Low Oxidative Phosphorylation Research Articles

Metabolism of primary high-grade serous ovarian carcinoma (HGSOC) cells under limited glutamine or glucose availability

BackgroundHigh-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive subtype of epithelial ovarian carcinoma. It is primarily diagnosed at stage III or IV when the 5-year survival rate ranges between 20% and 40%. Here, we aimed to validate the hypothesis, based on HGSOC cell lines, that proposed the existence of two distinct groups of HGSOC cells with high and low oxidative phosphorylation (OXPHOS) metabolism, respectively, which are associated with their responses to glucose and glutamine withdrawal.MethodsWe isolated and cultivated primary cancer cell cultures from HGSOC and nontransformed ovarian fibroblasts from the surrounding ovarium of 45 HGSOC patients. We tested the metabolic flexibility of the primary cells, particularly in response to glucose and glutamine depletion, analyzed and modulated endoplasmic reticulum stress, and searched for indices of the existence of previously reported groups of HGSOC cells with high and low OXPHOS metabolism.ResultsThe primary HGSOC cells did not form two groups with high and low OXPHOS that responded differently to glucose and glutamine availabilities in the cell culture medium. Instead, they exhibited a continuum of OXPHOS phenotypes. In most tumor cell isolates, the responses to glucose or glutamine withdrawal were mild and surprisingly correlated with those of nontransformed ovarian fibroblasts from the same patients. The growth of tumor-derived cells in the absence of glucose was positively correlated with the lipid trafficking regulator FABP4 and was negatively correlated with the expression levels of HK2 and HK1. The correlations between the expression of electron transport chain (ETC) proteins and the oxygen consumption rates or extracellular acidification rates were weak. ER stress markers were strongly expressed in all the analyzed tumors. ER stress was further potentiated by tunicamycin but not by the recently proposed ER stress inducers based on copper(II)-phenanthroline complexes. ER stress modulation increased autophagy in tumor cell isolates but not in nontransformed ovarian fibroblasts.ConclusionsAnalysis of the metabolism of primary HGSOC cells rejects the previously proposed hypothesis that there are distinct groups of HGSOC cells with high and low OXPHOS metabolism that respond differently to glutamine or glucose withdrawal and are characterized by ETC protein levels.

Insulin sensitivity and insulin secretion in adults with Friedreich's Ataxia: the role of skeletal muscle.

Friedreich's Ataxia (FRDA) is a multi-system disorder caused by frataxin deficiency. FRDA-related diabetes mellitus (DM) is common. Frataxin supports skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, a mediator of insulin sensitivity. Our objective was to test the association between skeletal muscle health and insulin sensitivity and secretion in adults with FRDA without DM. Case-control study (NCT02920671). Glucose and insulin metabolism (stable-isotope oral glucose tolerance tests), body composition (dual-energy x-ray absorptiometry), physical activity (self-report), and skeletal muscle OXPHOS capacity (creatine chemical exchange saturation transfer MRI) were assessed. Participants included 11 individuals with FRDA (4 female), median age 27y (IQR 23, 39), BMI 26.9kg/m2 (24.1, 29.4), and 24 controls (11 female), 29y (26, 39), 24.4kg/m2 (21.8, 27.0). Fasting glucose was higher in FRDA (91 vs. 83mg/dL (5.0 vs. 4.6mmol/L), p<0.05). Individuals with FRDA had lower insulin sensitivity (WBISI 2.8 vs. 5.3, p<0.01), higher post-prandial insulin secretion (insulin secretory rate iAUC 30-180 minutes, 24,652 vs. 17,858, p<0.05), and more suppressed post-prandial endogenous glucose production (-0.9% vs. 26.9% of fasting EGP, p<0.05). In regression analyses, lower OXPHOS and inactivity explained some of the difference in insulin sensitivity. More visceral fat contributed to lower insulin sensitivity independent of FRDA. Insulin secretion accounting for sensitivity (disposition index) was not different. Lower mitochondrial OXPHOS capacity, inactivity, and visceral adiposity contribute to lower insulin sensitivity in FRDA. Higher insulin secretion appears compensatory, and when inadequate, could herald DM. Further studies are needed to determine if muscle- or adipose-focused interventions could delay FRDA-related DM.

