bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2022‒04‒10
33 papers selected by
Kelsey Fisher-Wellman
East Carolina University

  1. Trends Pharmacol Sci. 2022 Apr 02. pii: S0165-6147(22)00057-8. [Epub ahead of print]
      Targeting metabolic reprogramming has proven successful in oncology, but this field requires better identification of drugs that inhibit mitochondrial metabolism in cancer cells. Recent work from Dr Wolf's group reveals that the primary target of the antitumor compound SMIP004-7 is mitochondrial complex I (NDUFS2 subunit), inhibition of which promotes anticancer immune surveillance.
    Keywords:  anticancer therapies; cancer metabolism; complex I; mitochondria; oxidative phosphorylation system; tumor microenvironment
  2. Curr Med Chem. 2022 Apr 01.
      Mitochondria are the main energy factory in living cells. To rapidly proliferate and metastasize, neoplastic cells increase their energy requirements. Thus, mitochondria become one of the most important organelles for them. Indeed, much research shows the interplay between cancer chemoresistance and altered mitochondrial function. In this review we focus on the differences in energy metabolism between cancer and normal cells, to better understand their resistance and how to develop drugs targeting energy metabolism and nucleotide synthesis. One of the differences between cancer and normal cells is the higher nicotinamide adenine dinucleotide (NAD+) level, a cofactor for the tricarboxylic acid cycle (TCA), which enhances their proliferation and helps cancer cells survive under hypoxic conditions. An important change is a metabolic switch, called the Warburg effect. This effect is based on the change of energy harvesting from oxygen-dependent transformation to oxidative phosphorylation (OXPHOS), adapt them to the tumor environment. Another mechanism is the high expression of one carbon (1C) metabolism enzymes. Again, this allows cancer cells to increase proliferation by producing precursors for the synthesis of nucleotides and amino acids. We reviewed drugs in clinical practice and in development targeting NAD+, OXPHOS, and 1C metabolism. Combinations of novel drugs with conventional antineoplastic agents may prove to be a promising new way of anticancer treatment.
    Keywords:  1C Metabolism; Cancer; Mitochondria; NAD+; Oxidative Phosphorylation (OXPHOS); Resistance
  3. FASEB Bioadv. 2022 Mar;4(3): 197-210
      Classically, mitochondrial respiration responds to decreased membrane potential (ΔΨ) by increasing respiration. However, we found that for succinate-energized complex II respiration in skeletal muscle mitochondria (unencumbered by rotenone), low ΔΨ impairs respiration by a mechanism culminating in oxaloacetate (OAA) inhibition of succinate dehydrogenase (SDH). Here, we investigated whether this phenomenon extends to far different mitochondria of a tissue wherein ΔΨ is intrinsically low, i.e., interscapular brown adipose tissue (IBAT). Also, to advance our knowledge of the mechanism, we performed isotopomer studies of metabolite flux not done in our previous muscle studies. In additional novel work, we addressed possible ways ADP might affect the mechanism in IBAT mitochondria. UCP1 activity, and consequently ΔΨ, were perturbed both by GDP, a well-recognized potent inhibitor of UCP1 and by the chemical uncoupler carbonyl cyanide m-chlorophenyl hydrazone (FCCP). In succinate-energized mitochondria, GDP increased ΔΨ but also increased rather than decreased (as classically predicted under low ΔΨ) O2 flux. In GDP-treated mitochondria, FCCP reduced potential but also decreased respiration. Metabolite studies by NMR and flux analyses by LC-MS support a mechanism, wherein ΔΨ effects on the production of reactive oxygen alters the NADH/NAD+ ratio affecting OAA accumulation and, hence, OAA inhibition of SDH. We also found that ADP-altered complex II respiration in complex fashion probably involving decreased ΔΨ due to ATP synthesis, a GDP-like nucleotide inhibition of UCP1, and allosteric enzyme action. In summary, complex II respiration in IBAT mitochondria is regulated by UCP1-dependent ΔΨ altering substrate flow through OAA and OAA inhibition of SDH.
    Keywords:  bioenergetics; brown adipose tissue; metabolism; metabolomics; mitochondria; mitochondrial metabolism; reactive oxygen species (ROS); uncoupling protein
  4. Mol Metab. 2022 Apr 04. pii: S2212-8778(22)00058-8. [Epub ahead of print] 101489
      OBJECTIVE: There is strong evidence that mitochondrial DNA mutations and mitochondrial dysfunction play a role in diabetes pathogenesis. The homozygous knock-in mtDNA mutator mouse is a model of premature aging due to the accumulation of mitochondrial DNA mutations. We used this mouse model to investigate the relationship between mitochondrial subunit expression and pancreatic islet cell composition.METHODS: Quadruple immunofluorescence was used to quantify mitochondrial subunit expression (complex I and IV) and cell composition in pancreatic islets from mitochondrial DNA mutator mice (PolgAmut/mut) and control C57BL/6 mice at 12 and 44 weeks of age.
    RESULTS: Mitochondrial complex I subunit expression was decreased in islets from 12 week PolgAmut/mut mice. This complex I deficiency persisted with age and was associated with decreased insulin staining intensity at 44 weeks. Complex I deficiency was greater in α-cells compared with β-cells in islets from 44 week PolgAmut/mut mice. Islet cell composition was normal in 12 week PolgAmut/mut mice, but the β: α cell ratio was decreased in islets from 44 week PolgAmut/mut mice. This was due to an increase in α-cell number linked to an increase in α-cell proliferation.
    CONCLUSION: Complex I deficiency promotes α-cell proliferation and alters islet cell composition.
