Glycolytic Enzymes Research Articles

Oncogenic alterations of metabolism associated with resistance to chemotherapy.

Metabolic reprogramming in cancer cells is a strategy to meet high proliferation rates, invasion, and metastasis. Also, several researchers indicated that the cellular metabolism changed during the resistance to chemotherapy. Since glycolytic enzymes play a prominent role in these alterations, the ability to reduce resistance to chemotherapy drugs is promising for cancer patients. Oscillating gene expression of these enzymes was involved in the proliferation, invasion, and metastasis of cancer cells. This review discussed the roles of some glycolytic enzymes associated with cancer progression and resistance to chemotherapy in the various cancer types.

Gamma-Aminobutyric Acid (GABA) Avoids Deterioration of Transport Water Quality, Regulates Plasma Biochemical Indices, Energy Metabolism, and Antioxidant Capacity of Tawny Puffer (Takifugui flavidus) under Transport Stress

Live fish transportation is crucial for managing aquaculture but can pose health risks to fish due to stressors encountered during transportation. Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that plays a crucial role in the central nervous system and is considered to exhibit anti-stress effects. This study aims to investigate the effects of GABA on the transport water quality, plasma biochemical indices, energy metabolism, and antioxidant capacity of tawny puffer (Takifugu flavidus) under transport stress. Tawny puffer were pretreated by immersing in aquariums containing GABA (final concentrations at 0, 5, 50, and 150 mg/L) seawater for 3 days; then, simulated transport was conducted using oxygen-filled polyethylene bags containing the same concentration of GABA seawater as the pretreatment period. Water samples, plasma, and liver were collected after 0, 6, and 12 h of transport. The results revealed that with the prolongation of transportation time, the control group’s water quality deteriorated, stress-related plasma biochemical indices increased, glycolytic substrate contents decreased, glycolytic enzyme activities and product contents increased, and aerobic metabolic enzyme activities exhibited initial increases followed by declines, ATPase activities decreased, antioxidant enzyme activities decreased, and the lipid peroxidation marker contents increased. It is noteworthy that GABA treatment could avoid water quality deterioration during transportation, inhibit an elevation in stress-related biochemical indicators, regulate energy metabolism, and reduce oxidative damage in tawny puffer, especially at 50 and 150 mg/L concentrations. In summary, GABA treatment can effectively alleviate the transport stress of tawny puffer.

Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer.

To identify drivers of malignancy in human pancreatic ductal adenocarcinoma (PDAC), we performed regulatory network analysis on a large collection of expression profiles from laser capture microdissected samples of PDAC and benign precursors. We discovered that BMAL2 plays a role in the initiation, progression, post resection survival, and KRAS activity in PDAC. Functional analysis of BMAL2 target genes led us to hypothesize that it plays a role in regulating the response to hypoxia, a critical but poorly understood feature of PDAC physiology. Knockout of BMAL2 in multiple human PDAC cell lines revealed effects on viability and invasion, particularly under hypoxic conditions. Loss of BMAL2 also affected glycolysis and other metabolic processes. We found that BMAL2 directly regulates hypoxia-responsive target genes. We also found that BMAL2 is necessary for the stabilization of HIF1A upon exposure to hypoxia, but destabilizes HIF2A under hypoxia. These data demonstrate that BMAL2 is a master transcriptional regulator of hypoxia responses in PDAC and may serve as a long-sought molecular switch that distinguishes HIF1A- and HIF2A-dependent modes of hypoxic metabolism.

Titanium dioxide nanoparticles induce energy perturbation by stimulating glycolytic metabolic profile in rats

