of transplanted tumors and liver and kidney in nude mice, which 3-BrPA caused. The expression of c-Myc in tumor tissues was detected by Immunohistochemistry (IHC). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was used to detect the apoptosis of tumor tissues. Besides, the expressions of Cytc, Bax, Bcl-2 and Caspase-9 were detected by Western blotting. Compared with the control group, intraperitoneal injection of 3-BrPA inhibited the growth of human TNBC transplant tumors, decreased HK activity and ATP production in tumor tissues, disrupted the tissue structure of transplant tumors, and did not significantly damage liver and kidney tissues. IHC staining and Western blotting showed that 3-BrPA could decrease the expression of c-Myc and Bcl-2, increase the expression of Cyt -c, Bax and Caspase-9 expression and promote apoptosis in tumor tissues. The above data indicate that 3-BrPA inhibits the growth of human TNBC transplanted tumors and promotes their apoptosis. Its anti-cancer mechanism might reduce HK activity by down-regulating c-Myc expression, eventually leading to decreased glycolytic pathway energy production and promoting apoptosis of transplanted tumors.

Phosphoglycerate dehydrogenase activates PKM2 to phosphorylate histone H3T11 and attenuate cellular senescence

Vascular endothelial cells (ECs) senescence correlates with the increase of cardiovascular diseases in ageing population. Although ECs rely on glycolysis for energy production, little is known about the role of glycolysis in ECs senescence. Here, we report a critical role for glycolysis-derived serine biosynthesis in preventing ECs senescence. During senescence, the expression of serine biosynthetic enzyme PHGDH is significantly reduced due to decreased transcription of the activating transcription factor ATF4, which leads to reduction of intracellular serine. PHGDH prevents premature senescence primarily by enhancing the stability and activity of pyruvate kinase M2 (PKM2). Mechanistically, PHGDH interacts with PKM2, which prevents PCAF-catalyzed PKM2 K305 acetylation and subsequent degradation by autophagy. In addition, PHGDH facilitates p300-catalyzed PKM2 K433 acetylation, which promotes PKM2 nuclear translocation and stimulates its activity to phosphorylate H3T11 and regulate the transcription of senescence-associated genes. Vascular endothelium-targeted expression of PHGDH and PKM2 ameliorates ageing in mice. Our findings reveal that enhancing serine biosynthesis could become a therapy to promote healthy ageing.

Increased Expression of the Mitochondrial Glucocorticoid Receptor Enhances Tumor Aggressiveness in a Mouse Xenograft Model

Mitochondria are important organelles for cellular physiology as they generate most of the energy requirements of the cell and orchestrate many biological functions. Dysregulation of mitochondrial function is associated with many pathological conditions, including cancer development. Mitochondrial glucocorticoid receptor (mtGR) is proposed as a crucial regulator of mitochondrial functions via its direct involvement in the regulation of mitochondrial transcription, oxidative phosphorylation (OXPHOS), enzymes biosynthesis, energy production, mitochondrial-dependent apoptosis, and regulation of oxidative stress. Moreover, recent observations revealed the interaction of mtGR with the pyruvate dehydrogenase (PDH), a key player in the metabolic switch observed in cancer, indicating direct involvement of mtGR in cancer development. In this study, by using a xenograft mouse model of mtGR-overexpressing hepatocarcinoma cells, we showed increased mtGR-associated tumor growth, which is accompanied by reduced OXPHOS biosynthesis, reduction in PDH activity, and alterations in the Krebs cycle and glucose metabolism, metabolic alterations similar to those observed in the Warburg effect. Moreover, autophagy activation is observed in mtGR-associated tumors, which further support tumor progression via increased precursors availability. Thus, we propose that increased mitochondrial localization of mtGR is associated with tumor progression possible via mtGR/PDH interaction, which could lead to suppression of PDH activity and modulation of mtGR-induced mitochondrial transcription that ends up in reduced OXPHOS biosynthesis and reduced oxidative phosphorylation versus glycolytic pathway energy production, in favor of cancer cells.

Dual roles of hexokinase 2 in shaping microglial function by gating glycolytic flux and mitochondrial activity.