    Keywords:  Mitochondria; mtDNA; mtDNA mutator mice; pancreatic islets
  5. Cell Death Differ. 2022 Apr 07.
      Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion, decreasing both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. The antineoplastic effect of NDI1 is mimicked by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3 and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes. These findings shed light on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.
  6. Cell Rep. 2022 Apr 05. pii: S2211-1247(22)00367-9. [Epub ahead of print]39(1): 110619
      The presequence translocase (TIM23 complex) imports precursor proteins into the mitochondrial inner membrane and matrix. The presequence translocase-associated motor (PAM) provides a driving force for transport into the matrix. The J-protein Pam18 stimulates the ATPase activity of the mitochondrial Hsp70 (mtHsp70). Pam16 recruits Pam18 to the TIM23 complex to ensure protein import. The Pam16-Pam18 module also associates with components of the respiratory chain, but the function of the dual localization of Pam16-Pam18 is largely unknown. Here, we show that disruption of the Pam16-Pam18 heterodimer causes redistribution of Pam18 to the respiratory chain supercomplexes, where it forms a homodimer. Redistribution of Pam18 decreases protein import into mitochondria but stimulates mtHsp70-dependent assembly of respiratory chain complexes. We conclude that coupling to Pam16 differentially controls the dual function of Pam18. It recruits Pam18 to the TIM23 complex to promote protein import but attenuates the Pam18 function in the assembly of respiratory chain complexes.
    Keywords:  CP: Cell biology; CP: Metabolism; Pam18; TIM23 complex; cytochrome c oxidase; mitochondria; mtHsp70; protein sorting; respiratory chain
  7. J Am Chem Soc. 2022 Apr 05.
      Respiratory complex I is an essential metabolic enzyme that uses the energy from NADH oxidation and ubiquinone reduction to translocate protons across an energy transducing membrane and generate the proton motive force for ATP synthesis. Under specific conditions, complex I can also catalyze the reverse reaction, Δp-linked oxidation of ubiquinol to reduce NAD+ (or O2), known as reverse electron transfer (RET). Oxidative damage by reactive oxygen species generated during RET underpins ischemia reperfusion injury, but as RET relies on several converging metabolic pathways, little is known about its mechanism or regulation. Here, we demonstrate Δp-linked RET through complex I in a synthetic proteoliposome system for the first time, enabling complete kinetic characterization of RET catalysis. We further establish the capability of our system by showing how RET in the mammalian enzyme is regulated by the active-deactive transition and by evaluating RET by complex I from several species in which direct assessment has not been otherwise possible. We thus provide new insights into the reversibility of complex I catalysis, an important but little understood mechanistic and physiological feature.
  8. Cell Rep. 2022 Apr 05. pii: S2211-1247(22)00355-2. [Epub ahead of print]39(1): 110607
      The mechanism by which redox metabolism regulates the fates of acute myeloid leukemia (AML) cells remains largely unknown. Using a highly sensitive, genetically encoded fluorescent sensor of nicotinamide adenine dinucleotide phosphate (NADPH), iNap1, we find three heterogeneous subpopulations of AML cells with different cytosolic NADPH levels in an MLL-AF9-induced murine AML model. The iNap1-high AML cells have enhanced proliferation capacities both in vitro and in vivo and are enriched for more functional leukemia-initiating cells than iNap1-low counterparts. The iNap1-high AML cells prefer localizing in the bone marrow endosteal niche and are resistant to methotrexate treatment. Furthermore, iNap1-high human primary AML cells have enhanced proliferation abilities both in vitro and in vivo. Mechanistically, the MTHFD1-mediated folate cycle regulates NADPH homeostasis to promote leukemogenesis and methotrexate resistance. These results provide important clues for understanding mechanisms by which redox metabolism regulates cancer cell fates and a potential metabolic target for AML treatments.
    Keywords:  CP: Cancer; NADPH metabolism; acute myeloid leukemia; endosteal niche; folate cycle; leukemia-initiating cells; metabolic sensor; methotrexate resistance; methylenetetrahydrofolate dehydrogenase; tetrahydrofolic acid; vascular niche
  9. Angew Chem Int Ed Engl. 2022 Apr 05.
      Metabolic adaptations can help cancer cells to escape from chemotherapeutics, mainly involving autophagy and ATP production. Herein, we report a new rhein-based cyclometalated Ir(III) complex, Ir-Rhein , that can accurately target mitochondria and effectively inhibit metabolic adaptations. The complex Ir-Rhein induces severe mitochondrial damage and initiates mitophagy to reduce the number of mitochondria and subsequently inhibit both mitochondrial and glycolytic bioenergetics, which eventually leads to ATP starvation death. Moreover, Ir-Rhein can overcome cisplatin resistance. In a co-incubation experiment, a 3D tumor spheroids experiment and transcriptome analysis reveal that Ir-Rhein shows promising antiproliferation performance for cisplatin-resistant cancer cells with the regulation of platinum resistance-related transporters. To the best of our knowledge, this is a new strategy to overcome metallodrug resistance with a mitochondria-relevant treatment.