Abstract 6234: Single-cell analysis of state-transition from health to leukemia and the leukemic cell heterogeneities in inv(16) acute myeloid leukemia (AML)

Abstract AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. Using a conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre) to mimic the somatic acquisition of fusion gene CBFB-MYH11 (CM), created in the recurrent chromosome translocation inv(16)(p13q22) of AML, we previously reported that the blood transcriptome undergoes state-transition from health to leukemia. In this study, we analyze the transcriptomic alterations and leukemic cell heterogeneities in a leukemia state-space using single-cell RNA sequencing (scRNA-seq). CM expression was induced in Cbfb56M/+/Mx1-Cre mice by poly (I:C) injection, and scRNA-seq analysis was performed on peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells from leukemic mice and age-matched wild-type (WT) controls. More than 105,000 cells were subjected to the following dimensionality reduction and cell type annotation. By performing principal component analysis (PCA), we observed a Y-shape cell distribution with emergence of a divergent branch occupied by leukemic cells which can be mapped to a leukemic state. The principal components 1 and 2 (PC1 and PC2) clearly distinguish the leukemic cells from normal cell types such as myeloid cells and lymphocytes. By accessing the eigenvalue of genes for PC1 and PC2, we quantified the crucial genes and pathways related to leukemia progression at a single-cell level. We characterized cKit+ leukemic cell clusters by transcriptome-based correlation and gene expression pattern and found that they resembled stem cells and megakaryocytic-erythroid progenitors (MEP) with dysregulated pathways, including upregulated "inflammatory response" and downregulated pathways related to Myc targets, oxidative phosphorylation, mitochondrial protein translation, and ribosomal biogenesis/assembly. Examining the 31 clusters (C0-C30) seen in cKit+ BM cells, we identified 9 leukemic clusters exhibiting distinct features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). In conclusion, our study unveils state-transition from health to leukemia at single-cell resolution, and uncovers diverse biological heterogeneities associated with the inv(16) AML state. Further refinement of transcriptome state-transition and single-cell analysis will provide an analytical framework for modeling the dynamic changes during disease evolution at the single-cell level. Citation Format: Yu-Hsuan Fu, Lianjun Zhang, Ying-Chieh Chen, David E. Frankhouser, Lisa Uechi, Denis O'Meally, Sergio Branciamore, Guido Marcucci, Russell Rockne, Ya-Huei Kuo. Single-cell analysis of state-transition from health to leukemia and the leukemic cell heterogeneities in inv(16) acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6234.

The Molecular Mechanisms behind Advanced Breast Cancer Metabolism: Warburg Effect, OXPHOS, and Calcium.

Altered metabolism represents a fundamental difference between cancer cells and normal cells. Cancer cells have a unique ability to reprogram their metabolism by deviating their reliance from primarily oxidative phosphorylation (OXPHOS) to glycolysis, in order to support their survival. This metabolic phenotype is referred to as the "Warburg effect" and is associated with an increase in glucose uptake, and a diversion of glycolytic intermediates to alternative pathways that support anabolic processes. These processes include synthesis of nucleic acids, lipids, and proteins, necessary for the rapidly dividing cancer cells, sustaining their growth, proliferation, and capacity for successful metastasis. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with the poorest patient outcome due to its high rate of metastasis. TNBC is characterized by elevated glycolysis and in certain instances, low OXPHOS. This metabolic dysregulation is linked to chemotherapeutic resistance in TNBC research models and patient samples. There is more than a single mechanism by which this metabolic switch occurs and here, we review the current knowledge of relevant molecular mechanisms involved in advanced breast cancer metabolism, focusing on TNBC. These mechanisms include the Warburg effect, glycolytic adaptations, microRNA regulation, mitochondrial involvement, mitochondrial calcium signaling, and a more recent player in metabolic regulation, JAK/STAT signaling. In addition, we explore some of the drugs and compounds targeting cancer metabolic reprogramming. Research on these mechanisms is highly promising and could ultimately offer new opportunities for the development of innovative therapies to treat advanced breast cancer characterized by dysregulated metabolism.

Hepatitis B virus e antigen induces atypical metabolism and differentially regulates programmed cell deaths of macrophages.

Macrophages can undergo M1-like proinflammatory polarization with low oxidative phosphorylation (OXPHOS) and high glycolytic activities or M2-like anti-inflammatory polarization with the opposite metabolic activities. Here we show that M1-like macrophages induced by hepatitis B virus (HBV) display high OXPHOS and low glycolytic activities. This atypical metabolism induced by HBV attenuates the antiviral response of M1-like macrophages and is mediated by HBV e antigen (HBeAg), which induces death receptor 5 (DR5) via toll-like receptor 4 (TLR4) to induce death-associated protein 3 (DAP3). DAP3 then induces the expression of mitochondrial genes to promote OXPHOS. HBeAg also enhances the expression of glutaminases and increases the level of glutamate, which is converted to α-ketoglutarate, an important metabolic intermediate of the tricarboxylic acid cycle, to promote OXPHOS. The induction of DR5 by HBeAg leads to apoptosis of M1-like and M2-like macrophages, although HBeAg also induces pyroptosis of the former. These findings reveal novel activities of HBeAg, which can reprogram mitochondrial metabolism and trigger different programmed cell death responses of macrophages depending on their phenotypes to promote HBV persistence.

