Bioenergetic Metabolism Research Articles

Is glycerol a good cryoprotectant for sperm cells? New exploration of its toxicity using avian model

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation.

Glucose oxidation drives trunk neural crest cell development and fate.

Bioenergetic metabolism is a key regulator of cellular function and signaling, but how it can instruct the behavior of cells and their fate during embryonic development remains largely unknown. Here, we investigated the role of glucose metabolism in the development of avian trunk neural crest cells (NCCs), a migratory stem cell population of the vertebrate embryo. We uncovered that trunk NCCs display glucose oxidation as a prominent metabolic phenotype, in contrast to what is seen for cranial NCCs, which instead rely on aerobic glycolysis. In addition, only one pathway downstream of glucose uptake is not sufficient for trunk NCC development. Indeed, glycolysis, mitochondrial respiration and the pentose phosphate pathway are all mobilized and integrated for the coordinated execution of diverse cellular programs, epithelial-to-mesenchymal transition, adhesion, locomotion, proliferation and differentiation, through regulation of specific gene expression. In the absence of glucose, the OXPHOS pathway fueled by pyruvate failed to promote trunk NCC adaptation to environmental stiffness, stemness maintenance and fate-decision making. These findings highlight the need for trunk NCCs to make the most of the glucose pathway potential to meet the high metabolic demands appropriate for their development.

MTA1, a Novel ATP Synthase Complex Modulator, Enhances Colon Cancer Liver Metastasis by Driving Mitochondrial Metabolism Reprogramming.

Liver metastasis is the most fatal event of colon cancer patients. Warburg effect has been long challenged by the fact of upregulated oxidative phosphorylation (OXPHOS), while its mechanism remains unclear. Here, metastasis-associated antigen 1 (MTA1) is identified as a newly identified adenosine triphosphate (ATP) synthase modulator by interacting with ATP synthase F1 subunit alpha (ATP5A), facilitates colon cancer liver metastasis by driving mitochondrial bioenergetic metabolism reprogramming, enhancing OXPHOS; therefore, modulating ATP synthase activity and downstream mTOR pathways. High-throughput screening of an anticancer drug shows MTA1 knockout increases the sensitivity of colon cancer to mitochondrial bioenergetic metabolism-targeted drugs and mTOR inhibitors. Inhibiting ATP5A enhances the sensitivity of liver-metastasized colon cancer to sirolimus in an MTA1-dependent manner. The therapeutic effects are verified in xenograft models and clinical cases. This research identifies a new modulator of mitochondrial bioenergetic reprogramming in cancer metastasis and reveals a new mechanism on upregulating mitochondrial OXPHOS as the reversal of Warburg effect in cancer metastasis is orchestrated.

Sodium-Glucose Cotransporter-2 Inhibitors in Depression.

Novel treatment strategies that refract existing treatment algorithms for depressive disorders are being sought. Abnormal brain bioenergetic metabolism may represent an alternative, therapeutically targetable neurobiological basis for depression. A growing body of research points to endogenous ketones as candidate neuroprotective metabolites with the potential to enhance brain bioenergetics and improve mood. Sodium-glucose cotransporter-2 (SGLT2) inhibitors, originally approved for the treatment of diabetes, induce ketogenesis and are associated with mood improvement in population-based studies. In this column, we highlight the rationale for the hypothesis that ketogenesis induced by SGLT2 inhibitors may be an effective treatment for depressive disorders.

Adenine nucleotide translocase: Current knowledge in post-translational modifications, regulations and pathological implications for human diseases.