The safety of titanium dioxide nanoparticles (TiO2-NPs) remains uncertain due to a scarcity of data regarding its absorption, distribution, elimination, and potential adverse effects following oral exposure. As emerging evidence suggests perturbations in energy metabolism play a pivotal role in the toxicity induced by various toxicants, this investigation aimed to assess the effect of TiO2-NPs on the activities of specific glycolytic enzymes in rats. In this study, seventy-two (72) male Wistar rats (200 ± 20g) were divided into 12 groups, each consisting of 6 animals. The rats were orally exposed to TiO2-NPs (8-12nm) at doses of 50, 150, and 250 mg/kg body weight (BW) for durations of 4, 8, and 12 weeks. The control groups received distilled water. The results showed that, except for the 150 mg/kg BW dose of 12 weeks, TiO2-NPs exposure led to an up-regulation in hexokinase activity in lymphocytes, plasma, erythrocytes, and the liver. Hepatic and lymphocyte aldolase activities were also up-regulated, except at 8 and 12 weeks for the 50 and 150 mg/kg BW doses, where slight decreases were observed. Plasma aldolase increased, except at 12 weeks for 150 mg/kg BW dose, while erythrocyte aldolase increased only during the 4 and 8-week exposure but decreased throughout the 12-week exposure. Notably, compared to erythrocyte lactate dehydrogenase activity, which decreased, a consistent pattern observed in lymphocytes, plasma, and hepatic enzymes was a down-regulation at 8 and 12 weeks for the 50 and 150 mg/kg BW doses of TiO2-NPs. In other instances, and time intervals, lactate dehydrogenase was up-regulated. The data from this study underscores that exposure to TiO2-NPs can disrupt the glycolytic pathway of energy metabolism, particularly during the initial 4 weeks of exposure. These perturbations are characterized by increased glycolytic enzymes activity in the lymphocytes, plasma, and the liver.

Catalysis-mediated dynamic ligand presentation regulates mechanosensing–metabolism coupling of stem cells

The dynamic presentation of ligands on biomaterial surfaces can greatly affect cellular behaviors. However, there have been no prior studies that utilize catalysis-mediated dynamic bonds for dynamic ligand presentation and explore its impact on cellular events. In this work we employ aniline as a catalyst to enhance the dynamics of the hydrazone bond in the tether structure of ligands attached to biomaterial substrate to study the impact of such controlled dynamic ligand presentation on the mechanosensing–metabolism coupling of adherent stem cells. Gold nanoparticles, decorated with polyethylene glycol (PEG)-hydrazide, are linked to benzaldehyde-modified glass substrates through hydrazone bonds. The catalytic aniline, combined with the cell adhesive arginyl-glycyl-aspartic acid (RGD) peptide, is conjugated on the nanoparticle surface to facilitate the disassociation and reassociation of hydrazone bonds in the RGD tether structure. This catalysis-mediated dynamic presentation of RGD effectively regulates the Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent mechanosensing and stimulates the actin cytoskeleton rearrangement in stem cells. The enhanced release of a glycolytic enzyme (Aldolase A) due to the cytoskeleton rearrangement leads to increased glycolysis, adenosine triphosphate (ATP) generation, oxidative phosphorylation, thereby contributing to the osteogenic differentiation of human mesenchymal stem cells (hMSCs). These findings highlight the importance of chemically manipulating the dynamic ligand presentation in regulating mechanosensing–metabolism coupling and the differentiation of stem cells.

Intrathecal Fumagillin Alleviates Chronic Neuropathy-Induced Nociceptive Sensitization and Modulates Spinal Astrocyte-Neuronal Glycolytic and Angiogenic Proteins.

Nociceptive sensitization is accompanied by the upregulation of glycolysis in the central nervous system in neuropathic pain. Growing evidence has demonstrated glycolysis and angiogenesis to be related to the inflammatory processes. This study investigated whether fumagillin inhibits neuropathic pain by regulating glycolysis and angiogenesis. Fumagillin was administered through an intrathecal catheter implanted in rats with chronic constriction injury (CCI) of the sciatic nerve. Nociceptive, behavioral, and immunohistochemical analyses were performed to evaluate the effects of the inhibition of spinal glycolysis-related enzymes and angiogenic factors on CCI-induced neuropathic pain. Fumagillin reduced CCI-induced thermal hyperalgesia and mechanical allodynia from postoperative days (POD) 7 to 14. The expression of angiogenic factors, vascular endothelial growth factor (VEGF) and angiopoietin 2 (ANG2), increased in the ipsilateral lumbar spinal cord dorsal horn (SCDH) following CCI. The glycolysis-related enzymes, pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) significantly increased in the ipsilateral lumbar SCDH following CCI on POD 7 and 14 compared to those in the control rats. Double immunofluorescence staining indicated that VEGF and PKM2 were predominantly expressed in the astrocytes, whereas ANG2 and LDHA were predominantly expressed in the neurons. Intrathecal infusion of fumagillin significantly reduced the expression of angiogenic factors and glycolytic enzymes upregulated by CCI. The expression of hypoxia-inducible factor-1α (HIF-1α), a crucial transcription factor that regulates angiogenesis and glycolysis, was also upregulated after CCI and inhibited by fumagillin. We concluded that intrathecal fumagillin may reduce the expression of ANG2 and LDHA in neurons and VEGF and PKM2 in the astrocytes of the SCDH, further attenuating spinal angiogenesis in neuropathy-induced nociceptive sensitization. Hence, fumagillin may play a role in the inhibition of peripheral neuropathy-induced neuropathic pain by modulating glycolysis and angiogenesis.

Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

The rheumatoid arthritis drug auranofin exerts potent anti-lymphoma effect by stimulating TXNRD-mediated ROS generation and inhibition of energy metabolism

Since the survival of lymphoma patients who experience disease progression or relapse remains very poor, new therapeutic approaches and effective drugs are urgently needed. Here we show that auranofin (AF), an anti-rheumatoid drug thought to inhibit thioredoxin reductases (TXNRDs) as its mechanism of action, exhibited potent activity against multiple cancer types, especially effective against B cell lymphoma. Surprisingly, a knockdown of TXNRD1 and TXNRD2 did not cause significant cytotoxicity, suggesting that abrogation of TXNRD enzyme per se was insufficient to cause cancer cell death. Further mechanistic study showed that the interaction of AF with TXNRD could convert this antioxidant enzyme to a ROS-generating molecule via disrupting its electron transport, leading to a leak of electrons that interact with molecular oxygen to form superoxide. AF also suppressed energy metabolism by inhibiting both mitochondria complex II and the glycolytic enzyme GAPDH, leading to a significant depletion of ATP and inhibition of cancer growth in vitro and in vivo. Importantly, we found that the AF-mediated ROS stress could induce PD-L1 expression, revealing an unwanted effect of AF in causing immune suppression. We further showed that a combination of AF with anti-PD-1 antibody could enhance the anticancer activity in a syngeneic immune-competent mouse B-cell lymphoma model. Our study suggests that AF could be a potential drug for lymphoma treatment, and its combination with immune checkpoint inhibitors would be a logical strategy to increase the therapeutic activity.

In-depth metaproteomics analysis reveals the protein profile and metabolism characteristics in pork during refrigerated storage

Alterations in microbiotas and endogenous enzymes have been implicated in meat deterioration. However, the factors that mediate the interactions between meat quality and microbiome profile were inadequately investigated. In this study, we collected pork samples throughout the refrigeration period and employed metaproteomics to characterize both the pork and microbial proteins. Our findings demonstrated that pork proteins associated with the catabolic process are upregulated during storage compared to the initial stage. Pseudomonas, Clostridium, Goodfellowiella, and Gonapodya contribute to the spoilage process. Notably, we observed an elevated abundance of microbial proteins related to glycolytic enzymes in refrigerated pork, identifying numerous proteins linked to biogenic amine production, thus highlighting their essential role in microbial decay. Further, we reveal that many of these microbial proteins from Pseudomonas are ribosomal proteins, promoting enzyme synthesis by enhancing transcription and translation. This study provides intrinsic insights into the underlying mechanisms by which microorganisms contribute to meat spoilage.

Exploring variations in glycolytic and gluconeogenic enzymes and isoforms across breast cancer cell lines and tissues

It is well established that tumour cells undergo metabolic changes to acquire biological advantage over normal cells with activation of the glycolytic pathway, a process termed “Warburg effect”. Enzyme isoforms are alternative enzymatic forms with the same function but with different biochemical or epigenetic features. Moreover, isoforms may have varying impacts on different metabolic pathways. We challenge ourselves to analyse the glycolytic and gluconeogenic enzymes and isoforms in breast cancer, a complex and heterogeneous pathology, associated with high incidence and mortality rates especially among women.We analysed epithelial and tumour cell lines by RT-PCR and compared values to a publicly available database for the expression profile of normal and tumour tissues (Gepia) of enzymes and enzymatic isoforms from glycolytic and gluconeogenic pathways. Additionally, GeneMANIA was used to evaluate interactions, pathways, and attributes of each glycolytic/gluconeogenic steps.The findings reveal that the enzymes and enzymatic isoforms expressed in cell culture were somewhat different from those in breast tissue. We propose that the tumor microenvironment plays a crucial role in the expression of glycolytic and gluconeogenic enzymes and isoforms in tumour cells. Nonetheless, they not only participate in glycolytic and gluconeogenic enzymatic activities but may also influence other pathways, such as the Pentose-Phosphate-Pathway, TCA cycle, as well as other carbohydrate, lipid, and amino acid metabolism.