    Keywords:  mitophagy, metabolic adaptation, mitochondria-targeting, metallodrug resistance
  10. Front Oncol. 2022 ;12 857686
      The ability of cancer cells to adjust their metabolism in response to environmental changes is a well-recognized hallmark of cancer. Diverse cancer and non-cancer cells within tumors compete for metabolic resources. Metabolic demands change frequently during tumor initiation, progression and metastasis, challenging our quest to better understand tumor biology and develop novel therapeutics. Vascularization, physical constraints, immune responses and genetic instability promote tumor evolution resulting in immune evasion, opportunities to breach basement membrane barriers and spread through the circulation and lymphatics. In addition, the unfolded protein response linked to the ubiquitin proteasome system is a key player in addressing stoichiometric imbalances between nuclear and mitochondrially-encoded protein subunits of respiratory complexes, and nuclear-encoded mitochondrial ribosomal protein subunits. While progressive genetic changes, some of which affect metabolic adaptability, contribute to tumorigenesis and metastasis through clonal expansion, epigenetic changes are also important and more dynamic in nature. Understanding the role of stromal and immune cells in the tumor microenvironment in remodeling cancer cell energy metabolism has become an increasingly important area of research. In this perspective, we discuss the adaptations made by cancer cells to balance mitochondrial and glycolytic energy metabolism. We discuss how hypoxia and nutrient limitations affect reductive and oxidative stress through changes in mitochondrial electron transport activity. We propose that integrated responses to cellular stress in cancer cells are central to metabolic flexibility in general and bioenergetic adaptability in particular and are paramount in tumor progression and metastasis.
    Keywords:  bioenergetic flexibility; glycolysis-OXPHOS continuum; mito-nuclear gene expression; tumor microenvironment (TME); tumor progression and metastasis
  11. Cell Death Dis. 2022 Apr 08. 13(4): 320
      Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD+) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD+ biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet.
  12. Biochem Biophys Res Commun. 2022 Mar 18. pii: S0006-291X(22)00421-1. [Epub ahead of print]607 131-137
      The mitochondrial enzyme SIRT3 is an NAD+-dependent deacetylase important in cell metabolism, and a decline in its protein expression or activity has been linked with insulin resistance in obesity, ageing and type 2 diabetes. While studies in SIRT3 knockout mice have dramatically improved our understanding of the function of SIRT3, the impact of increasing SIRT3 levels remains under-examined. In this study we investigated the effects of liver-specific SIRT3 overexpression in mice on mitochondrial function and metabolic profile in both isolated hepatocytes and in vivo. Primary hepatocytes overexpressing SIRT3 displayed increased oxygen consumption and a reduction in triglyceride accumulation. In mice with hepatic SIRT3 overexpression, increased fasting β-hydroxybutyrate levels were observed, coupled with an increase in oxygen consumption in isolated mitochondria and increased substrate utilization in liver homogenates. However, metabolic profiling of mice exposed to either chow or high-fat diet revealed no effect of hepatic SIRT3 overexpression on glucose tolerance, body composition or tissue triglyceride accumulation. These findings suggest limited whole-body benefit of increasing hepatic SIRT3 during the development of diet-induced insulin resistance.
    Keywords:  Liver; Mouse; Obesity; SIRT3; Sirtuin
  13. J Neurosci. 2022 Apr 06. pii: JN-RM-1463-21. [Epub ahead of print]
      Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand) dependent on Na+ and Ca2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production and OXPHOS in a Ca2+-dependent way. We find that Ca2+-upregulation of glycolysis, pyruvate levels and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production and respiration, a process inhibited in the presence of BAPTA-AM suggesting that the Ca2+ binding motifs in Aralar may be involved in the activation. MCU silencing had no effect indicating that none of these processes required MCU-dependent mitochondrial Ca2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+ flow maintaining basal cell bioenergetics in which Ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ release, and in which MCU does not participate. The results reveal that in neurons using glucose MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANT STATEMENTSNeuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production and neuronal respiration are stimulated upon neuronal activation in a Ca2+ dependent way, independently of effects of Ca2+ as workload inducer. MCU does not play a relevant role in Ca2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+ stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.
    Keywords:  Aralar/AGC1/Slc25a12; Neuronal metabolism; calcium regulation; glycolysis; malate aspartate shuttle; mitochondrial calcium uniporter
  14. Front Pharmacol. 2022 ;13 851832
      Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies and the main cause of cancer-related deaths. The multitarget tyrosine kinase inhibitors (TKIs) sorafenib and regorafenib are systemic therapeutic drugs approved for the treatment of HCC. Here, we found that sorafenib and regorafenib injured mitochondria by inducing mitochondrial Ca2+ (mtCa2+) overload and mitochondrial permeability transition pore (mPTP) opening, resulting in mitochondria-mediated cell death, which was alleviated by cyclosporin A (CsA), an inhibitor of mPTP. Meanwhile, mPTP opening caused PINK1 accumulation on damaged mitochondria, which recruited Parkin to mitochondria to induce mitophagy. Inhibition of autophagy by the lysosomal inhibitor chloroquine (CQ) or inhibition of mitochondrial fission by mdivi-1 aggravated sorafenib- and regorafenib-induced cell death. Moreover, knockdown of PINK1 also promotes sorafenib- and regorafenib-induced cell death. An in vivo study showed that sorafenib and regorafenib inhibited HepG2 cell growth more effectively in PINK1 knockdown cells than in shNTC cells in null mice. Thus, our data demonstrate that PINK1-Parkin-mediated mitophagy alleviates sorafenib and regorafenib antitumor effects in vitro and in vivo.