Adenine nucleotide translocases (ANTs) are central to mitochondrial integrity and bioenergetic metabolism. This review aims to integrate the progresses and knowledge on ANTs over the last few years, contributing to a potential implication of ANTs for various diseases. Structures, functions, modifications, regulators and pathological implications of ANTs for human diseases are intensively demonstrated here. ANTs have four isoforms (ANT1-4), responsible for exchanging ATP/ADP, possibly composing of pro-apoptotic mPTP as a major component, and mediating FA-dependent uncoupling of proton efflux. ANT can be modified by methylation, nitrosylation and nitroalkylation, acetylation, glutathionylation, phosphorylation, carbonylation and hydroxynonenal-induced modifications. Compounds, including bongkrekic acid, atractyloside calcium, carbon monoxide, minocycline, 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid, cardiolipin, free long-chain fatty acids, agaric acid, long chain acyl-coenzyme A esters, all have an ability to regulate ANT activities. ANT impairment leads to bioenergetic failure and mitochondrial dysfunction, contributing to pathogenesis of diseases, such as diabetes (deficiency), heart disease (deficiency), Parkinson's disease (reduction), Sengers Syndrome (decrease), cancer (isoform shifting), Alzheimer's Disease (coaggregation with Tau), Progressive External Opthalmoplegia (mutation), and Fascioscapulohumeral muscular dystrophy (overexpression). This review improves the understanding of the mechanism of ANT in pathogenesis of human diseases, and opens a window for novel therapeutic strategies targeted on ANT in diseases.

Oxidative phosphorylation in bone cells

The role of energy metabolism in bone cells is an active field of investigation. Bone cells are metabolically very active and require high levels of energy in the form of adenosine triphosphate (ATP) to support their function. ATP is generated in the cytosol via glycolysis coupled with lactic acid fermentation and in the mitochondria via oxidative phosphorylation (OXPHOS). OXPHOS is the final convergent metabolic pathway for all oxidative steps of dietary nutrients catabolism. The formation of ATP is driven by an electrochemical gradient that forms across the mitochondrial inner membrane through to the activity of the electron transport chain (ETC) complexes and requires the presence of oxygen as the final electron acceptor. The current literature supports a model in which glycolysis is the main source of energy in undifferentiated mesenchymal progenitors and terminally differentiated osteoblasts, whereas OXPHOS appears relevant in an intermediate stage of differentiation of those cells. Conversely, osteoclasts progressively increase OXPHOS during differentiation until they become multinucleated and mitochondrial-rich terminal differentiated cells. Despite the abundance of mitochondria, mature osteoclasts are considered ATP-depleted, and the availability of ATP is a critical factor that regulates the low survival capacity of these cells, which rapidly undergo death by apoptosis. In addition to ATP, bioenergetic metabolism generates reactive oxygen species (ROS) and intermediate metabolites that regulate a variety of cellular functions, including epigenetics changes of genomic DNA and histones. This review will briefly discuss the role of OXPHOS and the cross-talks OXPHOS-glycolysis in the differentiation process of bone cells.

Lnk/Sh2b3 modulates bioenergetic metabolism of activated CD8 T cells and control the development of Type 1 Diabetes

Abstract Recent genome-wide association studies (GWAS) have shown a strong correlation between a genetic polymorphism in the SH2B3gene and the development of autoimmune diseases, including type 1 diabetes (T1D). Lnk/Sh2b3 is an adaptor protein interacting with various intracellular proteins involved in JAK/STAT cytokine signaling of different cell types, including CD8 T cells. Additionally, Sh2b3-deficient CD8 T cells are hypersensitive to IL-15 stimulation. However, how reduced Sh2b3 function affects the molecular mechanisms that activate and sustain CD8 T cells promoting T1D development remains elusive. We hypothesized that genetic deletion of Sh2b3 in the non-obese diabetic (NOD) mouse would impact T1D incidence by altering T cell activation. NOD. Sh2b3−/− mice developed diabetes faster than wild-type controls. Additionally, Sh2b3-deficient CD8 T cells were hyperresponsive to IL-15 stimulation, promoting the generation of the activated CD44 high CD8 T cell population. Intriguingly, IL-15-stimulated Sh2b3−/− splenocytes possessed significantly higher mitochondrial respiratory capacity (SRC) and ATP synthesis than the wild-type counterparts. Furthermore, CD8 T cells of NOD. Sh2b3−/− mice showed increased expression of genes associated with mitochondrial biogenesis and oxidative metabolisms in response to in vitroIL-15 stimulation. Overall, our study supports a role of Sh2b3 in T1D development by regulating mitochondrial biogenesis and metabolism of IL-15-stimulated CD8 T cell. Our study provides new insight into the role of Sh2b3 in activating and sustaining the CD8 T cell response in the pathogenesis of T1D. Funded by NIH DK097605 Funded by NIH DK097605