Effect of Hydroxyurea on Morphology, Proliferation, and Protein Expression on Taenia crassiceps WFU Strain.

Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.

Erjingpill bionic cerebrospinal fluid alleviates LPS-induced inflammatory response in BV2 cells by inhibiting glycolysis via mTOR

Ethnopharmacological relevanceErjingpill, a well-known prescription documented in the classic Chinese medical text “Shengji Zonglu,” has been proven to have effective alleviating effects on neuroinflammation in Alzheimer's disease (AD). Although the alterations in microglial cell glycolysis are known to play a crucial role in the development of neuroinflammation, it remains unclear whether the anti-neuroinflammatory effects of Erjingpill are associated with its impact on microglial cell glycolysis. Aim of the studyThis study aims to determine whether Erjingpill exerts anti-neuroinflammatory effects by influencing microglial cell glycolysis. Materials and methodsFirstly, Erjingpill decoction was prepared into an Erjingpill bionic cerebrospinal fluid (EBCF) through a process of in vitro intestinal absorption, hepatocyte incubation, and blood-brain barrier (BBB) transcytosis. Subsequently, UPLC/Q-TOF-MS/MS technology was used to analyze the compounds in Erjingpill and EBCF. Next, an in vitro neuroinflammation model was established by LPS-induced BV2 cells. The impact of EBCF on BV2 cell proliferation activity was evaluated using the CCK-8 assay, while the NO release was assessed using the Griess assay. Additionally, mRNA levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α, and COX-2), anti-inflammatory factors (IL-10, IL-4, Arg-1, and TGF-β), M1 microglial markers (iNOS, CD86), M2 microglial markers (CD36, CD206), and glycolytic enzymes (HK2, GLUT1, PKM, and LDHA) were measured using qPCR. Furthermore, protein expression of microglial activation marker Iba-1, M1 marker iNOS, and M2 marker CD206 were identified through immunofluorescence, while concentrations of pro-inflammatory cytokines IL-1β and TNF-α were measured using ELISA. Enzymatic activity of glycolytic enzymes (HK, PK, and LDH) was assessed using assay kits, and the protein levels of pro-inflammatory factors (IL-1β, iNOS, and COX-2), anti-inflammatory factors (IL-10 and Arg-1), and key glycolytic proteins GLUT1 and PI3K/AKT/mTOR were detected by Western blot. ResultsThrough the analysis of Erjingpill and EBCF, 144 compounds were identified in Erjingpill and 40 compounds were identified in EBCF. The results demonstrated that EBCF effectively inhibited the elevation of inflammatory factors and glycolysis levels in LPS-induced BV2 cells, promoted polarization of M1 microglial cells towards the M2 phenotype, and suppressed the PI3K/AKT/mTOR inflammatory pathway. Moreover, EBCF alleviated LPS-induced BV2 cell inflammatory response by modulating mTOR to inhibit glycolysis. ConclusionsEBCF exhibits significant anti-neuroinflammatory effects, likely attributed to its modulation of mTOR to inhibit microglial cell glycolysis. This study furnishes experimental evidence supporting the clinical utilization of Erjingpill for preventing and treating AD.

A novel polysaccharide of Undaria pinnatifida: Structural characterization, carboxymethylation and hypoglycemic activity in vivo

A new polysaccharide (UPP) was isolated and purified from Undaria pinnatifida with a low molecular weight of 8.34 kDa. The UPP was modified to obtain carboxymethylated polysaccharide (CM-UPP). UPP and CM-UPP were characterized by FT-IR, Ion chromatography, Congo red, DSC, SEM and NMR, and their hypoglycemic activity in vivo was investigated using the STZ-induced diabetes mice model. UPP mainly contained Ara, Gal, Glc and Man at a molar ratio of 0.18: 0.13: 2.06: 1, and CM-UPP added two alduronic acids including GalA and GlcA. CM-UPP had been prepared successfully and the carboxymethylation reaction might occur partially at the C-2 and C-4 position. There were no triple helix structures in both UPP and CM-UPP. In vivo hypoglycemic activity studies showed that both UPP and CM-UPP could alleviate symptoms of high blood glucose, improve blood lipid level, oxidative stress state and liver glycolytic enzyme activity in diabetic mice. Under the same dose, CM-UPP showed a more significant effect than UPP. These results indicated that carboxymethylation was an effective way to enhance the hypoglycemic activitiy of polysaccharide. UPP and CM-UPP could be potential therapeutic agents of T2DM.