Single-Cell RNA-Seq Reveals Intermediate Cell States and Identifies Features Defining Cellular Heterogeneity in Inv(16) Acute Myeloid Leukemia (AML)

AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. One recurrent chromosomal translocation common in AML is chromosome 16 inversion, inv(16)(p13.1q22), which results in the leukemogenic fusion gene CBFB- MYH11. Using a conditional Cbfb-MYH11 (CM) knock-in mouse model to mimic the somatic expression of the CM fusion gene, we demonstrated that CM expression leads to impaired hematopoietic differentiation as well as accumulation of phenotypic hematopoietic stem cells (HSC) and pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitors, which are predisposed to leukemic transformation. Flow cytometry-based phenotypic analysis revealed that the transformed leukemic blasts are predominantly characterized as pre-granulocyte-macrophage progenitor (pre-GM) (Lin −cKit +Sca1 −CD16/32 -/loCD150 −CD105 −). However, cell type characterization via conventional surface markers is limited by known cell markers and is incapable of resolving pathologic heterogeneities. We previously reported that modeling AML in a continuum of differentiation states using single-cell RNA-seq (scRNA-seq) data predicts intermediate states of differentiation in AML. Herein, we utilized scRNA-seq to characterize CM leukemic blasts, and identified transcriptomic alternations and features that defined the leukemic cell states and heterogeneities. CM expression was induced by poly (I:C) and mice (n=3) were monitored for leukemia progression. At the leukemic end-stage (detection of &amp;gt;50% of circulating cKit + cells), we collected unfractionated and ckit + sorted peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells. Similarly poly(I:C)More than 105,000 cells harvested from the WT and CM leukemic mice were analyzed by scRNA-seq. We applied uniform manifold approximation and projection (UMAP) for dimensionality reduction and performed cell type annotation by SingleR. First, we mapped mature hematopoietic cell types in the WT PB and hematopoietic stem/progenitor cell populations (HSPC) in the WT BM, and in contrast, leukemic PB and BM were highly similar and showed distinct cell clusters compared to WT. To characterize HSPC subpopulations, we focused on cKit+ sorted WT and leukemic BM cells, visualized them via UMAP, and performed clustering and cell type annotation. The leukemic cKit+ cells were mostly identified as megakaryocytic-erythroid progenitor (MEP)-like HSC, revealing a discrepancy between flow cytometry-based phenotypic characterization and transcriptome-based cell type assignment. These leukemic MEP-like HSCs showed up-regulation of erythroid differentiation genes, including Hba-a1, Hba-a2, Hbb-bs, Hbb-bt and Sox6, together with decreased expression of granulocytic markers such as Elane, Prtn3, S100a8, S100a9 and Mpo. Comparison of leukemic MEP-like HSCs vs WT MEP clusters via gene set enrichment analysis (GSEA) with a threshold of nominal p-value ≤ 0.05 and FDR ≤ 0.05 in 50 hallmark gene sets (MSigDB) indicated dysregulated pathways, including up-regulation of “TNFα signaling pathway via NFκB”, “inflammatory response”, and “UV responsive downregulated genes”; and down-regulation of “MYC target V1”, “MYC target V2”, “E2F targets”, “G2M checkpoint”, and “oxidative phosphorylation”. Similarly, GSEA in Gene Ontology (GO) showed downregulation of pathways relevant to mitochondrial protein translation and ribosomal biogenesis/assembly in leukemic MEP-like HSCs, compared to WT MEP. In addition, cKit+ BM cells can be separated into 31 clusters (C0-C30), of which 9 leukemic clusters were identified. Through gene expression-based cell cycle scoring, single sample gene set enrichment (ssGSEA), and marker identification, these 9 leukemic clusters can be defined by several features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). Altogether, these data reveal intermediate differentiation cell states in inv(16) leukemic cells which are highly heterogeneous populations with distinct cell cycle stages, metabolic activities, and marker expression.