Immunometabolic modulation by targeting UCP2 and IL-17A boost anti-tumor immunity against pancreatic cancer

Abstract Metabolic reprograming of cytotoxic effectors by tumor microenvironment (TME) is one of the key mechanisms by which TME impairs the success of cancer immunotherapy. Therapeutic strategies that mitigate the immunosuppressive TME and selectively enhance bioenergetic fitness of cytotoxic effectors may enhance benefits of immunotherapy, particularly in TME-abundant pancreatic cancer. IL-17 is a key TME cytokine engaged in a feed-forward loop promoting the immunosuppressive milieu that facilitates cancer progression. Mitochondrial uncoupling protein 2 (UCP2) acts as an inhibitor of T cell metabolic fitness and directly promotes tumorigenesis. Therefore, we hypothesized that targeting IL-17 and UCP2 could act additionally to enhance antitumor immune response and directly inhibit pancreatic cancer growth. UCP2 deficiency regulated bioenergetic metabolism and mTOR signaling in cytotoxic T cells to enhance IFN-γ production and anti-tumor immune responses. UCP2 inhibition increased anti-tumor immunity in PBMCs and tumors of patients with PDAC. The dual inhibition of IL-17 and UCP2 boosted antitumor immunity and suppressed tumorigenesis in tumor-bearing mice. Combination of UCP2 and IL-17 blockade may offer additional complimentary and cooperative mechanisms of antitumor immunity against pancreatic cancer. 1. Ministry of Science and Technology (MOST 108-2320-B-039-024-MY3) 2. China Medical University Hospital (DMR-110-186) 3. Chinese Medicine Research Center, China Medical University from the Featured Areas Research 4. Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (CMRC-CHM-2)

The histone demethylase KDM5C controls female bone mass by promoting energy metabolism in osteoclasts.

Women experience osteoporosis at higher rates than men. Aside from hormones, the mechanisms driving sex-dependent bone mass regulation are not well understood. Here, we demonstrate that the X-linked H3K4me2/3 demethylase KDM5C regulates sex-specific bone mass. Loss of KDM5C in hematopoietic stem cells or bone marrow monocytes increases bone mass in female but not male mice. Mechanistically, loss of KDM5C impairs the bioenergetic metabolism, resulting in impaired osteoclastogenesis. Treatment with the KDM5 inhibitor reduces osteoclastogenesis and energy metabolism of both female mice and human monocytes. Our report details a sex-dependent mechanism for bone homeostasis, connecting epigenetic regulation to osteoclast metabolism and positions KDM5C as a potential target for future treatment of osteoporosis in women.

Abstract 3969: Screening of kinase inhibitors as bioenergetic metabolism modulator using the XF Real-Time ATP Rate Assay