Microglia continuously survey the brain parenchyma and actively shift status following stimulation. These processes demand a unique bioenergetic programme; however, little is known about the metabolic determinants in microglia. By mining large datasets and generating transgenic tools, here we show that hexokinase 2 (HK2), the most active isozyme associated with mitochondrial membrane, is selectively expressed in microglia in the brain. Genetic ablation of HK2 reduced microglial glycolytic flux and energy production, suppressed microglial repopulation, and attenuated microglial surveillance and damage-triggered migration in male mice. HK2 elevation is prominent in immune-challenged or disease-associated microglia. In ischaemic stroke models, however, HK2 deletion promoted neuroinflammation and potentiated cerebral damages. The enhanced inflammatory responses after HK2 ablation in microglia are associated with aberrant mitochondrial function and reactive oxygen species accumulation. Our study demonstrates that HK2 gates both glycolytic flux and mitochondrial activity to shape microglial functions, changes of which contribute to metabolic abnormalities and maladaptive inflammation in brain diseases.

Regulation of Metabolic Plasticity in Cancer Stem Cells and Implications in Cancer Therapy.

Cancer stem cells (CSCs), a subpopulation of tumor cells with self-renewal capacity, have been associated with tumor initiation, progression, and therapy resistance. While the bulk of tumor cells mainly use glycolysis for energy production, CSCs have gained attention for their ability to switch between glycolysis and oxidative phosphorylation, depending on their energy needs and stimuli from their microenvironment. This metabolic plasticity is mediated by signaling pathways that are also implicated in the regulation of CSC properties, such as the Wnt/β-catenin, Notch, and Hippo networks. Two other stemness-associated processes, autophagy and hypoxia, seem to play a role in the metabolic switching of CSCs as well. Importantly, accumulating evidence has linked the metabolic plasticity of CSCs to their increased resistance to treatment. In this review, we summarize the metabolic signatures of CSCs and the pathways that regulate them; we especially highlight research data that demonstrate the metabolic adaptability of these cells and their role in stemness and therapy resistance. As the development of drug resistance is a major challenge for successful cancer treatment, the potential of specific elimination of CSCs through targeting their metabolism is of great interest and it is particularly examined.

Lysosomes and Their Role in Regulating the Metabolism of Hematopoietic Stem Cells.

Simple SummaryHematopoietic stem cells (HSCs) are an essential component of the cellular architecture of the body. These cells are responsible for self-renewal as well as the production of hematopoietic progenitors and adult blood cells. HSCs have been successfully used as a treatment for a variety of hematological disorders, including lymphoma, leukemia, anemia, and anomalies in the immune system. However, owing to the difficulties associated with the limited supply of HSCs, the majority of hematological conditions cannot be treated in clinical settings. It is possible that understanding the intrinsic and extrinsic regulators of HSC stemness could pave the way for developing novel methods for accessing alternative HSC sources. This would enable researchers to get around the limitations that currently exist in clinical applications.Hematopoietic stem cells (HSCs) have the capacity to renew blood cells at all stages of life and are largely quiescent at a steady state. It is essential to understand the processes that govern quiescence in HSCs to enhance bone marrow transplantation. It is hypothesized that in their quiescent state, HSCs primarily use glycolysis for energy production rather than mitochondrial oxidative phosphorylation (OXPHOS). In addition, the HSC switch from quiescence to activation occurs along a continuous developmental path that is driven by metabolism. Specifying the metabolic regulation pathway of HSC quiescence will provide insights into HSC homeostasis for therapeutic application. Therefore, understanding the metabolic demands of HSCs at a steady state is key to developing innovative hematological therapeutics. Lysosomes are the major degradative organelle in eukaryotic cells. Catabolic, anabolic, and lysosomal function abnormalities are connected to an expanding list of diseases. In recent years, lysosomes have emerged as control centers of cellular metabolism, particularly in HSC quiescence, and essential regulators of cell signaling have been found on the lysosomal membrane. In addition to autophagic processes, lysosomal activities have been shown to be crucial in sustaining quiescence by restricting HSCs access to a nutritional reserve essential for their activation into the cell cycle. Lysosomal activity may preserve HSC quiescence by altering glycolysis-mitochondrial biogenesis. The understanding of HSC metabolism has significantly expanded over the decade, revealing previously unknown requirements of HSCs in both their dividing (active) and quiescent states. Therefore, understanding the role of lysosomes in HSCs will allow for the development of innovative treatment methods based on HSCs to fight clonal hematopoiesis and HSC aging.

