bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2020‒03‒22
forty-five papers selected by
Kelsey Fisher-Wellman
East Carolina University


  1. Nat Commun. 2020 Mar 20. 11(1): 1487
    Schöpf B, Weissensteiner H, Schäfer G, Fazzini F, Charoentong P, Naschberger A, Rupp B, Fendt L, Bukur V, Giese I, Sorn P, Sant'Anna-Silva AC, Iglesias-Gonzalez J, Sahin U, Kronenberg F, Gnaiger E, Klocker H.
      Rewiring of energy metabolism and adaptation of mitochondria are considered to impact on prostate cancer development and progression. Here, we report on mitochondrial respiration, DNA mutations and gene expression in paired benign/malignant human prostate tissue samples. Results reveal reduced respiratory capacities with NADH-pathway substrates glutamate and malate in malignant tissue and a significant metabolic shift towards higher succinate oxidation, particularly in high-grade tumors. The load of potentially deleterious mitochondrial-DNA mutations is higher in tumors and associated with unfavorable risk factors. High levels of potentially deleterious mutations in mitochondrial Complex I-encoding genes are associated with a 70% reduction in NADH-pathway capacity and compensation by increased succinate-pathway capacity. Structural analyses of these mutations reveal amino acid alterations leading to potentially deleterious effects on Complex I, supporting a causal relationship. A metagene signature extracted from the transcriptome of tumor samples exhibiting a severe mitochondrial phenotype enables identification of tumors with shorter survival times.
    DOI:  https://doi.org/10.1038/s41467-020-15237-5
  2. Mol Metab. 2020 Feb 04. pii: S2212-8778(19)30959-7. [Epub ahead of print]34 97-111
    Fan J, Du W, Kim-Muller JY, Son J, Kuo T, Larrea D, Garcia C, Kitamoto T, Kraakman MJ, Owusu-Ansah E, Cirulli V, Accili D.
      OBJECTIVE: Diabetes is characterized by pancreatic β-cell dedifferentiation. Dedifferentiating β cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the β-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear.METHODS: Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to β-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios.
    RESULTS: The expression of Cyb5r3 is decreased in FoxO1-deficient β cells. Mice with β-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient β cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient β cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability.
    CONCLUSIONS: The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate β-cell failure.
    Keywords:  Beta cell dedifferentiation; Diabetes genetics; Diabetes therapy; Endocrine pancreas; Glucose clamp; Hyperglycemia; Mitochondrial complex III failure; Transcription factor in beta cell function; Type 2 diabetes
    DOI:  https://doi.org/10.1016/j.molmet.2019.12.008
  3. Exp Gerontol. 2020 Mar 13. pii: S0531-5565(19)30701-6. [Epub ahead of print] 110924
    Özsoy M, Zimmermann FA, Feichtinger RG, Mayr JA, Kofler B, Neureiter D, Klieser E, Schütz S, Weghuber D, Schneider AM.
      OBJECTIVE: Mitochondria produce cellular energy via oxidative phosphorylation (OXPHOS), mediated by respiratory chain complexes I to IV and ATP synthase (complex V). Mitochondrial respiratory complexes have been shown to decline with age in several tissues. As the intestinal epithelium is a tissue with a high energy demand, the aim of the present study was to establish whether the expression profile of OXPHOS subunits in the intestinal mucosa changes during the aging process.DESIGN: Biopsies of intestinal mucosa with no evidence of endoscopic or histomorphologic abnormalities, taken from 55 patients (mean age 42 years, age range 4-82 years; 62% female), were divided into four age groups (4-19, 20-39, 40-59, ≥60 years). Sections from different intestinal segments (terminal ileum, ascending colon, and sigmoid colon/rectum) were stained immunohistochemically (IHC) for subunits of OXPHOS complexes I-V and the voltage-dependent anion-selective channel 1 protein (VDAC1, porin), a marker of mitochondrial mass. Scores for IHC staining were determined by multiplication of the staining intensity and the percentage of positive cells. In addition, the numbers of intestinal crypts staining positive, partly positive, and negative were assessed.
    RESULTS: The average protein expression levels of OXPHOS subunits increased continuously from childhood onward, peaked in persons aged 20 to 59 years, and declined thereafter. This was seen for complexes II to V in the terminal ileum, complexes I to V in the ascending colon, and complexes I to IV in the sigmoid colon/rectum. Across all age groups, no effect of age on expression of the porin subunit VDAC1 was detected. The number of complex I- and IV-negative crypts in different intestinal segments increased with age.
    CONCLUSION: The protein expression levels of OXPHOS complexes increases from childhood onward and declines in elderly individuals, while the numbers of crypts with partial or complete loss of expression of complexes I and IV increase continuously with age. These data suggest that the continued reductions in the levels of mitochondrial OXPHOS complexes in crypts might be compensated in adulthood, but that, ultimately, reduced expression levels occur in persons aged 60 years and older. These findings raise two important questions: first, can the process of aging could be delayed through (pharmacological) intervention of mitochondrial pathways, and second, pathophysiologically, are these findings associated with disorders of the intestinal mucosa, e.g. inflammation?
    Keywords:  Aging; Colonic crypt; Expression; Intestine; Mitochondria; OXPHOS
    DOI:  https://doi.org/10.1016/j.exger.2020.110924
  4. Cell Metab. 2020 Mar 16. pii: S1550-4131(20)30114-5. [Epub ahead of print]
    Yang L, Garcia Canaveras JC, Chen Z, Wang L, Liang L, Jang C, Mayr JA, Zhang Z, Ghergurovich JM, Zhan L, Joshi S, Hu Z, McReynolds MR, Su X, White E, Morscher RJ, Rabinowitz JD.
      NADH provides electrons for aerobic ATP production. In cells deprived of oxygen or with impaired electron transport chain activity, NADH accumulation can be toxic. To minimize such toxicity, elevated NADH inhibits the classical NADH-producing pathways: glucose, glutamine, and fat oxidation. Here, through deuterium-tracing studies in cultured cells and mice, we show that folate-dependent serine catabolism also produces substantial NADH. Strikingly, when respiration is impaired, serine catabolism through methylene tetrahydrofolate dehydrogenase (MTHFD2) becomes a major NADH source. In cells whose respiration is slowed by hypoxia, metformin, or genetic lesions, mitochondrial serine catabolism inhibition partially normalizes NADH levels and facilitates cell growth. In mice with engineered mitochondrial complex I deficiency (NDUSF4-/-), serine's contribution to NADH is elevated, and progression of spasticity is modestly slowed by pharmacological blockade of serine degradation. Thus, when respiration is impaired, serine catabolism contributes to toxic NADH accumulation.
    Keywords:  MTHFD2; NAD; NADH; SHMT2; complex I inhibitor; hypoxia; methylene tetrahydrofolate dehydrogenase; mitochondrial disease; redox; respiration inhibition; serine catabolism; serine hydroxymethyltransferase
    DOI:  https://doi.org/10.1016/j.cmet.2020.02.017
  5. Aging Cell. 2020 Mar 20. e13124
    Adelnia F, Ubaida-Mohien C, Moaddel R, Shardell M, Lyashkov A, Fishbein KW, Aon MA, Spencer RG, Ferrucci L.
      Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23-87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by 31 P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τPCr ) in the vastus lateralis muscle, with smaller τPCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC-MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.
    Keywords:  31P MRS; bioenergetic; mitochondria; proteomic; skeletal muscle
    DOI:  https://doi.org/10.1111/acel.13124
  6. Biomolecules. 2020 Mar 09. pii: E427. [Epub ahead of print]10(3):
    Szwed A, Miłowska K, Michlewska S, Moreno S, Shcharbin D, Gomez-Ramirez R, de la Mata FJ, Majoral JP, Bryszewska M, Gabryelak T.