    Keywords:  HCC; MPTP; PINK1; mitofission; mitophagy; regorafenib; sorafenib
  15. Nat Commun. 2022 Apr 04. 13(1): 1789
      The metabolic principles underlying the differences between follicular and marginal zone B cells (FoB and MZB, respectively) are not well understood. Here we show, by studying mice with B cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that glutathione synthesis affects homeostasis and differentiation of MZB to a larger extent than FoB, while glutathione-dependent redox control contributes to the metabolic dependencies of FoB. Specifically, Gclc ablation in FoB induces metabolic features of wild-type MZB such as increased ATP levels, glucose metabolism, mTOR activation, and protein synthesis. Furthermore, Gclc-deficient FoB have a block in the mitochondrial electron transport chain (ETC) due to diminished complex I and II activity and thereby accumulate the tricarboxylic acid cycle metabolite succinate. Finally, Gclc deficiency hampers FoB activation and antibody responses in vitro and in vivo, and induces susceptibility to viral infections. Our results thus suggest that Gclc is required to ensure the development of MZB, the mitochondrial ETC integrity in FoB, and the efficacy of antiviral humoral immunity.
  16. Nature. 2022 Apr 06.
      Mammalian embryogenesis requires rapid growth and proper metabolic regulation1. Midgestation features increasing oxygen and nutrient availability concomitant with fetal organ development2,3. Understanding how metabolism supports development requires approaches to observe metabolism directly in model organisms in utero. Here we used isotope tracing and metabolomics to identify evolving metabolic programmes in the placenta and embryo during midgestation in mice. These tissues differ metabolically throughout midgestation, but we pinpointed gestational days (GD) 10.5-11.5 as a transition period for both placenta and embryo. Isotope tracing revealed differences in carbohydrate metabolism between the tissues and rapid glucose-dependent purine synthesis, especially in the embryo. Glucose's contribution to the tricarboxylic acid (TCA) cycle rises throughout midgestation in the embryo but not in the placenta. By GD12.5, compartmentalized metabolic programmes are apparent within the embryo, including different nutrient contributions to the TCA cycle in different organs. To contextualize developmental anomalies associated with Mendelian metabolic defects, we analysed mice deficient in LIPT1, the enzyme that activates 2-ketoacid dehydrogenases related to the TCA cycle4,5. LIPT1 deficiency suppresses TCA cycle metabolism during the GD10.5-GD11.5 transition, perturbs brain, heart and erythrocyte development and leads to embryonic demise by GD11.5. These data document individualized metabolic programmes in developing organs in utero.
  17. Oxid Med Cell Longev. 2022 ;2022 5652586
      Metabolic changes have been suggested to be a hallmark of tumors and are closely associated with tumorigenesis. In a previous study, we demonstrated the role of lactate dehydrogenase in regulating abnormal glucose metabolism in pituitary adenomas (PA). As the key organelle of oxidative phosphorylation (OXPHOS), mitochondria play a vital role in the energy supply for tumor cells. However, few attempts have been made to elucidate mitochondrial metabolic homeostasis in PA. Dynamin-related protein 1 (Drp1) is a member of the dynamin superfamily of GTPases, which mediates mitochondrial fission. This study is aimed at investigating whether Drp1 affects the progression of PA through abnormal mitochondrial metabolism. We analyzed the expression of dynamin-related protein 1 (Drp1) in 20 surgical PA samples. The effects of Drp1 on PA growth were assessed in vitro and in xenograft models. We found an upregulation of Drp1 in PA samples with a low proliferation index. Knockdown or inhibition of Drp1 enhanced the proliferation of PA cell lines in vitro, while overexpression of Drp1 could reversed such effects. Mechanistically, overexpressed Drp1 damaged mitochondria by overproduction of reactive oxygen species (ROS), which induced mitochondrial OXPHOS inhibition and decline of ATP production. The energy deficiency inhibited proliferation of PA cells. In addition, overexpressed Drp1 promoted cytochrome c release from damaged mitochondria into the cytoplasm and then activated the downstream caspase apoptotic cascade reaction, which induced apoptosis of PA cells. Moreover, the decreased ATP production induced by Drp1 overexpressing activated the AMPK cellular energy stress sensor and enhanced autophagy through the AMPK-ULK1 pathway, which might play a protective role in PA growth. Furthermore, overexpression of Drp1 repressed PA growth in vivo. Our data indicates that Drp1-mediated mitochondrial metabolic dysfunction inhibits PA growth by affecting cell proliferation, apoptosis, and autophagy. Selectively targeting mitochondrial metabolic homeostasis stands out as a promising antineoplastic strategy for PA therapy.
  18. Mol Metab. 2022 Mar 30. pii: S2212-8778(22)00047-3. [Epub ahead of print] 101478
      OBJECTIVE: Profound metabolic alterations characterize cancer development and, beyond glucose addiction, amino acid (AA) dependency is now recognized as a hallmark of tumour growth. Therefore, targeting the metabolic addiction of tumours by reprogramming their substrate utilization is an attractive therapeutic strategy. We hypothesized that a dietary approach targeted to stimulate oxidative metabolism could reverse the metabolic inflexibility of tumours and represent a proper adjuvant therapy.METHODS: We measured tumour development in xenografted mice fed with a designer, casein-deprived diet enriched in free essential amino acids (EAAs; SFA-EAA diet), or two control isocaloric, isolipidic, and isonitrogenous diets, identical to the SFA-EAA diet except for casein presence (SFA diet), or casein replacement by the free AA mixture designed on the AA profile of casein (SFA-CAA diet). Moreover, we investigated the metabolic, biochemical, and molecular effects of two mixtures that reproduce the AA composition of the SFA-EAA diet (i.e., EAAm) and SFA-CAA diet (i.e., CAAm) in diverse cancer and non-cancer cells.