CircRNA6448-14/miR-455-3p/OTUB2 axis stimulates glycolysis and stemness of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a gastrointestinal malignancy with high incidence. This study aimed to reveal the complete circRNA-miRNA-mRNA regulatory network in ESCC and validate its function mechanism. Expression of OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 2 (OTUB2) in ESCC was analyzed by bioinformatics to find the binding sites between circRNA6448-14 and miR-455-3p, as well as miR-455-3p and OTUB2. The binding relationships were verified by RNA Immunoprecipitation (RIP) and dual-luciferase assay. The expressions of circRNA6448-14, miR-455-3p, and OTUB2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). MTT assay measured cell viability, and the spheroid formation assay assessed the ability of stem cell sphere formation. Western blot (WB) determined the expression of marker proteins of stem cell surface and rate-limiting enzyme of glycolysis. The Seahorse XFe96 extracellular flux analyzer measured the rate of extracellular acidification rate and cellular oxygen consumption. Corresponding assay kits assessed cellular glucose consumption, lactate production, and adenosine triphosphate (ATP) generation. In ESCC, circRNA6448-14 and OTUB2 were highly expressed in contrast to miR-455-3p. Knocking down circRNA6448-14 could prevent the glycolysis and stemness of ESCC cells. Additionally, circRNA6448-14 enhanced the expression of OTUB2 by sponging miR-455-3p. Overexpression of OTUB2 or silencing miR-455-3p reversed the inhibitory effect of knockdown of circRNA6448-14 on ESCC glycolysis and stemness. This research demonstrated that the circRNA6448-14/miR-455-3p/OTUB2 axis induced the glycolysis and stemness of ESCC cells. Our study revealed a novel function of circRNA6448-14, which may serve as a potential therapeutic target for ESCC.

Pyruvate kinase deficiency in 29 Turkish patients with two novel intronic variants.

Pyruvate kinase (PK) is a key enzyme of anaerobic glycolysis. The genetic heterogeneity of PK deficiency (PKD) is high, and over 400 unique variants have been identified. Twenty-nine patients who had been diagnosed as PKD genetically in seven distinct paediatric haematology departments were evaluated. Fifteen of 23 patients (65.2%) had low PK levels. The PK:hexokinase ratio had 100% sensitivity for PKD diagnosis, superior to PK enzyme assay. Two novel intronic variants (c.695-1G>A and c.694+43C>T) have been described. PKD should be suspected in patients with chronic non-spherocytic haemolytic anaemia, even if enzyme levels are falsely normal. Total PKLR gene sequencing is necessary for the characterization of patients with PKD and for genetic counselling.

AMPK Drives both Glycolytic and Oxidative Metabolism in Murine and Human T Cells During Graft-versus-host Disease

Allogeneic T cells reprogram their metabolism during acute graft-versus-host disease (GVHD) in a process involving the cellular energy sensor AMP-activated protein kinase (AMPK). Deletion of AMPK in donor T cells limits GVHD but still preserves homeostatic reconstitution and graft-versus-leukemia (GVL) effects. In the current studies, murine AMPK KO T cells decreased oxidative metabolism at early timepoints post-transplant and lacked a compensatory increase in glycolysis following inhibition of the electron transport chain. Immunoprecipitation using an antibody specific to phosphorylated targets of AMPK determined that AMPK modified interactions of several glycolytic enzymes including aldolase, enolase, pyruvate kinase M (PKM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and enzyme assays indicated impaired aldolase and GAPDH activity in AMPK KO T cells. Importantly, these changes in glycolysis correlated with both an impaired ability of AMPK KO T cells to produce significant amounts of interferon gamma (IFNγ) upon antigenic re-stimulation and a decrease in the total number of donor CD4 T cells recovered at later time points post-transplant. Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following expansion in vivo. GVHD results also mirrored those of the murine model, with reduced CD4/CD8 ratios and a significant improvement in disease severity. Together these data highlight a significant role for AMPK in controlling oxidative and glycolytic metabolism in both murine and human T cells and endorse further study of AMPK inhibition as a potential clinical target for future GVHD therapies.