      Dendrimers as drug carriers can be utilized for drugs and siRNA delivery in central nervous system (CNS) disorders, including various types of cancers, such as neuroblastomas and gliomas. They have also been considered as drugs per se, for example as anti-Alzheimer's disease (AD), anti-cancer, anti-prion or anti-inflammatory agents. Since the influence of carbosilane-viologen-phosphorus dendrimers (SMT1 and SMT2) on the basic cellular processes of nerve cells had not been investigated, we examined the impact of two generations of these hybrid macromolecules on two murine cell lines-cancer cell line N2a (mouse neuroblastoma) and normal immortalized cell line mHippoE-18 (embryonic mouse hippocampal cell line). We examined alterations in cellular responses including the activity of mitochondrial dehydrogenases, the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, and morphological modifications and fractions of apoptotic and dead cells. Our results show that both dendrimers at low concentrations affected the cancer cell line more than the normal one. Also, generation-dependent effects were found: the highest generation induced greater cytotoxic effects and morphological modifications. The most promising is that the changes in mitochondrial membrane potential and transmission electron microscopy (TEM) images indicate that dendrimer SMT1 can reach mitochondria. Thus, SMT1 and SMT2 seem to have potential as nanocarriers to mitochondria or anti-cancer drugs per se in CNS disorders.
    Keywords:  N2a; ROS; TEM; apoptosis; carbosilane–viologen–phosphorus dendrimers; cytotoxicity; mHippoE-18; ΔΨm
    DOI:  https://doi.org/10.3390/biom10030427
  7. Theranostics. 2020 ;10(6): 2571-2586
    Li T, Li Y, Liu T, Hu B, Li J, Liu C, Liu T, Li F.
      Rationale: P21-activated kinase 6 (PAK6) is a member of the class II PAKs family, which is a conserved family of serine/threonine kinases. Although the effects of PAK6 on many malignancies, especially in prostate cancer, have been studied for a long time, the role of PAK6 in mitochondria remains unknown. Methods: The expression of PAK6, SIRT4 and ANT2 in prostate cancer and adjacent non-tumor tissues was detected by immunohistochemistry. Immunofuorescence and immunoelectron microscopy were used to determine the subcellular localization of PAK6. Immunoprecipitation, immunofuorescence and ubiquitination assays were performed to determine how PAK6 regulates SIRT4, how SIRT4 regulates ANT2, and how PAK6 regulates ANT2. Flow cytometry detection and xenograft models were used to evaluate the impact of ANT2 mutant expression on the prostate cancer cell cycle and apoptosis regulation. Results: The present study revealed that the PAK6-SIRT4-ANT2 complex is involved in mitochondrial apoptosis in prostate cancer cells. It was found that PAK6 is mainly located in the mitochondrial inner membrane, in which PAK6 promotes SIRT4 ubiquitin-mediated proteolysis. Furthermore, SIRT4 deprives the ANT2 acetylation at K105 to promote its ubiquitination degradation. Hence, PAK6 adjusts the acetylation level of ANT2 through the PAK6-SIRT4-ANT2 pathway, in order to regulate the stability of ANT2. Meanwhile, PAK6 directly phosphorylates ANT2 atT107 to inhibit the apoptosis of prostate cancer cells. Therefore, the phosphorylation and deacetylation modifications of ANT2 are mutually regulated, leading to tumor growth in vivo. Consistently, these clinical prostate cancer tissue evaluations reveal that PAK6 is positively correlated with ANT2 expression, but negatively correlated with SIRT4. Conclusion: These present findings suggest the pivotal role of the PAK6-SIRT4-ANT2 complex in the apoptosis of prostate cancer. This complex could be a potential biomarker for the treatment and prognosis of prostate cancer.
    Keywords:  ANT2; PAK6; SIRT4; apoptosis; prostate cancer.
    DOI:  https://doi.org/10.7150/thno.42874
  8. Am J Transl Res. 2020 ;12(2): 428-446
    Chen L, Peng J, Wang Y, Jiang H, Wang W, Dai J, Tang M, Wei Y, Kuang H, Xu G, Xu H, Zhou F.
      Cancer cells reprogram their metabolism to adapt to fast growth and environmental demands, which differ them from normal cells. Mitochondria are central to the malignant metabolism reprogramming process. Here, we report that PPARα was highly expressed in gastric cancer tissues and negatively correlated with prognosis. Fenofibrate, a common drug used to treat severe hypertriglyceridemia and mixed dyslipidemia, reversed cellular metabolism and mitochondrial dysfunction in gastric cancer cells through PPARα. Our results show that fenofibrate altered glucose and lipid metabolism, inhibited gastric cancer cell proliferation, and promoted apoptosis in gastric cancer cells. We further show that fenofibrate induced mitochondrial reprogramming via CPT1 and the fatty acid oxidation pathway, as well as by activating the AMPK pathway and inhibiting the HK2 pathway. Additionally, fenofibrate inhibited subcutaneous gastric cancer cell tumor growth without obvious toxicity in mice. Collectively, our results indicate that fenofibrate exhibits anti-tumor activity in vitro and in vivo via the mitochondria and metabolic reprogramming, demonstrating that mitochondrial regulation and the normalization of cancer cell metabolism are novel therapeutic strategies for cancer.
    Keywords:  Fenofibrate; PPARα; gastric cancer; metabolic reprogramming; mitochondrial dysfunctions
  9. mBio. 2020 Mar 17. pii: e00268-20. [Epub ahead of print]11(2):
    Huang G, Docampo R.
      Mitochondrial Ca2+ transport mediated by the uniporter complex (MCUC) plays a key role in the regulation of cell bioenergetics in both trypanosomes and mammals. Here we report that Trypanosoma brucei MCU (TbMCU) subunits interact with subunit c of the mitochondrial ATP synthase (ATPc), as determined by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Mutagenesis analysis in combination with MYTH assays suggested that transmembrane helices (TMHs) are determinants of this specific interaction. In situ tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that T. brucei ATPc (TbATPc) coimmunoprecipitates with TbMCUC subunits and colocalizes with them to the mitochondria. Blue native PAGE and immunodetection analyses indicated that the TbMCUC is present together with the ATP synthase in a large protein complex with a molecular weight of approximately 900 kDa. Ablation of the TbMCUC subunits by RNA interference (RNAi) significantly increased the AMP/ATP ratio, revealing the downregulation of ATP production in the cells. Interestingly, the direct physical MCU-ATPc interaction is conserved in Trypanosoma cruzi and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed in vitro by mutagenesis and MYTH assays and in vivo by coimmunoprecipitation. In summary, our study has identified that MCU complex physically interacts with mitochondrial ATP synthase, possibly forming an MCUC-ATP megacomplex that couples ADP and Pi transport with ATP synthesis, a process that is stimulated by Ca2+ in trypanosomes and human cells.IMPORTANCE The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in Trypanosoma brucei, the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that Trypanosoma brucei MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in T. cruzi and human cells.
    Keywords:  ATP synthase; Trypanosoma ; c ring; mitochondrial calcium uniporter
    DOI:  https://doi.org/10.1128/mBio.00268-20
  10. Autophagy. 2020 Mar 18. 1-13
    Li C, Zhang Y, Liu J, Kang R, Klionsky DJ, Tang D.
      Pancreatic cancer tends to be highly resistant to current therapy and remains one of the great challenges in biomedicine with very low 5-year survival rates. Here, we report that zalcitabine, an antiviral drug for human immunodeficiency virus infection, can suppress the growth of primary and immortalized human pancreatic cancer cells through the induction of ferroptosis, an iron-dependent form of regulated cell death. Mechanically, this effect relies on zalcitabine-induced mitochondrial DNA stress, which activates the STING1/TMEM173-mediated DNA sensing pathway, leading to macroautophagy/autophagy-dependent ferroptotic cell death via lipid peroxidation, but not a type I interferon response. Consequently, the genetic and pharmacological inactivation of the autophagy-dependent ferroptosis pathway diminishes the anticancer effects of zalcitabine in cell culture and animal models. Together, these findings not only provide a new approach for pancreatic cancer therapy but also increase our understanding of the interplay between autophagy and DNA damage response in shaping cell death.Abbreviations: ALOX: arachidonate lipoxygenase; ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATM: ATM serine/threonine kinase; ATG: autophagy-related; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; ER: endoplasmic reticulum; FANCD2: FA complementation group D2; GPX4: glutathione peroxidase 4; IFNA1/IFNα: interferon alpha 1; IFNB1/IFNβ: interferon beta 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MDA: malondialdehyde; mtDNA: mitochondrial DNA; NCOA4: nuclear receptor coactivator 4; PDAC: pancreatic ductal adenocarcinoma; POLG: DNA polymerase gamma, catalytic subunit; qRT-PCR: quantitative polymerase chain reaction; RCD: regulated cell death; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; STING1/TMEM173: stimulator of interferon response cGAMP interactor 1; TFAM: transcription factor A, mitochondrial.