Stathmin-1 Regulates Hematopoietic Stem Cell Mitochondrial and Cellular Function

Mitochondrial function is important to control HSC quiescence and self-renewal. HSCs are known to have low oxidative phosphorylation (OXPHOS), and low mitochondrial reactive oxygen species, however, studies have shown that HSCs have high mitochondrial membrane potential (MMP), which supports their function (Hinge et al 2020; Mansell et al 2021). Microtubules are important for normal mitochondrial function, acting as the main pathway through which mitochondria transport occurs. Stathmin 1 (Stmn1) is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of free alpha/beta-tubulin dimers. It is highly expressed in normal hematopoietic stem cells (HSCs) and neural tissues. Also known as oncoprotein 18 (OP18) or leukemia-associated protein 18 (LAP18), Stmn1 is overexpressed in multiple cancers, including both myeloid and lymphoid leukemias. Despite its robust expression in normal HSCs and its elevated expression in leukemias, little is known about Stmn1's role in either normal or malignant HSCs. Herein, we found that Stmn1 is a critical regulator of HSC mitochondrial and cellular function. To determine the role of Stmn1 in HSCs, we first assessed HSC numbers and function in Stmn1 -/-mice. We determined HSC and progenitor numbers, as well as peripheral blood lineages, in young adult Stmn1 -/-mice and WT littermates. Compared to WT, Stmn1 -/- mice have mild anemia and thrombocytopenia, however, HSC numbers are similar. Despite normal HSC numbers, Stmn1 -/-HSCs had markedly impaired repopulating activity in competitive transplantation experiments (10% vs. 60% chimerism at 24 weeks p&amp;lt;0.0001). Similar defects in repopulating activity were found in non-competitive transplants and competitive intra-tibial transplants (done to circumvent any potential homing defects). In addition, Stmn1 -/- HSCs formed fewer colonies when plated in methocult, and were not capable of serial replating. Finally, Stmn1 -/- mice had delayed hematopoietic recovery following a single dose of the chemotherapeutic agent 5-fluorouracil (5-FU). We next assessed the cycling and apoptosis of HSCs. Stmn1 -/- HSCs are hyper-quiescent at baseline, although they can be stimulated to cycle in response to cytokine stimulation (1.3% vs. 3.4% S/G 2/M phase at baseline and 3.4% vs. 7% S/G 2/M phase following stimulation). In addition, they have elevated levels of apoptosis compared to WT (18.5% vs. 9.90%). Consistent with its role as a microtubule destabilizer, Stmn1 expression in HSCs coincides with tubulin expression by confocal immunofluorescent microscopy, and Stmn1 -/- HSCs have decreased tubulin compared to WT HSCs. To gain further insight into their impaired function, we next performed RNA-seq on sorted Stmn1 -/- and WT HSCs. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function (OXPHOS pathway) in the Stmn1 -/- HSCs compared to WT. Indeed, measurement of mitochondrial function using the Seahorse analyzer showed that Stmn1 -/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT (251.29 vs 411.84 pmol/min). In addition, Stmn1 -/- HSCs showed reduced mitochondrial membrane potential and increased mtROS as assessed by flow-based assays. Finally, the mitochondria of Stmn1 -/- HSCs showed markedly abnormal cristae morphology by TEM, consistent with their impaired function, and Stmn1 -/- HSCs have decreased mitochondria number compared to WT (medium 12.8 vs 17.3 per 2D slice cell; p&amp;lt;0.0250). As elevated ROS can impair HSC function, we assessed Stmn1 -/- HSCs following treatment with MitoTEMPO, a specific scavenger of mitochondrial superoxide. Treatment of Stmn1 -/- mice with 4mg/Kg of MitoTEMPO or vehicle control for two weeks resulted in improved HSC colony formation and serial replating capacity in Methocult. Together, these data identify Stmn1 as a critical regulator of HSC function and suggest a model whereby Stmn1 promotes normal microtubule dynamics and mitochondrial health in HSCs. Ongoing experiments are aimed at understanding Stmn1's role in regulating mitochondrial quality control processes such as mitophagy and fission in both normal and malignant HSCs, and in elucidating the effects of Stmn1 loss on leukemic cell growth and viability.

AMPK is a mechano-metabolic sensor linking cell adhesion and mitochondrial dynamics to Myosin-dependent cell migration

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.