Abstract Cancer cells are metabolically reprogrammed through tumor initiation and progression, and there are increasing developments in cancer therapies exploiting the potential metabolic vulnerability. A better understanding of cancer cell metabolic regulation can also contribute to modulating the harsh tumor microenvironment, which strongly affects the efficacy of emerging anti-cancer immunotherapies. As one of the critical contributors to cancer therapy design, there is increasing demand for time-efficient and reliable analytic tools when searching for drugs and genes modulating cancer cell metabolism. The XF Real-Time ATP Rate Assay measures ATP production from glycolysis and mitochondrial respiration simultaneously and provides a quantitative overview of the bioenergetic phenotype of the cells. By using ATP production rate as a universal unit of bioenergetic metabolism, this assay quickly detects metabolic modulations induced by chemical stimuli or genetic modifications. It allows the identification of potential targets for therapy design. In this study, we used the Seahorse XF Pro Analyzer combined with the XF ATP Rate assay to screen 80 kinase inhibitors for the suppressive effects on energy metabolism and/or inducing a metabolic phenotype shift in THP-1 cells, a monocytic leukemia cell line, using PBMC as the control. We identified four mitochondrial and two glycolytic suppressors using the screening and dose-response views available in Seahorse Analytics, a cloud-based data analysis tool for Seahorse XF assays. These compounds showed moderate or no effects on the net ATP production rates but induced a significant shift in the metabolic phenotype of THP-1 cells. Among the selected kinase inhibitors, AG-879 (ErbB2 inhibitor), SU-1498 (VEGFR inhibitor), and U-0126 (MEK1/2 inhibitor) showed the most potent inhibitory effect on mitochondrial energy production in THP-1 cells. Dose-response studies indicated that SU-1498 and U-0126 have higher potency in THP-1 than PBMC, and thus they can be considered good cancer-cell targets for mitochondrial suppressors. In contrast, AG-879 showed a similar or higher inhibitory effect on PBMC even though it is the most potent mitochondrial suppressor among the selected compounds. These results demonstrate that the XF Real-Time ATP Rate Assay can be used as a primary assay for screening and validating drug candidate(s) targeting the bioenergetic metabolism of cancer cells. Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. Screening of kinase inhibitors as bioenergetic metabolism modulator using the XF Real-Time ATP Rate Assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3969.

Resting mitochondrial complex I from Drosophila melanogaster adopts a helix-locked state.

Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deactive' state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.

Synthetic Analogues of Gibbilimbol B Induce Bioenergetic Damage and Calcium Imbalance in Trypanosoma cruzi

Chagas disease is an endemic tropical disease caused by the protozoan Trypanosoma cruzi, which affects around 7 million people worldwide, mostly in development countries. The treatment relies on only two available drugs, with severe adverse effects and a limited efficacy. Therefore, the search for new therapies is a legitimate need. Within this context, our group reported the anti-Trypanosoma cruzi activity of gibbilimbol B, a natural alkylphenol isolated from the plant Piper malacophyllum. Two synthetic derivatives, LINS03018 (1) and LINS03024 (2), demonstrated a higher antiparasitic potency and were selected for mechanism of action investigations. Our studies revealed no alterations in the plasma membrane potential, but a rapid alkalinization of the acidocalcisomes. Nevertheless, compound 1 exhibit a pronounced effect in the bioenergetics metabolism, with a mitochondrial impairment and consequent decrease in ATP and reactive oxygen species (ROS) levels. Compound 2 only depolarized the mitochondrial membrane potential, with no interferences in the respiratory chain. Additionally, no macrophages response of nitric oxide (NO) was observed in both compounds. Noteworthy, simple structure modifications in these derivatives induced significant differences in their lethal effects. Thus, this work reinforces the importance of the mechanism of action investigations at the early phases of drug discovery and support further developments of the series.

Glial Cell Metabolic Profile Upon Iron Deficiency: Oligodendroglial and Astroglial Casualties of Bioenergetic Adjustments.