    Keywords:  Antiviral drug; CGAS; DNA damage; POLG; STING1; TFAM; autophagy; ferroptosis; mitochondria; tumor therapy
    DOI:  https://doi.org/10.1080/15548627.2020.1739447
  11. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30160-8. [Epub ahead of print]155 157-180
    Agrawal RR, Tamucci KA, Pera M, Larrea D.
      Mitochondria are responsible for the generation of ATP by oxidative phosphorylation; however, these multifaceted organelles regulate many other key cellular functions as well, such as calcium homeostasis, apoptosis, and biosynthesis of steroid hormones, heme and phospholipids. In order to carry out these functions, mitochondria establish physical and functional connections with other organelles such as the plasma membrane, lipid droplets/vesicles, peroxisomes, endosomes, and the endoplasmic reticulum. Dysregulation of any of the aforementioned processes or inter-organelle contacts can lead to mitochondrial dysfunction and subsequent changes in oxygen consumption and ATP production. Seahorse technology has become a critical tool for quantification of mitochondrial oxygen consumption and can help differentiate primary mitochondrial disorders from disorders where alterations in mitochondrial metabolism are consequences of a prior, upstream insult. In this chapter, we describe the application of Seahorse technology for assaying mitochondrial respiration in whole cells, permeabilized cells and isolated mitochondria. We leave it to the researcher's discretion to determine which of these approaches will generate the most physiologically relevant data based on the model system and research question at hand.
    Keywords:  Mitochondria; Oxygen consumption; Respiration
    DOI:  https://doi.org/10.1016/bs.mcb.2019.12.005
  12. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30137-2. [Epub ahead of print]155 121-156
    Frazier AE, Vincent AE, Turnbull DM, Thorburn DR, Taylor RW.
      Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease. Considerations for adaption of these techniques to other cells, tissues, and organisms are presented, as well as comparisons to alternate methods for analysis of respiratory chain function. In this context, a quantitative immunofluorescence protocol is also provided that is suitable for measurement of the amount of multiple respiratory chain complexes in small diagnostic tissue samples. The analysis and interpretation of OXPHOS enzyme activities are then placed in the context of mitochondrial disease tissue pathology and diagnosis.
    Keywords:  Citrate synthase; Enzymology; Mitochondria; Mitochondrial disease; Oxidative phosphorylation; Quantitative immunofluorescence; Respiratory chain
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.007
  13. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30124-4. [Epub ahead of print]155 199-219
    Morciano G, Imamura H, Patergnani S, Pedriali G, Giorgi C, Pinton P.
      Adenosine 5'-triphosphate (ATP) is the central metabolite in the energy metabolism of cells and is hydrolyzed to ADP and inorganic phosphate to provide free energy in various cellular processes. ATP also functions as an intracellular signaling molecule. Thus, it is important to know the ATP concentration within cells to understand cellular activities. Here, we describe two methods to detect ATP concentrations in the cytoplasm and mitochondrial matrix using genetically encoded luminescent or fluorescent biosensors. These methods enable quantitative investigation of ATP concentration dynamics in living cells, single cells and cell populations.
    Keywords:  ATP; Bioluminescence; Biosensor; FRET; Fluorescence; Live cell imaging; Luciferase assay; Mitochondria
    DOI:  https://doi.org/10.1016/bs.mcb.2019.10.007
  14. Redox Biol. 2020 Mar 07. pii: S2213-2317(19)31503-4. [Epub ahead of print]32 101472
    Mailloux RJ.
      The pathogenesis of many human diseases has been attributed to the over production of reactive oxygen species (ROS), particularly superoxide (O2●-) and hydrogen peroxide (H2O2), by-products of metabolism that are generated by the premature reaction of electrons with molecular oxygen (O2) before they reach complex IV of the respiratory chain. To date, there are 32 known ROS generators in mammalian cells, 16 of which reside inside mitochondria. Importantly, although these ROS are deleterious at high levels, controlled and temporary bursts in H2O2 production is beneficial to mammalian cells. Mammalian cells use sophisticated systems to take advantage of the second messaging properties of H2O2. This includes controlling its availability using antioxidant systems and negative feedback loops that inhibit the genesis of ROS at sites of production. At its core, ROS production depends on fuel metabolism. Therefore, desensitizing H2O2 signals would also require the temporary inhibition of fuel combustion and fluxes through metabolic pathways that promote ROS production. Additionally, this would also demand the diversion of fuels and nutrients into pathways that generate NADPH and other molecules required to maintain cellular redox buffering capacity. Therefore, fuel selection and metabolic flux plays an integral role in dictating the strength and duration of cellular redox signals. In the present review I provide an updated view on the function of protein S-glutathionylation, a ubiquitous redox sensitive modification involving the formation of a disulfide between the small molecular antioxidant glutathione and a cysteine residue, in the regulation of cellular metabolism on a global scale. To date, these concepts have mostly been reviewed at the level of mitochondrial bioenergetics in the contexts of health and disease. Careful examination of the literature revealed that glutathionylation is a temporary inhibitor of most metabolic pathways including glycolysis, the Krebs cycle, oxidative phosphorylation, amino acid metabolism, and fatty acid combustion, resulting in the diversion of fuels towards NADPH-producing pathways and the inhibition of ROS production. Armed with this information, I propose that protein S-glutathionylation reactions desensitize H2O2 signals emanating from catabolic pathways using a three-pronged regulatory mechanism; 1) inhibition of metabolic flux through pathways that promote ROS production, 2) diversion of metabolites towards pathways that support antioxidant defenses, and 3) direct inhibition of ROS-generating enzymes.
    Keywords:  ROS; glutathoinylation; metabolic regulation; redox signaling
    DOI:  https://doi.org/10.1016/j.redox.2020.101472
  15. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30138-4. [Epub ahead of print]155 295-319
    Liao PC, Franco-Iborra S, Yang Y, Pon LA.
      The redox state of mitochondria is determined by the levels of reducing and oxidizing species in the organelle, which reflects mitochondrial metabolic activity and overall fitness. Mitochondria are also the primary endogenous source of reactive oxygen species (ROS). This chapter describes methods to measure the mitochondrial superoxide levels and the redox state of the organelle in mammalian cells and yeast. We describe the use of dihydroethidium (DHE) and MitoSOX (a derivative of dihydroethidium bound to a lipophilic cation) to detect mitochondrial superoxide in yeast and mammalian cells, respectively. We also describe the use of genetically encoded fluorescent biosensors for quantitative analysis of mitochondrial NADPH levels (iNap) in mammalian cells and mitochondrial redox state (mito-roGFP) in yeast.
    Keywords:  Dihydroethidium (DHE); MitoSOX; NADPH; Oxidative stress; Reactive oxygen species (ROS); iNap3; roGFP
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.008
  16. Cancer Res. 2020 Mar 16. pii: canres.2852.2019. [Epub ahead of print]
    Xie J, Ye J, Cai Z, Luo Y, Zhu X, Deng Y, Feng Y, Liang Y, Liu R, Han Z, Liang Y, Zheng Y, Mo R, Zhuo Y, Wu Y, Jiang F, Zhu J, Wu CL, Zhong W.