Proteomic profiling reveals higher presence of glycolytic enzymes in vulnerable atherosclerotic lesions

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq PlaqOmics Background Our definition of vulnerable plaques is predominantly driven by histological analysis. The molecular mechanisms behind the vulnerable histological characteristics are not fully understood. Here we use mass spectrometry-based proteomics data, to identify molecular mechanisms linked to vulnerable plaques. Methods Atherosclerotic lesions from two hundred patients undergoing endarterectomy surgery were collected (149 male and 51 female plaques). From a total of two hundred plaque samples, extracellular matrix proteins were extracted and processed for untargeted proteomics analysis. The resulting protein levels were linked to existing histological characteristics, transcriptomic, and clinical data. Results We detected 1499 proteins in atherosclerotic lesions, which were mostly affiliated with biological processes including extracellular matrix organization, coagulation, and peptidase activity. Linear regression identified 240 proteins associated with plaque vulnerability index (a semi-quantitative score summarizing the presence of SMC cells, macrophages, plaque hemorrhage, and fat in the plaque). Among the proteins associated with high plaque vulnerability index we identified an overrepresentation of enzymes involved in glycolysis – including the rate limiting enzymes HK3 (beta =0.40 p =0.003) and PKM (beta =0.19 p =0.009). This was driven by macrophage numbers and fat content in the plaque. Furthermore, data from a validation cohort of 120 patients from the Medical University of Vienna, Austria, revealed that HK3 and PKM were associated with plaque progression (p =<0.001, p =0.017) and clinical symptoms (p = <0.001, p = <0.001). Conclusion Enzymes involved in the glycolysis process show higher presence in vulnerable plaques. This suggests an important role for energy metabolism in plaque vulnerability.

Exploring the mechanisms of metabolic adaptations to cardiotoxic doxorubicin

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq Foundation Background Doxorubicin (DOX) is an anthracycline chemotherapeutic agent whose clinical application is hindered by the emergence of early and late cardiotoxic effects. Recent findings demonstrated that DOX damages cardiac mitochondria, with subsequent metabolic perturbation and energetic imbalance. We previously observed that phosphoinositide 3-kinase γ (PI3Kγ) contributes to DOX-induced cardiotoxicity, mediating mitophagy inhibition and accumulation of damaged mitochondria into cardiac cells. Objective Here we intend to describe the cardiotoxic metabolic phenotype of DOX-treated hearts, unravelling the contribution of PI3Kγ signalling to this process. Methods Wild-type (WT) and knock-in mice expressing a kinase-inactive PI3Kγ (kinase-dead; KD) were treated with DOX at day 0, 7 and 14 (cumulative dose 12 mg/kg). To explore the contribution of PI3Kγ on the early cardiac metabolic rewiring, metabolomic analysis, mitochondrial respiration capacity and glycolytic enzymes activity were evaluated at day 3. To investigate the role of autophagy, mice were infected with an adeno-associated virus 9 (AAV9) carrying a vector expressing a short-hairpin RNA against ATG7 (ATG7sh). To test the role of PI3Kγ in glycolysis regulation, glucose uptake and plasma membrane exposure of GLUT-4 were measured in neonatal mouse cardiomyocytes with DOX acute treatment (1µM for 3h). To evaluate the contribution of different substrates to oxygen consumption, OROBOROS analysis were performed on myocardial slices from mice sacrificed at day 3 and 42. Results DOX-treated cardiomyocytes exhibited a metabolic phenotype characterized by glucose as the primary energy substrate, with increased uptake and augmented glycolytic enzymes activity. Metabolomic analysis of DOX-treated hearts indicated diminished pyruvate levels, the glycolysis end-product, unaltered lactate but increased Acetyl-CoA quantity, suggesting enhanced glucose processing for the TCA cycle. Accordingly, oxygen consumption after pyruvate supplementation significantly increased in DOX-treated condition, resulting in the generation of cytotoxic ROS rather than energy production. This was mainly due to DOX-induced mitochondrial damage, with impaired fatty acid oxidation and electron transport chain activity, resulting in TCA cycle slow-down, with accumulation of cardiotoxic glucose-derived intermediates. These metabolic alterations were completely prevented in KD hearts. In vitro experiments demonstrated that inhibiting PI3Kγ reduced the activity of pyruvate dehydrogenase (PDH), the key enzyme of Randle cycle, regulating the switch from fatty acids to glucose usage, while decreasing DOX-induced mobilization of GLUT-4-carrying vesicles to the plasma membrane, limiting subsequent glucose uptake. Conclusion These results demonstrate that DOX promotes an early metabolic rewiring with PI3Kγ-dependent increased glucose uptake and modulation of the Randle cycle towards augmented glucose utilization.