Iron deficiency (ID) represents one of the most prevalent nutritional deficits, affecting almost two billion people worldwide. Gestational iron deprivation induces hypomyelination due to oligodendroglial maturation deficiencies and is thus a useful experimental model to analyze oligodendrocyte (OLG) requirements to progress to a mature myelinating state. A previous proteomic study in the adult ID brain by our group demonstrated a pattern of dysregulated proteins involved in the tricarboxylic acid cycle and mitochondrial dysfunction. The aim of the present report was to assess bioenergetics metabolism in primary cultures of OLGs and astrocytes (ASTs) from control and ID newborns, on the hypothesis that the regulation of cell metabolism correlates with cell maturation. Oxygen consumption and extracellular acidification rates were measured using a Seahorse extracellular flux analyzer. ID OLGs and ASTs both exhibited decreased spare respiratory capacity, which indicates that ID effectively induces mitochondrial dysfunction. A decrease in glycogen granules was observed in ID ASTs, and an increase in ROS production was detected in ID OLGs. Immunolabeling of structural proteins showed that mitochondrial number and size were increased in ID OLGs, while an increased number of smaller mitochondria was observed in ID ASTs. These results reflect an unfavorable bioenergetic scenario in which ID OLGs fail to progress to a myelinating state, and indicate that the regulation of cell metabolism may impact cell fate decisions and maturation.

EFFECT OF GLYCOLYSIS SUPRESSION ON ULTRASTRUCTURE OF THE HIPPOCAMPAL GIANT SYNAPSES

Suppression of glycolysis in the rat hippocampal formations was induced by daily intracerebroventricular injection of 2-deoxy-D-glucose for 10 days. Using electron microscopy, a disturbance of morphological interactions of axonal terminals of granular neurons with perisynaptic astrocytic processes was found. At the same time, both the determinant ultrastructural characteristics of giant synapses and two types of functional contacts (asymmetric chemical active zones with dendritic spines and symmetric adhesive junctions with dendrites) were retained. However, they showed morphological signs of a weakening of synaptic processes, which were expressed in a decrease in the number of intraterminal active zones, clustering of synaptic vesicles, and their removal from neurotransmitter release sites. Compared to the norm, an increase in the number of small mitochondria with an electron-dense matrix and the evidence of initiation of glycogen synthesis in the form of glycosomes were found in the giant terminals. The data obtained indicate that the giant synapses of the hippocampal mossy fibers are a plastic, self-correcting system that is able to function by adapting its own bioenergetic metabolism, when brain glycolysis is suppressed.

Lethal action of Licarin A derivatives in Leishmania (L.) infantum: Imbalance of calcium and bioenergetic metabolism

Natural metabolites present an extraordinary chemo-diversity and have been used as the inspiration for new drugs. Considering the need for new treatments against the neglected parasitic disease leishmaniasis, three semi-synthetic derivatives of natural neolignane licarin A were prepared: O-acetyl (1a), O-allyl (1b), and 5-allyl (1c). Using an ex vivo assay, compounds 1a, 1b, and 1c showed activity against the intracellular amastigotes of Leishmania (L.) infantum, with IC50 values of 9, 13, and 10 μM, respectively. Despite no induction of hemolytic activity, only compound 1b resulted in mammalian cytotoxicity (CC50 = 64 μM). The most potent compounds (1a and 1c) resulted in selectivity indexes >18. The mechanism of action of compound 1c was evaluated by fluorescent/luminescent based techniques and MALDI-TOF/MS. After a short incubation period, increased levels of the cytosolic calcium were observed in the parasites, with alkalinization of the acidocalcisomes. Compound 1c also induced mitochondrial hyperpolarization, resulting in decreased levels of ATP without altering the reactive oxygen species (ROS). Neither plasma membrane damages nor DNA fragmentation were observed after the treatment, but a reduction in the cellular proliferation was detected. Using MALDI-TOF/MS, mass spectral alterations of promastigote proteins were observed when compared to untreated and miltefosine-treated groups. This chemically modified neolignan induced lethal alterations of the bioenergetic and protein metabolism of Leishmania. Future PKPD and animal efficacy studies are needed to optimize this promising natural-derived compound.

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis.