Enhancing oxidative phosphorylation over glycolysis for energy production in cultured mesenchymal stem cells.

ObjectiveStrokes represent as one of the leading causes of death and disability in the USA, however, there is no optimal treatment to reduce the occurrence or improve prognosis. Preconditioning of tissues triggers ischemic tolerance, a physiological state that may involve a metabolic switch (i.e. from glycolysis to oxidative phosphorylation or OxPhos) to preserve tissue viability under an ischemic insult. Here, we hypothesized that metabolic switching of energy source from glucose to galactose in cultured mesenchymal stem cells (MSCs) stands as an effective OxPhos-enhancing strategy.MethodsMSCs were grown under ambient condition (normal MSCs) or metabolic switching paradigm (switched MSCs) and then assayed for oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) using the Seahorse technology to assess mitochondrial respiration.ResultsNormal MSCs showed a lower OCR/ECAR ratio than switched MSCs at baseline (P < 0.0001), signifying that there were greater levels of OxPhos compared to glycolysis in switched MSCs. By modulating the mitochondrial metabolism with oligomycin (time points 4–6), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (7–9), and rotenone and antimycin (time points 10–12), switched MSCs greater reliance on OxPhos was further elucidated (time points 5–12; P < 0.0001; time point 4; P < 0.001).ConclusionThe metabolic switch from glycolytic to oxidative metabolism amplifies the OxPhos potential of MSCs, which may allow these cells to afford more robust therapeutic effects against neurological disorders that benefit from ischemic tolerance.

Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer

Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current–based MS1 workflow to quantify ∼6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and ‘drug response’ mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.

Cholesterol trapping by SOAT1 induces mitochondrial cholesterol accumulation and decrease oxidative metabolism

Background and Aims : Normal hepatocytes in-vivo express only Sterol O-Acyltransferase2 (SOAT2), whereas in pathologic conditions (cancer) acquire SOAT1 expression. Hepatocytes mainly use mitochondrial oxidative metabolism for energy production, whereas cancer cells rely more on glycolysis for energy production. Currently, it is not known how cholesterol in mitochondria affects their metabolism, and whether SOAT2 and SOAT1 could play a role. Thus, we studied HepG2 cells and a unique cell model in which SOAT1 was genetically depleted: SOAT2-only-HepG2 cells.

2-Deoxy-D-glucose and combined 2-Deoxy-D-glucose/albendazole exhibit therapeutic efficacy against Echinococcus granulosus protoscoleces and experimental alveolar echinococcosis.

2-Deoxy-D-glucose (2-DG) is a glucose analog used as a promising anticancer agent. It exerts its effects by inhibiting the glycolytic energy metabolism to deplete cells of energy. The larval stage of Echinococcus relies on glycolysis for energy production. Therefore, in this study, we investigated the in vitro and in vivo efficacy of 2-DG against the larval stage of Echinococcus granulosus and E. multilocularis. 2-DG exhibited significant time- and dose-dependent effects against in vitro cultured E. granulosus protoscoleces and E. multilocularis metacestodes. A daily oral administration of 500 mg/kg 2-DG in E. multilocularis-infected mice effectively reduced the weight of metacestodes. Notably, the combination treatment, either 2-DG (500 mg/kg/day) + albendazole (ABZ) (200 mg/kg/day) or 2-DG (500 mg/kg/day) + half-dose of ABZ (100 mg/kg/day), exhibited a potent therapeutic effect against E. multilocularis, significantly promoting the reduction of metacestodes weight compared with the administration of 2-DG or ABZ alone. Furthermore, the combination significantly promoted apoptosis of the cells of metacestodes and inhibited glycolysis in metacestodes, compared with the administration of 2-DG or ABZ alone. In conclusion, 2-DG exerts an effective activity against the larval stage of Echinococcus. Thus, it may be a promising anti-Echinococcus drug, and its combination with ABZ may provide a new strategy for the treatment of echinococcosis in humans.