      Metformin is an oral drug widely used for the treatment of type 2 diabetes mellitus. Numerous studies have demonstrated the value of metformin in cancer treatment. However, for metformin to elicit effects on cancer this often requires a high dosage, and any underlying mechanism for how to improve its inhibitory effects remains unknown. Here we found that low mRNA expression of glycerol-3-phosphate dehydrogenase 1 (GPD1) may predict a poor response to metformin treatment in 15 cancer cell lines. In vitro and in vivo, metformin treatment alone significantly suppressed cancer cell proliferation, a phenotype enhanced by GPD1 overexpression. Total cellular glycerol-3-phosphate concentration was significantly increased by the combination of GPD1 overexpression and metformin treatment, which suppressed cancer growth via inhibition of mitochondrial function. Eventually, increased reactive oxygen species and mitochondrial structural damage was observed in GPD1-overexpressing cell lines treated with metformin, which may contribute to cell death. In summary, this study demonstrates that GPD1 overexpression enhances the anti-cancer activity of metformin and that patients with increased GPD1 expression in tumor cells may respond better to metformin therapy.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-19-2852
  17. Liver Int. 2020 Mar 16.
    Geng X, Geng Z, Li H, Zhang Y, Li J, Chang H.
      BACKGROUND & AIMS: Human TFB2M (mitochondrial transcription factor B2) is a key regulator of mitochondria transcription. Our bioinformatic analysis based on the cancer genome atlas (TCGA) data revealed an aberrant over-expression of TFB2M in hepatocellular carcinoma (HCC). However, the functional roles of TFB2M in tumorigenesis remains unexplored, including HCC.METHODS: The expression and clinical significance of TFB2M were evaluated by qRT-PCR and western blot analysis. The biological effects and underlying mechanisms of TFB2M in HCC were determined by cell proliferation, colony formation, cell cycle, apoptosis, migration, and invasion assays.
    RESULTS: TFB2M was commonly up-regulated in HCC mainly due to the down-regulation of miR101-3p, which significantly correlated with poor survival of HCC patients. Functional experiments revealed that TFB2M significantly promoted HCC cell proliferation, migration, and invasion, while inhibited apoptosis in vitro and promoted xenograft tumorigenesis and lung metastasis in nude mice models in vivo. Mechanistically, increased production of reactive oxygen species (ROS) and subsequently activated Akt/NF-κB signaling was found to be involved in the promotion of growth and metastasis by TFB2M in HCC cells.
    CONCLUSIONS: these findings suggest that TFB2M plays a pivotal oncogenic role in HCC cells through activating ROS-Akt-NF-κB signaling pathway.
    Keywords:  HCC; TFB2M; growth; hepatocellular carcinoma; metastasis; mitochondrial transcription factor B2; reactive oxygen species
    DOI:  https://doi.org/10.1111/liv.14440
  18. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30139-6. [Epub ahead of print]155 383-400
    Naini A, Gilkerson R, Shanske S, Pang J.
      The maternally inherited mitochondrial DNA (mtDNA) is a circular 16,569bp double stranded DNA that encodes 37 genes, 24 of which (2 rRNAs and 22 tRNAs) are necessary for transcription and translation of 13 polypeptides that are all subunits of respiratory chain. Pathogenic mutations in mtDNA cause respiratory chain dysfunction, and are the underlying defect in an ever-increasing number of mtDNA-related encephalomyopathies with distinct phenotypes. In this chapter, we present an overview of mtDNA mutations and describe the molecular techniques currently employed in our laboratory to detect two types of mtDNA mutations: single-large-scale rearrangements and point mutations.
    Keywords:  Mitochondria; Mutations; Next generation sequencing; Real-time PCR; mtDNA
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.009
  19. Science. 2020 Mar 19. pii: eaay2494. [Epub ahead of print]
    Zhou Z, Torres M, Sha H, Halbrook CJ, Van den Bergh F, Reinert RB, Yamada T, Wang S, Luo Y, Hunter AH, Wang C, Sanderson TH, Liu M, Taylor A, Sesaki H, Lyssiotis CA, Wu J, Kersten S, Beard DA, Qi L.
      The endoplasmic reticulum (ER) engages mitochondria at specialized ER domains known as mitochondria-associated membranes (MAMs). Here, we used three-dimensional high-resolution imaging to investigate the formation of pleomorphic "megamitochondria" with altered MAMs in brown adipocytes lacking the Sel1L-Hrd1 protein complex of ER-associated protein degradation (ERAD). Mice with ERAD deficiency in brown adipocytes were cold sensitive and exhibited mitochondrial dysfunction. ERAD deficiency affected ER-mitochondria contacts and mitochondrial dynamics, at least in part, by regulating the turnover of the MAM protein, sigma receptor 1 (SigmaR1). Thus, our study provides molecular insights into ER-mitochondrial crosstalk and expands our understanding of the physiological importance of Sel1L-Hrd1 ERAD.
    DOI:  https://doi.org/10.1126/science.aay2494
  20. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30159-1. [Epub ahead of print]155 441-487
    Bacman SR, Gammage PA, Minczuk M, Moraes CT.
      Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences. We have taken advantage of these enzymes to develop reagents to selectively eliminate mutant mtDNA. The replication of intact mitochondrial genomes normalizes mtDNA levels and consequently mitochondrial function. In this chapter, we describe the methodology used to design and express these nucleases in mammalian cells in culture and in vivo.
    Keywords:  Gene therapy; Heteroplasmy; Mitochondrial diseases; mitoTALEN; mtDNA; mtZFN
    DOI:  https://doi.org/10.1016/bs.mcb.2019.12.004
  21. J Gerontol A Biol Sci Med Sci. 2020 Mar 16. pii: glaa059. [Epub ahead of print]
    Cordeiro AV, Brícola RS, Braga RR, Lenhare L, Silva VRR, Anaruma CP, Katashima CK, Crisol BM, Simabuco FM, Silva ASR, Cintra DE, Moura LP, Pauli JR, Ropelle ER.
      The impairment of the mitochondrial functions is a hallmark of aging. During aging, there is a downregulation of two mechanisms strictly associated with mitochondrial integrity, including the mitonuclear imbalance (e.g. imbalance in mitochondrial- versus nuclear-encoded mitochondrial proteins) and the mitochondrial Unfolded Protein Response (UPRmt). Here, we evaluated the effects of aerobic exercise in the mitonuclear imbalance and UPRmt markers in the skeletal muscle of old mice. We combined the physiological tests, molecular and bioinformatic analyzes to evaluate the effects of 4 weeks of Aerobic Exercise Training on mitonuclear imbalance and UPRmt markers in the skeletal muscle of young (2 mo.) and aged (24 mo.) C57BL/6J mice. Initially, we found that aging reduced several mitochondrial genes in the gastrocnemius muscle, and it was accompanied by the low levels of UPRmt markers, including Yme1l1 and Clpp mRNA. As expected, physical training improved the whole-body metabolism and physical performance of aged mice. The aerobic exercise increased key proteins involved in the mitochondrial biogenesis/functions (VDAC and SIRT1) along with mitochondrial-encoded genes (mtNd1, mtCytB, and mtD-Loop) in the skeletal muscle of old mice. Interestingly, aerobic exercise induced the mitonuclear imbalance, increasing MTCO1/ATP5a ratio and UPRmt markers in the skeletal muscle, including HSP60, Lonp1, and Yme1L1 protein levels in the gastrocnemius muscle of aged mice. These data demonstrate that aerobic exercise training induced mitonuclear imbalance and UPRmt in the skeletal muscle during aging. These phenomena could be involved in the improvement of the mitochondrial metabolism and oxidative capacity in aged individuals.
    Keywords:  Aging; Mitonuclear Imbalance; Physical Exercise; Skeletal Muscle; UPRmt
    DOI:  https://doi.org/10.1093/gerona/glaa059
  22. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30140-2. [Epub ahead of print]155 491-518
    Hoppins S, Lackner LL, Lee JE, Mears JA.
      Mitochondria are required for cell survival and are best known for their role in energy production. These organelles also participate in many other biological processes that are critical for cellular function, and thus, play a central role in cellular life and death decisions. In a majority of cell types, mitochondria form highly dynamic, reticular networks. Maintaining the shape of these complex, ever-changing networks is critical for mitochondrial and cellular function, and requires the conserved activities of mitochondrial fission and fusion. Great advances in our knowledge about the molecular machines that mediate these dynamic activities have been made over the past 2 decades. These advances have been driven by the use of highly complementary in vitro and in vivo approaches that have proven extremely powerful for studying the complex membrane remodeling processes that drive fission and fusion of the organelle. In this chapter, we detail current methods used to examine the mechanisms and regulation of mitochondrial fission and fusion in vitro and in vivo.