The protoplast experimental system eis an effective tool for functional genomics and cell fusion breeding. However, the physiological and molecular mechanisms of protoplast response to enzymolysis are not clear, which has become a major obstacle to protoplast regeneration. Here, we used physiological, cytological, proteomics and gene expression analysis to compare the young leaves of sugarcane and enzymolized protoplasts. After enzymatic digestion, we obtained protoplasts with viability of > 90%. Meanwhile, the content of malondialdehyde, an oxidation product, increased in the protoplasts following enzymolysis, and the activity of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), acid peroxidase (APX), and O2-, significantly decreased. Cytologic analysis results showed that, post enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed several short rod-like structures in protoplasts. In this study, a proteomics approaches was used to identify proteins of protoplasts in response to the enzymatic digestion process. GO, KEGG, and KOG enrichment analyses revealed that the abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which allow for protein biosynthesis or degradation. RT-qPCR analysis revealed that the expression of osmotic stress resistance genes, such as DREB, WRKY, MAPK4, and NAC, was upregulated, while that of key regeneration genes, such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA, and GAUT, was significantly downregulated in the protoplasts. Hierarchical clustering and identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. Our findings can facilitate the development of a standard system to produce regenerated protoplasts using molecular markers and antibody detection of enzymolysis.

1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases

The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 μM 3a or 0.5 μM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.

Parallel Detection of Single-Cells and Extracellular Vesicles from Bone Marrow and Peripheral Blood Reveals Asymmetric Immunometabolic Profile in Acute Myeloid Leukemia

Introduction. Acute myeloid leukemia (AML) is an aggressive disease with a high relapse rate. Alteration in cell metabolism and metabolic adaptation are crucial hallmarks in AML. Although several studies have addressed the metabolic profile of leukemic cells, the metabolic cross-modulation of immune cells according to their resident (bone marrow, BM) and circulating (peripheral blood, PB) status has been poorly investigated. Growing evidence has highlighted a key role for extracellular vesicles (EVs), shed from a broad variety of cells and cargo of crucial factors, in modulating several functions of leukemic cells, including bioenergetic metabolism. Again, few studies have addressed the impact of EVs in regulating the metabolism of immune cells from leukemic microenvironment. Therefore, in our work, we dissected the immunometabolic profile of immune cells and EV signature profile in parallel from BM versus PB from AML patients. Methods. BM/PB samples and plasma were collected from AML patients at diagnosis (n=40). Leukemic EVs (herein referred to as AML EVs) were purified from platelet-poor plasma BM/PB by size exclusion chromatography and ultrafiltration. MACSPlex Exosome Kit was used for screening surface EV markers. Flow cytometry-based Single Cell ENergetic metabolism by profilIng Translation inhibition (SCENITH) method was applied in fresh whole blood. In addition, BM/PB mononuclear cells from AML patients were characterized by SCENITH after co-culture with circulating AML EVs. Results. To explore the metabolic reprogramming within the leukemic immune microenvironment, we firstly applied the SCENITH method in multiple immune cell subpopulations from AML patients. In comparison with the paired PB, we observed a significant increase in glucose dependence of AML BM CD8+ T cells. By contrast, the capacity to use fatty acids and amino acids as sources for ATP production when glucose oxidation is inhibited was prominently reduced in AML BM CD8+ T cells and increased in AML PB CD8+ T cells. Also, mitochondrial dependence of CD8+ T cells was higher in the BM compartment, suggesting a switch from oxidative phosphorylation to glycolysis in circulation. Interestingly, in comparison to paired PB, only BM CD3+ and CD4+ T cells from female AML patients revealed a marked increase in their glucose dependence reporting gender-dependent metabolic differences. Towards clinically relevant implication, according to European LeukemiaNet (ELN) 2017 risk classification, SCENITH analysis showed a critical reduction in mitochondrial dependence at the expense of glycolytic capacity in circulating CD4+ T cells from favorable-risk AML compared to intermediate/adverse-risk patients. Given the immunometabolic profile between BM and PB cell compartments, we hypothesized that EVs may differentially contribute to immunometabolism according to their BM versus PB localization. Preliminarily, we explored the immune profiling of plasma-derived EVs from AML BM versus PB. We found that BM EVs exhibited higher expressions of specific markers including CD4, CD40, ROR-1 and HLA-II/-ABC compared to PB equivalents. Of note, we observed a novel link between metabolic regulation and EV profiling. Indeed, the glucose dependence of PB CD8+ T cells was negatively correlated to the expression of CD133-1 (stem cell marker) and CD209 (dendritic marker) depicted on circulating (PB) EVs. Finally, we co-cultured AML circulating EVs with BM/PB mononuclear cells from AML patients in pairs to investigate the EV's role in metabolic regulation. Notably, BM CD8+ T cells in monocultures showed a reduction in glucose dependence compared to their PB equivalents. In parallel, mitochondrial dependence was more pronounced in PB CD4+ T cells compared to other circulating immune subsets. However, PB EVs caused a metabolic shift in the PB compartment towards high glycolytic capacity in CD8+ T cells and high mitochondrial dependency in CD34+ cells suggesting an EV-driven metabolic rewiring. Apparently, the co-cultures with PB EVs did not influence the energy profile of resident (BM) immune subsets. Conclusions. Overall, this study shows the immunometabolic asymmetry orchestrated in AML at diagnosis, driven by distinct immune cell subsets according to their location (BM versus PB). We also describe the putative role of AML EVs in subverting the immune cell metabolism revealing critical implications for immunotherapy in AML.