    RESULTS: The SFA-EAA diet reduced tumour growth in vivo, promoted endoplasmic reticulum (ER) stress, and inhibited mechanistic/mammalian target of rapamycin (mTOR) activity in the tumours. Accordingly, in culture, the EAAm, but not the CAAm, activated apoptotic cell death in cancer cells without affecting the survival and proliferation of non-cancer cells. The EAAm increased branched-chain amino acid (BCAA) oxidation and decreased glycolysis, ATP levels, redox potential, and intracellular content of selective non-essential amino acids (NEAA) in cancer cells. The EAAm-induced NEAA starvation activated the GCN2-ATF4 stress pathway, leading to ER stress, mTOR inactivation, and apoptosis in cancer cells, unlike non-cancer cells.
    CONCLUSION: Together, these results confirm the efficacy of specific EAA mixtures in promoting cancer cells' death and suggest that manipulation of dietary EAA content and profile could be a valuable support to the standard chemotherapy for specific cancers.
    Keywords:  Branched-chain amino acids; Cancer metabolism; Essential amino acids; Glycolysis; Mechanistic/mammalian target of rapamycin; Mitochondria
  19. Expert Rev Mol Diagn. 2022 Apr 08.
      INTRODUCTION: Adaptations of eukaryotic cells to environmental changes are important for their survival. However, under some circumstances, microenvironmental changes promote that eukaryotic cells utilize a metabolic signature resembling a unicellular organism named the Warburg effect. Most cancer cells share the Warburg effect displaying lactic fermentation and high glucose uptake. The Warburg effect also induces a metabolic rewiring stimulating glutamine consumption and lipid synthesis, also considered cancer hallmarks. Amino acid metabolism alteration due to the Warburg effect increases plasma levels of proline and branched-chain amino acids in several cancer types. Proline and lipids are probably used as electron transfer molecules in carcinogenic cells. In addition, branched-chain amino acids fuel the Krebs cycle, protein synthesis, and signaling in cancer cells.AREAS COVERED: This review covers how metabolomics studies describe changes in some metabolites and proteins associated with the Warburg effect and related metabolic pathways.
    EXPERT OPINION: In this review, we analyze the metabolic signature of the Warburg effect and related phenotypes and propose some Warburg effect-related metabolites and proteins (lactate, glucose uptake, glucose transporters, glutamine, branched-chain amino acids, proline, and some lipogenic enzymes) as promising cancer biomarkers.
    Keywords:  Biomarker; Warburg effect; cancer; diagnosis; metabolism; molecular prognosis
  20. Kidney360. 2020 Dec 31. 1(12): 1353-1362
      Background: Kidney risk variants (KRVs) in the APOL1 gene are associated with mitochondrial dysfunction. However, the molecular spectrum of metabolites affected by the G1 and G2 KRVs, and the downstream mitochondrial pathways they affect, remain unknown.Methods: We performed a metabolomics analysis using HEK293 Tet-on cells conditionally expressing APOL1 G0, G1, and G2 KRVs to determine the patterns of metabolites and pathways potentially involved in nephropathy. The Welch two-sample t test, matched-pairs t test, and two-way repeated measures ANOVA were used to identify differential metabolites. Random forest, a supervised classification algorithm that uses an ensemble of decision trees, and the mean-decrease-accuracy metric were applied to prioritize top metabolites.
    Results: Alterations in the tricarboxylic acid cycle, increased fatty acid oxidation, and compromised redox homeostasis were the major pathways affected by overexpression of APOL1 KRVs.
    Conclusions: Impairment of mitochondrial membrane respiratory chain complex I appeared to account for critical metabolic consequences of APOL1 KRVs. This finding supports depletion of the mitochondrial membrane potential, as has been reported.
    Keywords:  APOL1; African Americans; CKD; FSGS; chronic kidney disease; genetics; kidney; metabolomics; mitochondria
  21. J Exp Clin Cancer Res. 2022 Apr 05. 41(1): 125
      BACKGROUND: The development of castration-resistant prostate cancer (CRPC) remains a major obstacle in the treatment of prostate cancer (PCa). Dysregulated mitochondrial function has been linked to the initiation and progression of diverse human cancers. Deciphering the novel molecular mechanisms underlying mitochondrial function may provide important insights for developing novel therapeutics for CRPC.METHODS: We investigate the expression of the protein tyrosine phosphatase receptor type F polypeptide interacting protein alpha 4 (PPFIA4) using public datasets and tumor specimens from PCa cases by immunohistochemistry. Gain- and loss-of-function studies are performed in PCa cell lines and mouse models of subcutaneous xenograft to characterize the role of PPFIA4 in CRPC. Gene expression regulation is evaluated by a series of molecular and biochemical experiments in PCa cell lines. The therapeutic effects of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) inhibitor combined enzalutamide are assessed using in vitro functional assays and in vivo mouse models.
    RESULTS: We show that the increase of PPFIA4 exacerbates aggressive phenotype resembling CRPC. A fraction of PPFIA4 localizes to mitochondria and interacts with MTHFD2, a key enzyme for one-carbon metabolism. Androgen deprivation increases the translocation of PPFIA4 into mitochondria and increases the interaction between PPFIA4 and MTHFD2, which result in the elevation of tyrosine phosphorylated MTHFD2. Consequently, the levels of NADPH synthesis increase, resulting in protection against androgen deprivation-induced mitochondrial dysfunction, as well as promotion of tumor growth. Clinically, PPFIA4 expression is significantly increased in CRPC tissues compared with localized PCa ones. Importantly, an MTHFD2 inhibitor, DS18561882, combined with enzalutamide can significantly inhibit CRPC cell proliferation in vitro and tumor growth in vivo.
    CONCLUSION: Overall, our findings reveal a PPFIA4-MTHFD2 complex in mitochondria that links androgen deprivation to mitochondrial metabolism and mitochondrial dysfunction, which suggest a potential strategy to inhibit CRPC progression.