    Keywords:  Mitochondrial division; Mitochondrial dynamics; Mitochondrial fission; Mitochondrial fusion
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.010
  23. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30163-3. [Epub ahead of print]155 273-293
    Stepanova A, Galkin A.
      Mitochondria-derived reactive oxygen species (ROS) play an important role in the development of several pathologies and are also involved in physiological signaling. Molecular oxygen is the direct substrate of complex IV of the respiratory chain, and at the same time, its partial reduction in mitochondria results in the formation of ROS, mainly H2O2. The accurate knowledge of the dependence of H2O2 production on oxygen concentration is vital for the studies of tissue ischemia/reperfusion, where the relationship between oxygen availability, respiration, and ROS production is critical. In this chapter, we describe a straightforward and reliable protocol for the assessment of H2O2 release by mitochondria at varying oxygen concentrations. This method can be used for any ROS-generating system where the effect of oxygen level on H2O2 production needs to be assessed.
    Keywords:  Ischemia-reperfusion; Mitochondria; Oxygen tension; Reactive oxygen species (ROS)
    DOI:  https://doi.org/10.1016/bs.mcb.2019.12.008
  24. Endocr Rev. 2020 Mar 16. pii: bnaa005. [Epub ahead of print]
    Diaz-Vegas A, Sanchez-Aguilera P, Krycer JR, Morales PE, Monsalves-Alvarez M, Cifuentes M, Rothermel BA, Lavandero S.
      Mitochondrial damage is implicated as a major contributing factor for a number of noncommunicable chronic diseases such as cardiovascular diseases, cancer, obesity, and insulin resistance/type 2 diabetes. Here, we discuss the role of mitochondria in maintaining cellular and whole-organism homeostasis, the mechanisms that promote mitochondrial dysfunction and the role of this phenomenon in noncommunicable chronic diseases. We also review the state of the art regarding the pre-clinical evidence associated with the regulation of the mitochondrial function and the development of current mitochondrial-targeted therapeutics to treat non communicable chronic diseases. Finally; we give an integrated vision of how the mitochondrial damage is implicated in these metabolic diseases.
    Keywords:  Mitochondria; cancer; cardiovascular diseases; insulin resistance; obesity
    DOI:  https://doi.org/10.1210/endrev/bnaa005
  25. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30119-0. [Epub ahead of print]155 3-31
    Liao PC, Bergamini C, Fato R, Pon LA, Pallotti F.
      Isolated mitochondria are useful to study fundamental processes including mitochondrial respiration, metabolic activity, protein import, membrane fusion, protein complex assembly, as well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs, and other organelles. In addition, studies of the mitochondrial proteome, phosphoproteome, and lipidome are dependent on preparation of highly purified mitochondria (Boldogh, Vojtov, Karmon, & Pon, 1998; Cui, Conte, Fox, Zara, & Winge, 2014; Marc et al., 2002; Meeusen, McCaffery, & Nunnari, 2004; Reinders et al., 2007; Schneiter et al., 1999; Stuart & Koehler, 2007). Most methods to isolate mitochondria rely on differential centrifugation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts followed by high speed centrifugation to concentrate mitochondria and separate them from other organelles. However, methods to disrupt cells and tissue vary. Moreover, density gradient centrifugation or affinity purification of the organelle are used to further purify mitochondria or to separate different populations of the organelle. Here, we describe protocols to isolate mitochondria from different cells and tissues as well as approaches to assess the purity and integrity of isolated organelles.
    Keywords:  Affinity purification; Mitochondria; Subcellular fractionation; Yeast
    DOI:  https://doi.org/10.1016/bs.mcb.2019.10.002
  26. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30157-8. [Epub ahead of print]155 181-197
    Acín-Pérez R, Hernansanz-Agustín P, Enríquez JA.
      This review focuses on three independent and complementary approaches to obtain information on the combined function of respiratory complexes when present in different structural situations, either as individual complexes or when superassembled with other complexes. We review the utility of in-gel activity after blue native electrophoresis, integrated oxygen consumption of supercomplexes containing complex IV, and spectrophotometric activity measurements.
    Keywords:  Blue native gel electrophoresis; Electron transport chain; Mitochondria respiration; Oxidative phosphorylation; Supercomplexes
    DOI:  https://doi.org/10.1016/bs.mcb.2019.12.002
  27. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30120-7. [Epub ahead of print]155 221-245
    Esteras N, Adjobo-Hermans MJW, Abramov AY, Koopman WJH.
      Assessment of the mitochondrial membrane potential (Δψ) in living cells, although not trivial, can be used to estimate mitochondrial bioenergetic state. For this purpose, fluorescent lipophilic cations are broadly applied. These cations enter cells and accumulate primarily in the mitochondrial matrix in a Δψ-dependent manner. Here, we describe the use of the cations tetramethylrhodamine methyl ester (TMRM) and rhodamine 123 (Rhod123) for semi-quantitative Δψ analysis in living mammalian cells. Two different strategies are presented: (1) steady-state measurements that are suited to compare Δψ between different conditions (i.e., for comparing disease states or treatments) and (2) dynamic measurements allowing temporal monitoring of Δψ changes (i.e., to assess the effect of acute perturbations). We discuss the rationale for the use of TMRM and Rhod123 in their non-quenching/redistribution and quenching mode, how these modes are associated with different fluorescence responses, and how data can be interpreted. Practically, three experimental protocols are provided describing the use of TMRM and/or Rhod123 to assess Δψ in primary human skin fibroblasts (PHSFs) and neuron/astrocyte co-cultures by live-cell fluorescence microscopy.
    Keywords:  Fluorescence; Microscopy; Quenching; Rhodamine 123; TMRM
    DOI:  https://doi.org/10.1016/bs.mcb.2019.10.003
  28. Diabetologia. 2020 Mar 17.
    Bilet L, Phielix E, van de Weijer T, Gemmink A, Bosma M, Moonen-Kornips E, Jorgensen JA, Schaart G, Zhang D, Meijer K, Hopman M, Hesselink MKC, Ouwens DM, Shulman GI, Schrauwen-Hinderling VB, Schrauwen P.
      AIMS/HYPOTHESIS: Physical inactivity, low mitochondrial function, increased intramyocellular lipid (IMCL) deposition and reduced insulin sensitivity are common denominators of chronic metabolic disorders, like obesity and type 2 diabetes. Yet, whether low mitochondrial function predisposes to insulin resistance in humans is still unknown.METHODS: Here we investigated, in an intervention study, whether muscle with low mitochondrial oxidative capacity, induced by one-legged physical inactivity, would feature stronger signs of lipid-induced insulin resistance. To this end, ten male participants (age 22.4 ± 4.2 years, BMI 21.3 ± 2.0 kg/m2) underwent a 12 day unilateral lower-limb suspension with the contralateral leg serving as an active internal control.
    RESULTS: In vivo, mitochondrial oxidative capacity, assessed by phosphocreatine (PCr)-recovery half-time, was lower in the inactive vs active leg. Ex vivo, palmitate oxidation to 14CO2 was lower in the suspended leg vs the active leg; however, this did not result in significantly higher [14C]palmitate incorporation into triacylglycerol. The reduced mitochondrial function in the suspended leg was, however, paralleled by augmented IMCL content in both musculus tibialis anterior and musculus vastus lateralis, and by increased membrane bound protein kinase C (PKC) θ. Finally, upon lipid infusion, insulin signalling was lower in the suspended vs active leg.
    CONCLUSIONS/INTERPRETATION: Together, these results demonstrate, in a unique human in vivo model, that a low mitochondrial oxidative capacity due to physical inactivity directly impacts IMCL accumulation and PKCθ translocation, resulting in impaired insulin signalling upon lipid infusion. This demonstrates the importance of mitochondrial oxidative capacity and muscle fat accumulation in the development of insulin resistance in humans.
    TRIAL REGISTRATION: ClinicalTrial.gov NCT01576250.