Association between skeletal muscle mitochondrial dysfunction and insulin resistance in patients with rheumatoid arthritis: a case–control study

BackgroundInsulin resistance affects a substantial proportion of patients with rheumatoid arthritis (RA). Skeletal muscle mitochondrial dysfunction results in the accumulation of lipid intermediates that interfere with insulin signaling. We therefore sought to determine if lower oxidative phosphorylation and muscle mitochondrial content are associated with insulin resistance in patients with RA.MethodsThis was a cross-sectional prospective study of RA patients. Matsuda index from the glucose tolerance test was used to estimate insulin sensitivity. Mitochondrial content was measured by citrate synthase (CS) activity in snap-frozen muscle samples. Mitochondrial function was measured by using high-resolution respirometry of permeabilized muscle fibers and electron transport chain complex IV enzyme kinetics in isolated mitochondrial subpopulations.ResultsRA participants demonstrated lower insulin sensitivity as measured by the Matsuda index compared to controls [median 3.95 IQR (2.33, 5.64) vs. 7.17 (5.83, 7.75), p = 0.02]. There was lower muscle mitochondrial content among RA vs. controls [median 60 mU/mg IQR (45, 80) vs. 79 mU/mg (65, 97), p = 0.03]. Notably, OxPhos normalized to mitochondrial content was higher among RA vs. controls [mean difference (95% CI) = 0.14 (0.02, 0.26), p = 0.03], indicating a possible compensatory mechanism for lower mitochondrial content or lipid overload. Among RA participants, the activity of muscle CS activity was not correlated with the Matsuda index (ρ = − 0.05, p = 0.84), but it was positively correlated with self-reported (IPAQ) total MET-minutes/week (ρ = 0.44, p = 0.03) and Actigraph-measured time on physical activity (MET rate) (ρ = 0.47, p = 0.03).ConclusionsMitochondrial content and function were not associated with insulin sensitivity among participants with RA. However, our study demonstrates a significant association between muscle mitochondrial content and physical activity level, highlighting the potential for future exercise interventions that enhance mitochondrial efficiency in RA patients.

Abstract 3704: The essential role of an alternate mRNA translation initiation in the regulation of breast cancer cell metabolism

Abstract Translational control and metabolic reprogramming are hallmarks of advanced cancers. Important genes involved in cancer progression express mRNAs that are selectively translated, including regulators of cancer cell metabolism. Cancer cells acquire an altered metabolism, switching from oxidative phosphorylation (OXPHOS) to a glycolytic phenotype (Warburg effect), to increase reliance on alternate metabolic pathways to support growth, proliferation, and metastasis. Triple-negative breast cancer (TNBC), one of the most aggressive and highly metastatic subtypes with the poorest outcome, is characterized by elevated glycolysis and low OXPHOS. Still, the exact mechanism for this metabolic switch is largely unknown. Using a TNBC cell model it has been shown an alternate mechanism of cap-dependent but mTORC1/eIF4Eindependent mRNA translation by the eIF4G homolog, DAP5, which directly binds the cap-binding protein eIF3d. Our research indicates that this alternate translation initiation is essential in regulating several mRNAs involved in breast cancer metabolism. DAVID analysis from genome-wide transcriptomic and translatomic studies, metabolomic assays including Glycolysis Assay [Extracellular Acidification], Oxidative Phosphorylation, and Mitotracker Assay, as well as RT-PCR and western blot analysis was performed from well-characterized triple-negative aggressive breast cancer cell lines,4T1 and MDA-231, Non-silencing (Nsi) control and silencing DAP5 (shDAP5) samples. Several mRNAs related to glucose metabolism decreased after silencing DAP5 and on the opposite, critical mRNAs associated with OXPHOS increased, independent of their steady-state mRNA abundance. Silencing DAP5 increased Oxidative Phosphorylation activity and decrease the glycolytic rate in TNBC breast cancer cells. Proteins correlated with aberrant glycolytic metabolism including the E-cadherin transcriptional repressors Snail (SNAI1) were reduced after silencing of DAP5. Our data indicates that the alternate mRNA translation initiation mechanism by DAP5 of specific mRNAs correlated to glycolysis and OXPHOS could play an essential role in the mechanism by which TNBC breast cancer cells orchestrate a metabolic switch required for malignant progression and metastasis. Citation Format: Hanna Rosenstock, Erna Mitaishvili, Columba de la Parra. The essential role of an alternate mRNA translation initiation in the regulation of breast cancer cell metabolism. [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 3704.

Loss of fatty acid degradation by astrocytic mitochondria triggers neuroinflammation and neurodegeneration.