    Keywords:  CRPC; MTHFD2; PPFIA4; mitochondrial function
  22. Nat Commun. 2022 Apr 06. 13(1): 1850
      Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.
  23. FEBS J. 2022 Apr 03.
      Age-related impairment of coordination of the processes of maintaining mitochondrial homeostasis is associated with a decrease in the functionality of cells and leads to degenerative processes. Mitochondrial DNA (mtDNA) can be a marker of oxidative stress and tissue degeneration. However, the mechanism of accumulation of age-related damage in mtDNA remains unclear. In this study, we analyzed the accumulation of mtDNA damage in several organs of rats during aging, as well as the possibility of reversing these alterations by dietary restriction (DR). We showed that mtDNA of brain compartments (with the exception of the cerebellum), along with kidney mtDNA, was the most susceptible to accumulation of age-related damage, while liver, testis, and lung were the least susceptible organs. DR prevented age-related accumulation of mtDNA damage in the cortex and led to its decrease in the lung and testis. Changes in mtDNA copy number and expression of genes involved in the regulation of mitochondrial biogenesis and mitophagy were also tissue-specific. There was a tendency for an age-related decrease in the copy number of mtDNA in the striatum and its increase in the kidney. DR promoted an increase in the amount of mtDNA in the cerebellum and hippocampus. mtDNA damage may be associated not only with the metabolic activity of organs but also with the lipid composition and activity of processes associated with the isoprostanes pathway of lipid peroxidation. The comparison of polyunsaturated fatty acids (PUFAs) and oxylipins profiles in old rats showed that DR decreased the synthesis of arachidonic acid and its metabolites synthesized by the cyclooxygenase (COX), cytochrome P450 monooxygenases (CYP), and lipoxygenase (LOX) metabolic pathways.
    Keywords:  caloric restriction; mitochondria; oxidative stress; oxylipins; quality control
  24. Angew Chem Int Ed Engl. 2022 Apr 05.
      Identification of different mitochondrial reactive oxygen species (ROS) simultaneously in living cells is vital for understanding the critical roles of different ROS in biological processes. To date, it remains a great challenge to develop ROS probes for direct and simultaneous identification of multiple ROS with high specificity. Herein, we report a SERS-borrowing-strategy-based nanoprobe (Au@Pt core-shell nanoparticles) for simultaneous and direct identification of different ROS by their distinct Raman fingerprints. Isotope substitution experiments and DFT calculations confirmed the ability of Au@Pt nanoprobe to capture and identify different mitochondrial ROS (i.e. •OOH, H2O2, and •OH). When functionalized with triphenylphosphine (TPP), the Au@Pt-TPP nanoprobe located to mitochondria and detected multiple ROS simultaneously in living cells under oxidative stimulation. Our method offers a new tool for the study of the functions of various ROS in biological processes.
    Keywords:  Core-shell nanoprobe; Living cells; SERS borrowing; Spectroscopic identification; reactive oxygen species
  25. Elife. 2022 Apr 07. pii: e75244. [Epub ahead of print]11
      Changes in DNA methylation (DNAm) are linked to aging. Here, we profile highly conserved CpGs in 339 predominantly female mice belonging to the BXD family for which we have deep longevity and genomic data. We use a 'pan-mammalian' microarray that provides a common platform for assaying the methylome across mammalian clades. We computed epigenetic clocks and tested associations with DNAm entropy, diet, weight, metabolic traits, and genetic variation. We describe the multifactorial variance of methylation at these CpGs, and show that high fat diet augments the age-associated changes. Entropy increases with age. The progression to disorder, particularly at CpGs that gain methylation over time, was predictive of genotype-dependent life expectancy. The longer-lived BXD strains had comparatively lower entropy at a given age. We identified two genetic loci that modulate rates of epigenetic age acceleration (EAA): one on chromosome (Chr) 11 that encompasses the Erbb2/Her2 oncogenic region, and a second on Chr19 that contains a cytochrome P450 cluster. Both loci harbor genes associated with EAA in humans including STXBP4, NKX2-3, and CUTC. Transcriptome and proteome analyses revealed associations with oxidation-reduction, metabolic, and immune response pathways. Our results highlight concordant loci for EAA in humans and mice, and demonstrate a tight coupling between the metabolic state and epigenetic aging.
    Keywords:  chromosomes; gene expression; genetics; genomics; mouse
  26. Nat Commun. 2022 Apr 06. 13(1): 1853
      Protein homeostatic control of mitochondria is key to age-related diseases and organismal decline. However, it is unknown how the diverse types of stress experienced by mitochondria can be integrated and appropriately responded to in human cells. Here we identify perturbations in the ancient conserved processes of mitochondrial protein import and processing as sources of DELE1 activation: DELE1 is continuously sorted across both mitochondrial membranes into the matrix and detects different types of perturbations along the way. DELE1 molecules in transit can become licensed for mitochondrial release and stress signaling through proteolytic removal of N-terminal sorting signals. Import defects that occur at the mitochondrial surface allow DELE1 precursors to bind and activate downstream factor HRI without the need for cleavage. Genome-wide genetics reveal that DELE1 additionally responds to compromised presequence processing by the matrix proteases PITRM1 and MPP, which are mutated in neurodegenerative diseases. These mechanisms rationalize DELE1-dependent mitochondrial stress integration in the human system and may inform future therapies of neuropathies.