    FUNDING: PS was supported by a 'VICI' Research Grant for innovative research from the Netherlands Organization for Scientific Research (Grant 918.96.618).
    Keywords:  Fat oxidation; Insulin resistance; Intramyocellular lipid content; Mitochondrial function; Mitochondrial oxidative capacity; Physical inactivity; Unilateral lower-limb suspension
    DOI:  https://doi.org/10.1007/s00125-020-05128-1
  29. Trends Endocrinol Metab. 2020 Apr;pii: S1043-2760(20)30032-1. [Epub ahead of print]31(4): 269-271
    Martín-Rodríguez S, de Pablos-Velasco P, Calbet JAL.
      Metformin has antidiabetic, anticancer, and prolongevity effects, but seems to interfere with aerobic training mitochondrial adaptations. The primary mechanism of action has been suggested to be the inhibition of mitochondrial complex I. Recent papers (Wang et al. and Cameron et al.), however, provide evidence to deny the hypothesis of a direct action of metformin on complex I.
    Keywords:  exercise; metformin; mitochondrial complex I
    DOI:  https://doi.org/10.1016/j.tem.2020.02.003
  30. Cells. 2020 Mar 14. pii: E712. [Epub ahead of print]9(3):
    Eldarov CM, Vangely IM, Vays VB, Sheval EV, Holtze S, Hildebrandt TB, Kolosova NG, Popkov VA, Plotnikov EY, Zorov DB, Bakeeva LE, Skulachev VP.
      Electron microscopic study of cardiomyocytes taken from healthy Wistar and OXYS rats and naked mole rats (Heterocephalus glaber) revealed mitochondria in nuclei that lacked part of the nuclear envelope. The direct interaction of mitochondria with nucleoplasm is shown. The statistical analysis of the occurrence of mitochondria in cardiomyocyte nuclei showed that the percentage of nuclei with mitochondria was roughly around 1%, and did not show age and species dependency. Confocal microscopy of normal rat cardiac myocytes revealed a branched mitochondrial network in the vicinity of nuclei with an organization different than that of interfibrillar mitochondria. This mitochondrial network was energetically functional because it carried the membrane potential that responded by oscillatory mode after photodynamic challenge. We suggest that the presence of functional mitochondria in the nucleus is not only a consequence of certain pathologies but rather represents a normal biological phenomenon involved in mitochondrial/nuclear interactions.
    Keywords:  electron and confocal microscopy; healthy cells; heart; intranuclear mitochondria
    DOI:  https://doi.org/10.3390/cells9030712
  31. Proc Natl Acad Sci U S A. 2020 Mar 16. pii: 201922344. [Epub ahead of print]
    Luukkonen PK, Dufour S, Lyu K, Zhang XM, Hakkarainen A, Lehtimäki TE, Cline GW, Petersen KF, Shulman GI, Yki-Järvinen H.
      Weight loss by ketogenic diet (KD) has gained popularity in management of nonalcoholic fatty liver disease (NAFLD). KD rapidly reverses NAFLD and insulin resistance despite increasing circulating nonesterified fatty acids (NEFA), the main substrate for synthesis of intrahepatic triglycerides (IHTG). To explore the underlying mechanism, we quantified hepatic mitochondrial fluxes and their regulators in humans by using positional isotopomer NMR tracer analysis. Ten overweight/obese subjects received stable isotope infusions of: [D7]glucose, [13C4]β-hydroxybutyrate and [3-13C]lactate before and after a 6-d KD. IHTG was determined by proton magnetic resonance spectroscopy (1H-MRS). The KD diet decreased IHTG by 31% in the face of a 3% decrease in body weight and decreased hepatic insulin resistance (-58%) despite an increase in NEFA concentrations (+35%). These changes were attributed to increased net hydrolysis of IHTG and partitioning of the resulting fatty acids toward ketogenesis (+232%) due to reductions in serum insulin concentrations (-53%) and hepatic citrate synthase flux (-38%), respectively. The former was attributed to decreased hepatic insulin resistance and the latter to increased hepatic mitochondrial redox state (+167%) and decreased plasma leptin (-45%) and triiodothyronine (-21%) concentrations. These data demonstrate heretofore undescribed adaptations underlying the reversal of NAFLD by KD: That is, markedly altered hepatic mitochondrial fluxes and redox state to promote ketogenesis rather than synthesis of IHTG.
    Keywords:  carbohydrate restriction; citrate synthase; insulin resistance; pyruvate carboxylase; redox
    DOI:  https://doi.org/10.1073/pnas.1922344117
  32. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30151-7. [Epub ahead of print]155 83-120
    Bennett MJ, Sheng F, Saada A.
      This chapter focuses on the methods to measure unique metabolites, specific enzymes, and metabolic flux in fatty acid β-oxidation, and on biochemical assays of tricarboxylic acid (TCA) cycle enzymes and the pyruvate dehydrogenase complex. These assays play an important role in the diagnosis of genetic diseases, newborn screening, and in cancer and metabolism research. The rationale, protocol, pros and cons, and alternative methods are discussed. Nevertheless, each laboratory should adapt the preferred method optimizing sample preparation and assay conditions for linearity and a low signal-to-noise ratio. The reader is also referred to the additional literature citing methods and clinical descriptions of the various diseases.
    Keywords:  Acylcarnitine; Acylglycines; Carnitine; Fatty acid oxidation; Krebs cycle; Mitochondria; Organic acids; Pyruvate dehydrogenase; TCA cycle
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.021
  33. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30121-9. [Epub ahead of print]155 369-379
    Carraro M, Bernardi P.
      The mitochondrial permeability transition (PT) is an increase in the inner membrane permeability caused by the opening of a Ca2+-activated high-conductance channel, the so-called PT pore (PTP) or mitochondrial megachannel (MMC). Recent data indicate that F-ATP synthase contributes substantially to the generation of the PTP, yet this hypothesis is the matter of controversy. In this chapter, we will describe an approach to study the pore, i.e., the evaluation of mitochondrial swelling by means of a decrease in the absorbance at 540nm. This method should be useful to resolve apparent discrepancies in the literature and help solve emerging issues on the identity of mitochondrial pores.
    Keywords:  Absorbance; Mitochondria; Permeability transition; Swelling
    DOI:  https://doi.org/10.1016/bs.mcb.2019.10.004
  34. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30154-2. [Epub ahead of print]155 247-270
    Franco-Iborra S, Tanji K.
      We describe here reliable histochemical and immunohistochemical techniques to visualize mitochondria and respiratory chain dysfunction in tissue sections. These morphological methods have been widely used for years, and yet remain relevant to obtain insight into the pathogenesis of mitochondrial diseases. Today, mitochondrial medicine is changing rapidly and genetic information plays an increasing role in the diagnostic process, owing to advances in next-generation sequencing. However, tissue analysis and morphological categorization remain essential, especially when genetic abnormalities of unknown significance might complicate a diagnostic odyssey. Furthermore, tissue assessment is an essential step in laboratory investigation using animal or cell models, in order to assess the distribution, severity, and/or progression of the disease, and to evaluate the effects of possible treatments. Optimized and reproducible staining and imaging methodology are the key elements for accurate tissue assessment. When these methods are used properly and integrated with wisely chosen genetic and biochemical approaches, powerful information can be obtained about the structure and function of mitochondria in both animal model systems and human patients. While the described protocols refer to skeletal muscle and brain mitochondria, the methods described can be applied to any tissue type.
    Keywords:  Cytochrome c oxidase; Histochemistry; Immunofluorescence; Immunohistochemistry; Muscle; Succinate dehydrogenase
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.024
  35. Biochim Biophys Acta Bioenerg. 2020 Mar 11. pii: S0005-2728(20)30035-9. [Epub ahead of print] 148185
    Djurabekova A, Haapanen O, Sharma V.
      In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.
    Keywords:  Cell respiration; Mitochondria; Proton transfer; Redox reactions
    DOI:  https://doi.org/10.1016/j.bbabio.2020.148185
  36. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30161-X. [Epub ahead of print]155 545-555
    Wolf DM, Segawa M, Shirihai OS, Liesa M.