Astrocytes provide key neuronal support, and their phenotypic transformation is implicated in neurodegenerative diseases. Metabolically, astrocytes possess low mitochondrial oxidative phosphorylation (OxPhos) activity, but its pathophysiological role in neurodegeneration remains unclear. Here, we show that the brain critically depends on astrocytic OxPhos to degrade fatty acids (FAs) and maintain lipid homeostasis. Aberrant astrocytic OxPhos induces lipid droplet (LD) accumulation followed by neurodegeneration that recapitulates key features of Alzheimer's disease (AD), including synaptic loss, neuroinflammation, demyelination and cognitive impairment. Mechanistically, when FA load overwhelms astrocytic OxPhos capacity, elevated acetyl-CoA levels induce astrocyte reactivity by enhancing STAT3 acetylation and activation. Intercellularly, lipid-laden reactive astrocytes stimulate neuronal FA oxidation and oxidative stress, activate microglia through IL-3 signalling, and inhibit the biosynthesis of FAs and phospholipids required for myelin replenishment. Along with LD accumulation and impaired FA degradation manifested in an AD mouse model, we reveal a lipid-centric, AD-resembling mechanism by which astrocytic mitochondrial dysfunction progressively induces neuroinflammation and neurodegeneration.

Super-resolution analyzing spatial organization of lysosomes with an organic fluorescent probe.

Lysosomes are multifunctional organelles involved in macromolecule degradation, nutrient sensing and autophagy. Live imaging has revealed lysosome subpopulations with dynamics and characteristic cellular localization. An as-yet unanswered question is whether lysosomes are spatially organized to coordinate and integrate their functions. Combined with super-resolution microscopy, we designed a small organic fluorescent probe, TPAE, that targeted lysosomes with a large Stokes shift. When we analyzed the spatial organization of lysosomes against mitochondria in different cell lines with this probe, we discovered different distance distribution patterns between lysosomes and mitochondria during increased autophagy flux. By using SLC25A46 mutation fibroblasts derived from patients containing highly fused mitochondria with low oxidative phosphorylation, we concluded that unhealthy mitochondria redistributed the subcellular localization of lysosomes, which implies a strong connection between mitochondria and lysosomes.

Characterizing region-specific glucose metabolic profile of the rodent brain using Seahorse XFe96 analyzer

The brain is highly complex with diverse structural characteristics in accordance with specific functions. Accordingly, differences in regional function, cellular compositions, and active metabolic pathways may link to differences in glucose metabolism at different brain regions. In the current study, we optimized an acute biopsy punching method and characterized region-specific glucose metabolism of rat and mouse brain by a Seahorse XFe96 analyzer. We demonstrated that 0.5 mm diameter tissue punches from 180-µm thick brain sections allow metabolic measurements of anatomically defined brain structures using Seahorse XFe96 analyzer. Our result indicated that the cerebellum displays a more quiescent phenotype of glucose metabolism than cerebral cortex, basal ganglia, and hippocampus. In addition, the cerebellum has higher AMPK activation than other brain regions evidenced by the expression of pAMPK, upstream pLKB1, and downstream pACC. Furthermore, rodent brain has relatively low mitochondrial oxidative phosphorylation efficiency with up to 30% of respiration linked to proton leak. In summary, our study discovered region-specific glucose metabolic profile and relative high proton leak coupled respiration in the brain. Our study warrants future research on spatial mapping of the brain glucose metabolism in physiological and pathological conditions and exploring the mechanisms and significance of mitochondrial uncoupling in the brain.

Interactions between NLRP3 inflammasome and glycolysis in macrophages: New insights into chronic inflammation pathogenesis.

NLRP3 inflammasome activation in macrophages fuels sterile inflammation, which has been tied with metabolic reprogramming characterized by high glycolysis and low oxidative phosphorylation. The key enzymes in glycolysis and glycolysis‐related products can regulate and activate NLRP3 inflammasome. In turn, NLRP3 inflammasome is considered to affect glycolysis, as well. However, the exact mechanism remains ambiguous. On the basis of these findings, the focus of this review is mainly on the developments in our understanding of interaction between NLRP3 inflammasome activation and glycolysis in macrophages, and small molecule compounds that influence the activation of NLRP3 inflammasomes by regulating glycolysis in macrophages. The application of this interaction in the treatment of diseases is also discussed. This paper may yield valuable clues for development of novel therapeutic agent for NLRP3 inflammasome‐related diseases.

Pan-cancer characterization of metabolism-related biomarkers identifies potential therapeutic targets

BackgroundGenerally, cancer cells undergo metabolic reprogramming to adapt to energetic and biosynthetic requirements that support their uncontrolled proliferation. However, the mutual relationship between two critical metabolic pathways, glycolysis and oxidative phosphorylation (OXPHOS), remains poorly defined.MethodsWe developed a “double-score” system to quantify glycolysis and OXPHOS in 9668 patients across 33 tumor types from The Cancer Genome Atlas and classified them into four metabolic subtypes. Multi-omics bioinformatical analyses was conducted to detect metabolism-related molecular features.ResultsCompared with patients with low glycolysis and high OXPHOS (LGHO), those with high glycolysis and low OXPHOS (HGLO) were consistently associated with worse prognosis. We identified common dysregulated molecular features between different metabolic subgroups across multiple cancers, including gene, miRNA, transcription factor, methylation, and somatic alteration, as well as investigated their mutual interfering relationships.ConclusionOverall, this work provides a comprehensive atlas of metabolic heterogeneity on a pan-cancer scale and identified several potential drivers of metabolic rewiring, suggesting corresponding prognostic and therapeutic utility.