  27. Proc Natl Acad Sci U S A. 2022 Apr 12. 119(15): e2118740119
      SignificanceMultiple human genetic diseases are caused by mutations in the maternally transmitted DNA of mitochondria, the powerhouses of the cell. It is important to study how these mutations arise and accumulate with age, especially because humans in many societies now choose to have children at an older age. However, this is difficult to accomplish in humans, particularly for female germline cells, oocytes. To overcome this limitation, we studied mitochondrial mutation origins and accumulation with age in a primate model species, rhesus macaque. We found that new mutations accumulate the fastest in metabolically active liver and the slowest in oocytes. Thus, primate oocytes might have developed a mechanism to protect their mitochondrial DNA from excessive mutations, allowing reproduction later in life.
    Keywords:  duplex sequencing; heteroplasmy; mitochondria; mutations; oocytes
  28. J Biomol Struct Dyn. 2022 Apr 08. 1-20
      Complex V or FoF1-ATPase is a multimeric protein found in bioenergetic membranes of cells and organelles like mitochondria/chloroplasts. The popular perception on Complex V deems it as a reversible molecular motor, working bi-directionally (breaking or making ATP) via a conformation-change based chemiosmotic rotary ATP synthesis (CRAS) mechanism, driven by proton-gradients or trans-membrane potential (TMP). In continuance of our pursuits against the CRAS model of cellular bioenergetics, herein we demonstrate the validity of the murburn model based in diffusible reactive (oxygen) species (DRS/DROS). Supported by new in silico derived data (that there are ∼12 adenosine nucleotide binding sites on the F1 bulb and not merely 3 sites, as perceived earlier), available structural information, known experimental observations, and thermodynamic/kinetic considerations (that de-solvation of protons from hydronium ions is facile), we deduce that Complex V serves as a physiological chemostat and a murzyme (enzyme working via murburn scheme, employing DRS). That is- Complex V uses ATP (via consumption at ε or proteins of F1 module) as a Michaelis-Menten substrate to serve as a pH-stat by inletting protons via the c-ring of Fo module. Physiologically, Complex V also functions as a murzyme by presenting ADP/Pi (or their reaction intermediates) on the αβ bulb, thereby enabling greater opportunities for DRS/proton-assisted ATP formation. Thus, the murburn paradigm succeeds the CRAS hypothesis for explaining the role of oxygen in mitochondrial physiologies of oxidative phosphorylation, thermogenesis, TMP and homeostasis. Communicated by Ramaswamy H. Sarma.
    Keywords:  ATPase; ATPsynthase; Complex V; bioenergetic phosphorylation; molecular motor; murburn concept; murzyme; rotary enzyme
  29. Front Cell Dev Biol. 2022 ;10 836755
      Mitochondria are multifunctional organelles of which ultrastructure is tightly linked to cell physiology. Accumulating evidence shows that mitochondrial remodeling has an impact on immune responses, but our current understanding of the mitochondrial architecture, interactions, and morphological changes in immune cells, mainly in eosinophils, is still poorly known. Here, we applied transmission electron microscopy (TEM), single-cell imaging analysis, and electron tomography, a technique that provides three-dimensional (3D) views at high resolution, to investigate mitochondrial dynamics in mouse eosinophils developing in cultures as well as in the context of inflammatory diseases characterized by recruitment and activation of these cells (mouse models of asthma, H1N1 influenza A virus (IAV) infection, and schistosomiasis mansoni). First, quantitative analyses showed that the mitochondrial area decrease 70% during eosinophil development (from undifferentiated precursor cells to mature eosinophils). Mitophagy, a consistent process revealed by TEM in immature but not in mature eosinophils, is likely operating in mitochondrial clearance during eosinophilopoiesis. Events of mitochondria interaction (inter-organelle membrane contacts) were also detected and quantitated within developing eosinophils and included mitochondria-endoplasmic reticulum, mitochondria-mitochondria, and mitochondria-secretory granules, all of them significantly higher in numbers in immature compared to mature cells. Moreover, single-mitochondrion analyses revealed that as the eosinophil matures, mitochondria cristae significantly increase in number and reshape to lamellar morphology. Eosinophils did not change (asthma) or reduced (IAV and Schistosoma infections) their mitochondrial mass in response to inflammatory diseases. However, asthma and schistosomiasis, but not IAV infection, induced amplification of both cristae numbers and volume in individual mitochondria. Mitochondrial cristae remodeling occurred in all inflammatory conditions with the proportions of mitochondria containing only lamellar or tubular, or mixed cristae (an ultrastructural aspect seen just in tissue eosinophils) depending on the tissue/disease microenvironment. The ability of mitochondria to interact with granules, mainly mobilized ones, was remarkably captured by TEM in eosinophils participating in all inflammatory diseases. Altogether, we demonstrate that the processes of eosinophilopoiesis and inflammation-induced activation interfere with the mitochondrial dynamics within mouse eosinophils leading to cristae remodeling and inter-organelle contacts. The understanding of how mitochondrial dynamics contribute to eosinophil immune functions is an open interesting field to be explored.
    Keywords:  electron tomography; eosinophilopoiesis; eosinophils; mitochondria contact sites; mitochondria ultrastructure; mitochondrial architecture; mitochondrial dynamics; mouse
  30. Cell Rep. 2022 Apr 05. pii: S2211-1247(22)00357-6. [Epub ahead of print]39(1): 110609
      Tumor-associated macrophages (TAMs) are a major cellular component in the tumor microenvironment (TME). However, the relationship between the phenotype and metabolic pattern of TAMs remains poorly understood. We performed single-cell transcriptome profiling on hepatic TAMs from mice bearing liver metastatic tumors. We find that TAMs manifest high heterogeneity at the levels of transcription, development, metabolism, and function. Integrative analyses and validation experiments indicate that increased purine metabolism is a feature of TAMs with pro-tumor and terminal differentiation phenotypes. Like mouse TAMs, human TAMs are highly heterogeneous. Human TAMs with increased purine metabolism exhibit a pro-tumor phenotype and correlate with poor therapeutic efficacy to immune checkpoint blockade. Altogether, our work demonstrates that TAMs are developmentally, metabolically, and functionally heterogeneous and purine metabolism may be a key metabolic feature of a pro-tumor macrophage population.