      The emergence of diffraction-unlimited live-cell imaging technologies has enabled the examination of mitochondrial form and function in unprecedented detail. We recently developed an approach for visualizing the inner mitochondrial membrane and determined that cristae membranes possess distinct mitochondrial membrane potentials, representing unique bioenergetic subdomains within the same organelle. Here, we outline a methodology for resolving cristae and inner boundary membranes using the LSM880 with Airyscan. Furthermore, we demonstrate how to analyze TMRE fluorescence intensity using the Nernst equation to calculate membrane potentials of individual cristae. Altogether, using these new techniques to study the electrochemical properties of the cristae can help to gain deeper insight into the still elusive nature of the mitochondrion.
    Keywords:  Airyscan; Cristae; Live-cell imaging; Mitochondrial membrane potential; Super-resolution imaging
    DOI:  https://doi.org/10.1016/bs.mcb.2019.12.006
  37. J Cancer. 2020 ;11(7): 1828-1838
    Yue T, Zuo S, Bu D, Zhu J, Chen S, Ma Y, Ma J, Guo S, Wen L, Zhang X, Hu J, Wang Y, Yao Z, Chen G, Wang X, Pan Y, Wang P, Liu Y.
      Background: As the third confirmed gaseous transmitter, the role of hydrogen sulfide (H2S) in the pathogenesis of multiple types of cancer has been attracting increasing attention. Increased expression of cystathionine β-synthase (CBS) and H2S in colon cancer tissue samples has been validated and tumor-derived H2S, mainly produced by CBS, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer. Recently, the therapeutic manipulation of H2S has been proposed as a promising anticancer approach. However, the effect of aminooxyacetic acid (AOAA), which has been widely used as an inhibitor of CBS dependent synthesis of H2S, on the chemotherapeutic effect of oxaliplatin (OXA) and the underlying mechanisms remain to be illustrated. Methods: We examined the expression of CBS in human colorectal cancer specimens and matched normal mucosa by immunohistochemistry. The effect of AOAA on the sensitivity of colon cancer cells to OXA and the level of apoptosis induced by caspase cascade was investigated in both HCT116 and HT29 cell lines utilizing CCK-8 assays, flow cytometry analysis and western blot analysis. The endogenous levels of reactive oxygen species (ROS) were detected fluorescently by DCF-DA, and glutathione (GSH) levels were measured by a Total GSH Detection Kit. Tumor bearing xenograft mouse models and in vivo imaging systems were further used to investigate the effect of AOAA in vivo and immunohistochemistry (IHC) and TUNEL analysis were performed. Results: In the current study, we confirmed CBS, the main target of AOAA, is overexpressed in human colorectal cancer by immunohistochemistry. The inhibitory effect of AOAA on the synthesis of H2S was validated utilizing fluorescent probe and specific electrode. AOAA significantly reduced the IC50 values of OXA in both colon cancer cell lines. Co-incubation with AOAA elicited increased apoptosis induced by OXA, featured by increased activation of caspase cascade. Besides, AOAA further increased the levels of ROS induced by OXA and attenuated the synthesis of glutathione (GSH), which is a vital antioxidant. Besides, the results of in vivo imaging and following IHC and TUNEL analysis were in accordance with cellular experiments, indicating that AOAA sensitizes colon cancer cells to OXA via exaggerating intrinsic apoptosis. Conclusion: The results suggested that CBS is overexpressed in colorectal cancer tissues and AOAA sensitizes colon cancer cells to OXA via exaggerating apoptosis both in vitro and in vivo. Decreasing the endogenous level of GSH and consequently impaired detoxification of ROS might be one of the mechanisms underlying the effect of AOAA.
    Keywords:  AOAA; apoptosis.; colorectal cancer; hydrogen sulfide; oxaliplatin; reactive oxygen species
    DOI:  https://doi.org/10.7150/jca.35375
  38. J Exp Med. 2020 Jun 01. pii: e20191689. [Epub ahead of print]217(6):
    Ward NP, Kang YP, Falzone A, Boyle TA, DeNicola GM.
      Human lung tumors exhibit robust and complex mitochondrial metabolism, likely precipitated by the highly oxygenated nature of pulmonary tissue. As ROS generation is a byproduct of this metabolism, reducing power in the form of nicotinamide adenine dinucleotide phosphate (NADPH) is required to mitigate oxidative stress in response to this heightened mitochondrial activity. Nicotinamide nucleotide transhydrogenase (NNT) is known to sustain mitochondrial antioxidant capacity through the generation of NADPH; however, its function in non-small cell lung cancer (NSCLC) has not been established. We found that NNT expression significantly enhances tumor formation and aggressiveness in mouse models of lung tumor initiation and progression. We further show that NNT loss elicits mitochondrial dysfunction independent of substantial increases in oxidative stress, but rather marked by the diminished activities of proteins dependent on resident iron-sulfur clusters. These defects were associated with both NADPH availability and ROS accumulation, suggesting that NNT serves a specific role in mitigating the oxidation of these critical protein cofactors.
    DOI:  https://doi.org/10.1084/jem.20191689
  39. Biochem Biophys Res Commun. 2020 Mar 16. pii: S0006-291X(20)30550-7. [Epub ahead of print]
    Yamamoto T, Takakura H, Mitamura K, Taga A.
      Enhanced expression of cyclophilin A (CypA) in colorectal cancer (CRC) was reported; however, how CypA influences CRC progression is not clear. Therefore, we examine the effects of CypA on CRC cell progression. Knockdown of CypA in SW480 cells significantly inhibited cell migration and invasion but had no effect on cell proliferation. In addition, upregulation of E-cadherin and downregulation of N-cadherin and Snail expression were observed by CypA knockdown. These results suggested that CypA knockdown inhibited cell migration and invasion by suppressing epithelial-mesenchymal transition. CypA knockdown was also associated with increased p38 phosphorylation, and the p38 inhibitor treatment led to increase in the number of invasive CypA-knockdown SW480 cells. Therefore, CypA may be a potential therapeutic target in preventing CRC metastasis.
    Keywords:  Cell invasion; Cell migration; Colorectal cancer; Cyclophilin A; EMT; p38 MAPK
    DOI:  https://doi.org/10.1016/j.bbrc.2020.03.065
  40. Theranostics. 2020 ;10(6): 2832-2848
    Han B, Jiang W, Liu H, Wang J, Zheng K, Cui P, Feng Y, Dang C, Bu Y, Wang QM, Ju Z, Hao J.
      Rationale: Mitochondrial dysfunction and oxidative stress occur in vascular dementia (VaD), but the specific molecular mechanism regulating these events remains unclear. Peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) is a master regulator for mitochondrial function. This study aims to investigate whether PGC-1α is involved in the pathophysiology of VaD. Methods: We firstly generated PGC-1α f/f Eno2-Cre mice to induce neuron-specific overexpression of PGC-1α by crossbreeding PGC-1α f/f mice with Eno2-cre mice. Then, the mice were subjected to bilateral common carotid artery stenosis to induce chronic cerebral hypoperfusion. Neurological function and hippocampal PGC-1α expression was evaluated. Next, RNA-Seq analysis and Seahorse assay were performed on the hippocampal neurons. In addition, mitochondrial antioxidants, uncoupling proteins, ROS production and the activation of glial cells were also measured. Results: Our results showed that hippocampal PGC-1α expression is down-regulated in the mouse VaD model induced by chronic cerebral hypoperfusion. In contrast, neuronal PGC-1α overexpression significantly ameliorated cognitive deficits. RNA-Seq analysis indicated that PGC-1α improved energy metabolism of neurons under hypoxic condition, and Seahorse assay confirmed that PGC-1α increases the metabolic activity of neurons. Further study demonstrated that PGC-1α boosted the expressions of mitochondrial antioxidants and uncoupling proteins (UCPs), including SOD2, Prx3, GPx1, UCP2, UCP4 and UCP5, which in turn reduced reactive oxygen species (ROS) production. Moreover, the activation of microglia and astrocytes was also found to decrease in the hippocampus. All of these changes greatly contributed to protect hippocampal neurons against ischemic insults. Conclusions: PGC-1α could suppress the excessive ROS and neuroinflammation in the hippocampus, opening up a potential therapeutic target for cognitive impairment.