Microfluidic devices as model platforms of CNS injury-ischemia to study axonal regeneration by regulating mitochondrial transport and bioenergetic metabolism

Central nervous system (CNS) neurons typically fail to regenerate their axons after injury leading to neurological impairment. Axonal regeneration is a highly energy-demanding cellular program that requires local mitochondria to supply most energy within injured axons. Recent emerging lines of evidence have started to reveal that injury-triggered acute mitochondrial damage and local energy crisis contribute to the intrinsic energetic restriction that accounts for axon regeneration failure in the CNS. Characterizing and reprogramming bioenergetic signaling and mitochondrial maintenance after axon injury-ischemia is fundamental for developing therapeutic strategies that can restore local energy metabolism and thus facilitate axon regeneration. Therefore, establishing reliable and reproducible neuronal model platforms is critical for assessing axonal energetic metabolism and regeneration capacity after injury-ischemia. In this focused methodology article, we discuss recent advances in applying cutting-edge microfluidic chamber devices in combination with state-of-the-art live-neuron imaging tools to monitor axonal regeneration, mitochondrial transport, bioenergetic metabolism, and local protein synthesis in response to injury-ischemic stress in mature CNS neurons.

PGC-1β maintains mitochondrial metabolism and restrains inflammatory gene expression.

Metabolic programming of the innate immune cells known as dendritic cells (DCs) changes in response to different stimuli, influencing their function. While the mechanisms behind increased glycolytic metabolism in response to inflammatory stimuli are well-studied, less is known about the programming of mitochondrial metabolism in DCs. We used lipopolysaccharide (LPS) and interferon-β (IFN-β), which differentially stimulate the use of glycolysis and oxidative phosphorylation (OXPHOS), respectively, to identify factors important for mitochondrial metabolism. We found that the expression of peroxisome proliferator-activated receptor gamma co-activator 1β (PGC-1β), a transcriptional co-activator and known regulator of mitochondrial metabolism, decreases when DCs are activated with LPS, when OXPHOS is diminished, but not with IFN-β, when OXPHOS is maintained. We examined the role of PGC-1β in bioenergetic metabolism of DCs and found that PGC-1β deficiency indeed impairs their mitochondrial respiration. PGC-1β-deficient DCs are more glycolytic compared to controls, likely to compensate for reduced OXPHOS. PGC-1β deficiency also causes decreased capacity for ATP production at steady state and in response to IFN-β treatment. Loss of PGC-1β in DCs leads to increased expression of genes in inflammatory pathways, and reduced expression of genes encoding proteins important for mitochondrial metabolism and function. Collectively, these results demonstrate that PGC-1β is a key regulator of mitochondrial metabolism and negative regulator of inflammatory gene expression in DCs.