    Keywords:  CP: Cancer; CP: Metabolism; cancer; checkpoint; immunosuppression; immunotherapy; liver; macrophage; metabolism; purine; single-cell RNA sequencing; tumor microenvironment
  31. Blood. 2022 Apr 07. pii: blood.2021013983. [Epub ahead of print]
      Mutant TP53 is an adverse risk factor in acute myeloid leukemia (AML), but large-scale integrated genomic-proteomic analyses of p53 alterations in AML patients remain limited. We analyzed TP53 mutational status, copy number (CN), and protein expression data in AML (N=528) and provide a compilation of mutation sites and types across disease subgroups among treated and untreated patients. Our analysis shows differential hotspots in subsets of AML and uncovered novel pathogenic variants involving TP53 splice sites. In addition, we identified TP53 CN loss in 70.2% of TP53-mutated AML, which had more deleterious TP53 mutations and copy neutral loss of heterozygosity in 5/32 (15.6%) AML patients who had intact TP53 CN. Importantly, we demonstrate that mutant p53 protein expression patterns by immunohistochemistry evaluated using digital image-assisted analysis provide a robust readout that integrates TP53 mutation and allelic states in patients with AML (sensitivity=94.49%, specificity=90.48%). Protein expression of p53 by immunohistochemistry informed mutation status irrespective of TP53 CN status. Genomic analysis of co-mutations in TP53-mutant AML showed a muted landscape that encompassed primarily mutations in genes involved in epigenetic regulation (DNMT3A and TET2), RAS/MAPK signaling (NF1, KRAS/NRAS, PTPN11), and RNA splicing (SRSF2). In summary, our data provides a rationale to refine risk stratification of AML patients on the basis of integrated molecular and protein-level TP53 analyses.
  32. Biomark Res. 2022 Apr 02. 10(1): 16
      Acute myeloid leukemia (AML) has the lowest survival rate among the leukemias. Targeting intracellular metabolism and energy production in leukemic cells can be a promising therapeutic strategy for AML. Recently, we presented the successful use of vitamin D (1,25VD3) gene therapy to treat AML mouse models in vivo. In this study, recognizing the importance of 1,25VD3 as one of only 2 molecules (along with glucose) photosynthesized for energy during the beginning stage of life on this planet, we explored the functional role of 1,25VD3 in AML metabolism.Transcriptome database (RNA-seq) of four different AML cell lines revealed 17,757 genes responding to 1,25VD3-treatment. Moreover, we discovered that fructose-bisphosphatase 1 (FBP1) noticeably stands out as the only gene (out of 17,757 genes) with a 250-fold increase in gene expression, which is known to encode the key rate-limiting gluconeogenic enzyme fructose-1,6-bisphosphatase. The significant increased expression of FBP1 gene and proteins induced by 1,25VD3 was confirmed by qPCR, western blot, flow cytometry, immunocytochemistry and functional lactate assay. Additionally, 1,25VD3 was found to regulate different AML metabolic processes including gluconeogenesis, glycolysis, TCA, de novo nucleotide synthesis, etc. In summary, we provided the first evidence that 1,25 VD3-induced FBP1 overexpression might be a novel therapeutic target to block the "Warburg Effect" to reduce energy production in AML blasts.
    Keywords:  AML; FBP1; Glycolysis; Metabolism; Vitamin D; Warburg effect
  33. Cell Mol Biol Lett. 2022 Apr 05. 27(1): 32
      BACKGROUND: Autophagy plays an essential role in maintaining cellular homeostasis and in the response to cellular stress. Autophagy is also involved in cell cycle progression, yet the relationship between these processes is not clearly defined.RESULTS: In exploring this relationship, we observed that the inhibition of autophagy impaired the G2/M phase-arresting activity of etoposide but enhanced the G1 phase-arresting activity of palbociclib. We further investigated the connection of basal autophagy and cell cycle by utilizing the autophagosome tracer dye Cyto-ID in two ways. First, we established a double-labeling flow-cytometric procedure with Cyto-ID and the DNA probe DRAQ5, permitting the cell cycle phase-specific determination of autophagy in live cells. This approach demonstrated that different cell cycle phases were associated with different autophagy levels: G1-phase cells had the lowest level, and G2/M-phase cells had the highest one. Second, we developed a flow-cytometric cell-sorting procedure based on Cyto-ID that separates cell populations into fractions with low, medium, and high autophagy. Cell cycle analysis of Cyto-ID-sorted cells confirmed that the high-autophagy fraction contained a much higher percentage of G2/M-phase cells than the low-autophagy fraction. In addition, Cyto-ID-based cell sorting also proved to be useful for assessing other autophagy-related processes: extracellular flux analysis revealed metabolic differences between the cell populations, with higher autophagy being associated with higher respiration, higher mitochondrial ATP production, and higher glycolysis.
    CONCLUSION: This work provides clear evidence of high autophagy in G2/M-phase cells by establishing a novel cell sorting technique based on Cyto-ID.
    Keywords:  Autophagy; Cell cycle; Cell sorting; Cyto-ID; DRAQ5; Metabolic analysis