Stem Cell Metabolism: Powering Cell-Based Therapeutics.

Cell-based therapeutics for cardiac repair have been extensively used during the last decade. Preclinical studies have demonstrated the effectiveness of adoptively transferred stem cells for enhancement of cardiac function. Nevertheless, several cell-based clinical trials have provided largely underwhelming outcomes. A major limitation is the lack of survival in the harsh cardiac milieu as only less than 1% donated cells survive. Recent efforts have focused on enhancing cell-based therapeutics and understanding the biology of stem cells and their response to environmental changes. Stem cell metabolism has recently emerged as a critical determinant of cellular processes and is uniquely adapted to support proliferation, stemness, and commitment. Metabolic signaling pathways are remarkably sensitive to different environmental signals with a profound effect on cell survival after adoptive transfer. Stem cells mainly generate energy through glycolysis while maintaining low oxidative phosphorylation (OxPhos), providing metabolites for biosynthesis of macromolecules. During commitment, there is a shift in cellular metabolism, which alters cell function. Reprogramming stem cell metabolism may represent an attractive strategy to enhance stem cell therapy for cardiac repair. This review summarizes the current literature on how metabolism drives stem cell function and how this knowledge can be applied to improve cell-based therapeutics for cardiac repair.

Hemin Prevents Increased Glycolysis in Macrophages upon Activation: Protection by Microbiota-Derived Metabolites of Polyphenols.

Meat consumption plays a critical role in the development of several types of cancer. Hemin, a metabolite of myoglobin produced after meat intake, has been demonstrated to be involved in the cancer initiation phase. Macrophages are key components of the innate immunity, which, upon activation, can prevent cancer development by eliminating neoplastic cells. Metabolic reprogramming, characterized by high glycolysis and low oxidative phosphorylation, is critical for macrophage activation. 3,4-dihydroxyphenylacetic acid (3,4DHPAA) and 4-hydroxyphenylacetic acid (4HPAA), both microbiota-derived metabolites of flavonoids, have not been extensively studied although they exert antioxidant properties. The aim of this study was to determine the effect of hemin on the anticancer properties of macrophages and the role of 3,4DHPAA and 4HPAA in metabolic reprogramming and activation of macrophages leading to the elimination of cancer cells. The results showed that hemin inhibited glycolysis, glycolytic, and pentose phosphate pathway (PPP) enzyme activities and hypoxia-inducible factor-1 alpha (HIF-1α) stabilization, which interferes with macrophage activation (evidenced by decreased interferon-γ-inducible protein 10 (IP-10) release) and their ability to eliminate cancer cells (via cytotoxic mediators and phagocytosis). Hemin also reduced the mitochondrial membrane potential (MMP) and mitochondrial mass in macrophages. 3,4DHPAA and 4HPAA, by stimulating glycolysis and PPP, prevented the impairment of the macrophage anticancer activity induced by hemin. In conclusion, 3,4HPAA and 4HPAA administration could represent a promising strategy for preventing the reduction of macrophage activation induced by hemin.

Mitochondrial Spare Reserve Capacity : A New Predictive Metabolic Biomarker for Aggressiveness of Acute Myeloid Leukemia