    Keywords:  PGC-1α; ROS; neuroinflammation; vascular dementia
    DOI:  https://doi.org/10.7150/thno.37119
  41. Proc Natl Acad Sci U S A. 2020 Mar 17. pii: 201918216. [Epub ahead of print]
    Di Bartolomeo F, Malina C, Campbell K, Mormino M, Fuchs J, Vorontsov E, Gustafsson CM, Nielsen J.
      Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.
    Keywords:  Saccharomyces cerevisiae; absolute proteomics; diauxic shift; mitochondria
    DOI:  https://doi.org/10.1073/pnas.1918216117
  42. Front Oncol. 2020 ;10 176
    Li WC, Huang CH, Hsieh YT, Chen TY, Cheng LH, Chen CY, Liu CJ, Chen HM, Huang CL, Lo JF, Chang KW.
      To support great demand of cell growth, cancer cells preferentially obtain energy and biomacromolecules by glycolysis over mitochondrial oxidative phosphorylation (OxPhos). Among all glycolytic enzymes, hexokinase (HK), a rate-limiting enzyme at the first step of glycolysis to catalyze cellular glucose into glucose-6-phosphate, is herein emphasized. Four HK isoforms, HK1-HK4, were discovered in nature. It was shown that HK2 expression is enriched in many tumor cells and correlated with poorer survival rates in most neoplastic cells. HK2-mediated regulations for cell malignancy and mechanistic cues in regulating head and neck tumorigenesis, however, are not fully elucidated. Cellular malignancy index, such as cell growth, cellular motility, and treatment sensitivity, and molecular alterations were determined in HK2-deficient head and neck squamous cell carcinoma (HNSCC) cells. By using various cancer databases, HK2, but not HK1, positively correlates with HNSCC progression in a stage-dependent manner. A high HK2 expression was detected in head and neck cancerous tissues compared with their normal counterparts, both in mouse and human subjects. Loss of HK2 in HNSCC cells resulted in reduced cell (in vitro) and tumor (in vivo) growth, as well as decreased epithelial-mesenchymal transition-mediated cell movement; in contrast, HK2-deficient HNSCC cells exhibited greater sensitivity to chemotherapeutic drugs cisplatin and 5-fluorouracil but are more resistant to photodynamic therapy, indicating that HK2 expression could selectively define treatment sensitivity in HNSCC cells. At the molecular level, it was found that HK2 alteration drove metabolic reprogramming toward OxPhos and modulated oncogenic Akt and mutant TP53-mediated signals in HNSCC cells. In summary, the present study showed that HK2 suppression could lessen HNSCC oncogenicity and modulate therapeutic sensitivity, thereby being an ideal therapeutic target for HNSCCs.
    Keywords:  cellular malignancy; head and neck cancer; hexokinase 2; metabolic shift; therapeutic efficacy
    DOI:  https://doi.org/10.3389/fonc.2020.00176
  43. Biomolecules. 2020 Mar 13. pii: E447. [Epub ahead of print]10(3):
    Augsburger F, Randi EB, Jendly M, Ascencao K, Dilek N, Szabo C.
      3-mercaptopyruvate sulfurtransferase (3-MST) has emerged as one of the significant sources of biologically active sulfur species in various mammalian cells. The current study was designed to investigate the functional role of 3-MST's catalytic activity in the murine colon cancer cell line CT26. The novel pharmacological 3-MST inhibitor HMPSNE was used to assess cancer cell proliferation, migration and bioenergetics in vitro. Methods included measurements of cell viability (MTT and LDH assays), cell proliferation and in vitro wound healing (IncuCyte) and cellular bioenergetics (Seahorse extracellular flux analysis). 3-MST expression was detected by Western blotting; H2S production was measured by the fluorescent dye AzMC. The results show that CT26 cells express 3-MST protein and mRNA, as well as several enzymes involved in H2S degradation (TST, ETHE1). Pharmacological inhibition of 3-MST concentration-dependently suppressed H2S production and, at 100 and 300 µM, attenuated CT26 proliferation and migration. HMPSNE exerted a bell-shaped effect on several cellular bioenergetic parameters related to oxidative phosphorylation, while other bioenergetic parameters were either unaffected or inhibited at the highest concentration of the inhibitor tested (300 µM). In contrast to 3-MST, the expression of CBS (another H2S producing enzyme which has been previously implicated in the regulation of various biological parameters in other tumor cells) was not detectable in CT26 cells and pharmacological inhibition of CBS exerted no significant effects on CT26 proliferation or bioenergetics. In summary, 3-MST catalytic activity significantly contributes to the regulation of cellular proliferation, migration and bioenergetics in CT26 murine colon cancer cells. The current studies identify 3-MST as the principal source of biologically active H2S in this cell line.
    Keywords:  bioenergetics; gasotransmitters; hydrogen sulfide; migration; mitochondria; nitric oxide; proliferation
    DOI:  https://doi.org/10.3390/biom10030447
  44. Sci Rep. 2020 Mar 17. 10(1): 4905
    Cai Y, Rattray NJW, Zhang Q, Mironova V, Santos-Neto A, Hsu KS, Rattray Z, Cross JR, Zhang Y, Paty PB, Khan SA, Johnson CH.
      Women have a lower incidence of colorectal cancer (CRC) than men, however, they have a higher incidence of right-sided colon cancer (RCC). This is of concern as patients with RCC have the poorest clinical outcomes among all CRC patients. Aberrant metabolism is a known hallmark and therapeutic target for cancer. We propose that metabolic subphenotypes exist between CRCs due to intertumoral molecular and genomic variation, and differences in environmental milieu of the colon which vary between the sexes. Metabolomics analysis of patient colon tumors (n = 197) and normal tissues (n = 39) revealed sex-specific metabolic subphenotypes dependent on anatomic location. Tumors from women with RCC were nutrient-deplete, showing enhanced energy production to fuel asparagine synthesis and amino acid uptake. The clinical importance of our findings were further investigated in an independent data set from The Cancer Genomic Atlas, and demonstrated that high asparagine synthetase (ASNS) expression correlated with poorer survival for women. This is the first study to show a unique, nutrient-deplete metabolic subphenotype in women with RCC, with implications for tumor progression and outcomes in CRC patients.
    DOI:  https://doi.org/10.1038/s41598-020-61851-0
  45. J Intern Med. 2020 Mar 18.
    Wei W, Chinnery PF.
      The first draft human mitochondrial DNA (mtDNA) sequence was published in 1981, paving the way for two decades of discovery linking mtDNA variation with human disease. Severe pathogenic mutations cause sporadic and inherited rare disorders that often involve the nervous system. However, some mutations cause mild organ-specific phenotypes that have a reduced clinical penetrance, and polymorphic variation of mtDNA is associated with an altered risk of developing several late-onset common human diseases including Parkinson's disease. mtDNA mutations also accumulate during human life and are enriched in affected organs in a number of age-related diseases. Thus, mtDNA contributes to a wide range of human pathologies. For many decades, it has generally been accepted that mtDNA is inherited exclusively down the maternal line in humans. Although recent evidence has challenged this dogma, whole-genome sequencing has identified nuclear-encoded mitochondrial sequences (NUMTs) that can give the false impression of paternally inherited mtDNA. This provides a more likely explanation for recent reports of 'bi-parental inheritance', where the paternal alleles are actually transmitted through the nuclear genome. The presence of both mutated and wild-type variant alleles within the same individual (heteroplasmy) and rapid shifts in allele frequency can lead to offspring with variable severity of disease. In addition, there is emerging evidence that selection can act for and against specific mtDNA variants within the developing germ line, and possibly within developing tissues. Thus, understanding how mtDNA is inherited has far-reaching implications across medicine. There is emerging evidence that this highly dynamic system is amenable to therapeutic manipulation, raising the possibility that we can harness new understanding to prevent and treat rare and common human diseases where mtDNA mutations play a key role.
    Keywords:  human mitochondrial DNA; mitochondrial DNA mutation; mitochondrial bottleneck; mitochondrial disorders; mitochondrial inheritance
    DOI:  https://doi.org/10.1111/joim.13047