Abstract LB128: Oncolytic HSV-P10 and effects on metabolism as potential therapy for treatment of glioblastoma

Abstract Glioblastoma is an aggressive tumor of the central nervous system associated with a very poor prognosis even with surgery and chemoradiation. We have previously developed an oHSV that encodes for PTENα (HSV-P10) and stimulates anti-tumor immunity. This virus is currently licensed by and is being developed by Mesoblast to evaluate safety and efficacy in patients. To understand the effect of PTENα on tumor cell responses to oHSV therapy we performed RNA sequencing of primary GBM cells infected with control or oHSV-P10. We identified a dysregulation of glycolysis in cells infected with oHSV-P10 relative to control oHSV. Since cancer cells rely heavily on glycolysis for energy production (Warburg effect) the impact of PTENα on metabolic switch was significant. RNA sequencing PCR and western blot analysis showed a significant reduction in cellular Hexokinase II expression and downregulation of HIF-1α in HSV-P10 infected cells. Consistent with the function of HIF1 and hexokinase in driving glycolysis we also observed a significant reduction in glucose uptake and lactate release: implying a reduction in glycolysis in cells after being treated with oHSV-P10. Thus, cellular energetics of HSV-P10 infected cells was directed away from glycolysis relative to HSVQ infected cells. Consistent with this we observed increased, oxidative phosphorylation, production of reactive oxidative species and ATP production. Many cancers, including Glioblastoma are known to utilize the glutamine as a substrate for energy production through its entry into the TCA cycle. Blocking this shows a significant decrease in cell viability and ATP production when combined with HSV-P10. These results indicate that combination of HSV-P10 with radiation has a significant therapeutic potential and should be tested in patients for safety and efficacy. Citation Format: Matthew P. Mullarkey, Upasana Sahu, Bangxing Hong, Balveen Kaur. Oncolytic HSV-P10 and effects on metabolism as potential therapy for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB128.

Proteomic analysis of the mitochondrial glucocorticoid receptor interacting proteins reveals pyruvate dehydrogenase and mitochondrial 60 kDa heat shock protein as potent binding partners

Glucocorticoids are steroid hormones that regulate plethora biological actions such as growth and metabolism, immune response, and apoptosis. Glucocorticoids actions are mediated via glucocorticoid receptors which act mainly as transcription factors, but it is also found to be localized in mitochondria. Mitochondrial localization of the receptor indicates novel functions of the receptor. Characterization of the mitochondrial glucocorticoid receptor (mtGR) interacting proteins will shed light on these actions and the biochemical mechanisms that underlie mitochondrial glucocorticoid receptor import and functions. In this study, applying immunoprecipitation, mass spectrometry and Western blot analysis of the GR interacting proteins in total or mitochondrial extracts of HepG2 cells and of HepG2 cells overexpressing a mitochondrial targeted GR we found pyruvate dehydrogenase (PDH), chaperones such as and heat shock protein (HSP) −60, −70, −75 and −90, and 78 kDa glucose-regulated protein, mitochondrial transcription factors and enzymes involved in the regulation of the mitochondrial protein biosynthesis, lipid metabolism, ATP production and apoptosis as glucocorticoid receptor interacting proteins. Our results uncover potential novel mitochondrial partners of the receptor, suggesting possible new regulatory roles of mtGR in the control of mitochondrial-associated functions of the cell. SignificanceIn this study the mitochondrial GR interacting proteins were characterized highlighting novel regulatory roles of the receptor in mitochondria. Detection of the mtGR/PDH and mtGR/HSP60 interaction in almost all the analyses performed uncovered PDH and HSP60 proteins as potent mtGR binding partners. The interesting finding of the PDH/mtGR interaction possibly indicates involvement of mtGR in the regulation of the balance between glycolytic and oxidative phosphorylation energy production. Characterization of the mitochondrial heat shock −60, −70, −75 and 78 proteins as mtGR binding partners contribute to the characterization of the biochemical mechanisms of the mitochondrial import of the receptor. Moreover, identification of mitochondrial heat shock proteins, metabolic enzymes, transcription factors, OXPHOS, and regulatory molecules in mitochondrial protein biosynthesis as mtGR binding partners indicates possible new regulatory roles of mtGR in the glucocorticoids-induced regulation and orchestration of nuclear and mitochondrial functions, the exact biochemical mechanism of which remain to be established. The study discloses potential new regulatory roles of the receptor in mitochondria, pointing out its importance as a promising target molecule for the control of the mitochondria-associated pathophysiology of the cell.