Introduction The persistence of leukemic cells after treatment limits the effectiveness of anticancer drugs and is the cause of relapse in patients with acute myeloid leukemia (AML). After exposure to chemotherapeutic drugs, the survival of leukemic cells is mainly supported by mitochondrial energy metabolism. Several preclinical studies have shown that the combination of mitochondrial oxidative phosphorylation inhibitors with various anticancer treatments constitutes an effective therapeutic combination in vitro to eradicate the surviving leukemic cells. Evaluating the mitochondrial bioenergetic activity of blasts from AML patients could therefore provide predictive information on treatment response. The basal oxygen consumption of cells varies according to hematopoietic differentiation and depends on the energy needs in the in vitro condition of measurement. But it is necessary to treat the cells with uncoupling agents (eg FCCP) to assess the maximum activity that the respiratory chain could reach to respond to energy stress. Then, the switch from a basal level of oxygen consumption to a maximum level defines the mitochondrial spare reserve capacity (SRC). In this study, we propose to determine whether spare reserve capacity of blasts is a potential biomarker of AML aggressiveness in patients and to characterize the biochemical processes involved in the control of SRC in leukemic cells. Results Using the XFe24 Seahorse fluorometric oximeter, we first determined the mitochondrial oxygen consumption and glycolytic activity in hematopoietic cells (monocytes, lymphocytes, dendritic cells) of healthy donors, in AML patient blasts at diagnosis or at relapse and in AML cell lines (HL-60, MOLM-13, THP-1, KG1, OCI-AML3, MV-4-11, U-937). All measures have been assessed from freshly collected samples of peripheral blood and of bone marrow. As expected, AMLs are characterized by low oxidative phosphorylation activity compared to normal hematopoietic cells. From all the OXPHOS values obtained we defined a SRC threshold above which the SRC is considered high. This threshold has been set at a capacity to increase basal respiration by 250%. From patients blasts, we have therefore defined two groups characterized by high (n=14) or low (n=21) mitochondrial spare reserve capacity. Blasts with high SRC exhibit high glycolytic activity suggesting a link between spare reserve capacity and glucose metabolism. Using U-13C6 glucose and pharmacological inhibitors, we have demonstrated that the utilization of the mitochondrial spare reserve capacity of leukemic cells is supported through glycolysis and that mitochondrial oxidation of pyruvate is a key element for SRC recruitment. Mitochondrial pyruvate carrier inhibitors (as UK-5099) or gene silencing of BRP44 abolish the SRC of leukemic cells highlighting the importance of pyruvate oxidation to increase oxygen consumption. Since high mutation rate is recognized as an unfavorable prognostic factor in AML, we have also sequenced 45 commonly genes mutated in AMLs characterized by high or low SRC blasts. Interestingly, DNA sequencing analysis showed that AML with low SRC blasts have a higher mutation rate than high SRC blasts and also exhibited exclusive mutations such as ASXL1 (25%), IDH2 (25%), NPM1 (25%), IDH1 (13%), JAK2 (13%) and SF3B1 (13%). Conclusion Currently, most of the clinical biomarkers used to predict AML aggressiveness are based on DNA analysis, but the emergence of mutations is not always associated with phenotypic changes. This study shows that the mitochondrial spare reserve capacity of blasts represents a new functional biomarker based on the assessment of the energetic phenotype and could help the clinicians to determine the prognosis of AML. Moreover we have showed that altering pyruvate metabolism highly decrease spare reserve capacity of blasts and then could be evaluated as metabolic strategies to improve the therapeutic response in patients with AML. Disclosures Kluza: Daiichi-Sankyo: Research Funding.

Nuclear-Encoded lncRNA MALAT1 Epigenetically Controls Metabolic Reprogramming in HCC Cells through the Mitophagy Pathway

Mitochondrial dysfunction is a metabolic hallmark of cancer cells. In search of molecular factors involved in this dysregulation in hepatocellular carcinoma (HCC), we found that the nuclear-encoded long noncoding RNA (lncRNA) MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was aberrantly enriched in the mitochondria of hepatoma cells. Using RNA reverse transcription-associated trap sequencing (RAT-seq), we showed that MALAT1 interacted with multiple loci on mitochondrial DNA (mtDNA), including D-loop, COX2, ND3, and CYTB genes. MALAT1 knockdown induced alterations in the CpG methylation of mtDNA and in mitochondrial transcriptomes. This was associated with multiple abnormalities in mitochondrial function, including altered mitochondrial structure, low oxidative phosphorylation (OXPHOS), decreased ATP production, reduced mitophagy, decreased mtDNA copy number, and activation of mitochondrial apoptosis. These alterations in mitochondrial metabolism were associated with changes in tumor phenotype and in pathways involved in cell mitophagy, mitochondrial apoptosis, and epigenetic regulation. We further showed that the RNA-shuttling protein HuR and the mitochondria transmembrane protein MTCH2 mediated the transport of MALAT1 in this nuclear-mitochondrial crosstalk. This study provides the first evidence that the nuclear genome-encoded lncRNA MALAT1 functions as a critical epigenetic player in the regulation of mitochondrial metabolism of hepatoma cells, laying the foundation for further clarifying the roles of lncRNAs in tumor metabolic reprogramming.

GLUT1 inhibition blocks growth of RB1-positive triple negative breast cancer

Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data suggests that the functionality of the E2F pathway may reflect to some extent OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) strongly correlate with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.