Transcriptional regulation of hexokinase 2 by BRD4 drives perivascular adipose tissue meta-inflammation in cardiometabolic disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Holcim Stiftung SwissLife Stiftung Background/Introduction BRD4 is an epigenetic reader that modulates inflammatory transcriptional programmes implicated in cancer. The therapeutic potential of BRD4 modulation in cardiometabolic disorders remains elusive. Purpose We investigate BRD4-related transcriptional programmes and therapeutic modulation in mouse and human models of cardiometabolic disease. Methods Small arteries (100-300 μM) from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were dissected from visceral fat biopsies and mounted on a pressurised myograph to assess ex-vivo vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. Vasorelaxation was assessed in the presence or in the absence of perivascular adipose tissue (PVAT). An experimental mouse model of cardiometabolic disease (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) or vehicle for 10 days (n=6 each group) to test in vivo effect of chronic BRD4 inhibition on endothelial and PVAT functions. Vascular and PVAT levels of cytosolic and mitochondrial ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression were measured by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array and confirmed by ChIP, qPCR and Western blot and further characterised by metabolomics, lipidomics and mitochondrial swelling assays. Results Endothelial-dependent vasorelaxation and tissue levels of TNF-α, IL-1β and IL-6 were altered in the vessel wall and PVAT from cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction. Distinct ex-vivo modulation of well-established inflammatory pathways showed that the effect observed with BRD4 inhibition was stronger than with anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. (Figure 1). Gene expression profiling in PVAT from cardiometabolic mice unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validated the findings, showing a PVAT glycolytic shift in condition of disease. Finally, ex-vivo selective inhibition of HK2 restored vascular dysfunction (Figure 2). Conclusions Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic-related vascular dysfunction reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammatory pathways might represent a promising strategy to prevent and vascular dysfunction, key signature of cardiometabolic disease.Figure 1Figure 2

Exploring the therapeutic potential of rutin through investigating its inhibitory mechanism on lactate dehydrogenase: Multi-spectral methods and computer simulation

Lactate dehydrogenase (LDH), a crucial enzyme in anaerobic glycolysis, plays a pivotal role in the energy metabolism of tumor cells, positioning it as a promising target for tumor treatment. Rutin, a plant-based flavonoid, offers benefits like antioxidant, antiapoptotic, and antineoplastic effects. This study employed diverse experiments to investigate the inhibitory mechanism of rutin on LDH through a binding perspective. The outcomes revealed that rutin underwent spontaneous binding within the coenzyme binding site of LDH, leading to the formation of a stable binary complex driven by hydrophobic forces, with hydrogen bonds also contributing significantly to sustaining the stability of the LDH-rutin complex. The binding constant (Ka) for the LDH-rutin system was 2.692 ± 0.015 × 104 M−1 at 298 K. Furthermore, rutin induced the alterations in the secondary structure conformation of LDH, characterized by a decrease in α-helix and an increase in antiparallel and parallel β-sheet, and β-turn. Rutin augmented the stability of coenzyme binding to LDH, which could potentially hinder the conversion process among coenzymes. Specifically, Arg98 in the active site loop of LDH provided essential binding energy contribution in the binding process. These outcomes might explain the dose-dependent inhibition of the catalytic activity of LDH by rutin. Interestingly, both the food additives ascorbic acid and tetrahydrocurcumin could reduce the binding stability of LDH and rutin. Meanwhile, these food additives did not produce positive synergism or antagonism on the rutin binding to LDH. Overall, this research could offer a unique insight into the therapeutic potential and medicinal worth of rutin.