bims-celmim Biomed News
on Cellular and mitochondrial metabolism
Issue of 2024‒06‒30
twenty papers selected by
Marc Segarra Mondejar
  1. Mitochondrial One-Carbon Metabolism and Alzheimer's Disease.
  2. Metabolic flexibility ensures proper neuronal network function in moderate neuroinflammation.
  3. Implications of mitochondrial membrane potential gradients on signaling and ATP production analyzed by correlative multi-parameter microscopy.
  4. Mitochondrial metabolic reprogramming in diabetic kidney disease.
  5. Fluorescence Lifetime Super-Resolution Imaging Unveil the Dynamic Relationship between Mitochondrial Membrane Potential and Cristae Structure Using the Förster Resonance Energy Transfer Strategy.
  6. Interplay between mTOR and Purine Metabolism Enzymes and Its Relevant Role in Cancer.
  7. Endothelial Drp1 Couples VEGF-induced Redox Signaling with Glycolysis Through Cysteine Oxidation to Drive Angiogenesis.
  8. Methionine Sulfoxide Speciation in Mouse Hippocampus Revealed by Global Proteomics Exhibits Age- and Alzheimer's Disease-Dependent Changes Targeted to Mitochondrial and Glycolytic Pathways.
  9. HIF-2α expression and metabolic signaling require ACSS2 in clear cell renal cell carcinoma.
  10. Endoplasmic reticulum and mitochondrial calcium handling dynamically shape slow afterhyperpolarizations in vasopressin magnocellular neurons.
  11. Raman micro-spectroscopy reveals the spatial distribution of fumarate in cells and tissues.
  12. Ferroptotic stress drives liver aging and metabolic dysfunction.
  13. Lactate transported by MCT1 plays an active role in promoting mitochondrial biogenesis and enhancing TCA flux in skeletal muscle.
  14. A metabolic redox relay supports ER proinsulin export in pancreatic islet β-cells.
  15. ATP Citrate Lyase Supports Cardiac Function and NAD+/NADH Balance And Is Depressed in Human Heart Failure.
  16. FADS1/2 control lipid metabolism and ferroptosis susceptibility in triple-negative breast cancer.
  17. Visualizing Metabolism in Biotechnologically Important Yeasts with dDNP NMR Reveals Evolutionary Strategies and Glycolytic Logic.
  18. Intra-organ cell specific mitochondrial quantitative interactomics.
  19. Using Fluorescent GAP Indicators to Monitor ER Ca2.
  20. Integrated multi-omic analyses uncover the effects of aging on cell-type regulation in glucose-responsive tissues.
  1. Int J Mol Sci. 2024 Jun 07. pii: 6302. [Epub ahead of print]25(12):
    Mitochondrial One-Carbon Metabolism and Alzheimer's Disease.
    Yizhou Yu, L Miguel Martins.
      Mitochondrial one-carbon metabolism provides carbon units to several pathways, including nucleic acid synthesis, mitochondrial metabolism, amino acid metabolism, and methylation reactions. Late-onset Alzheimer's disease is the most common age-related neurodegenerative disease, characterised by impaired energy metabolism, and is potentially linked to mitochondrial bioenergetics. Here, we discuss the intersection between the molecular pathways linked to both mitochondrial one-carbon metabolism and Alzheimer's disease. We propose that enhancing one-carbon metabolism could promote the metabolic processes that help brain cells cope with Alzheimer's disease-related injuries. We also highlight potential therapeutic avenues to leverage one-carbon metabolism to delay Alzheimer's disease pathology.
    Keywords:  Alzheimer’s disease; folate; mitochondria; one-carbon metabolism
    DOI:  https://doi.org/10.3390/ijms25126302
  2. Sci Rep. 2024 06 22. 14(1): 14405
    Metabolic flexibility ensures proper neuronal network function in moderate neuroinflammation.
    Bruno Chausse, Nikolai Malorny, Andrea Lewen, Gernot Poschet, Nikolaus Berndt, Oliver Kann.
      Microglia, brain-resident macrophages, can acquire distinct functional phenotypes, which are supported by differential reprogramming of cell metabolism. These adaptations include remodeling in glycolytic and mitochondrial metabolic fluxes, potentially altering energy substrate availability at the tissue level. This phenomenon may be highly relevant in the brain, where metabolism must be precisely regulated to maintain appropriate neuronal excitability and synaptic transmission. Direct evidence that microglia can impact on neuronal energy metabolism has been widely lacking, however. Combining molecular profiling, electrophysiology, oxygen microsensor recordings and mathematical modeling, we investigated microglia-mediated disturbances in brain energetics during neuroinflammation. Our results suggest that proinflammatory microglia showing enhanced nitric oxide release and decreased CX3CR1 expression transiently increase the tissue lactate/glucose ratio that depends on transcriptional reprogramming in microglia, not in neurons. In this condition, neuronal network activity such as gamma oscillations (30-70 Hz) can be fueled by increased ATP production in mitochondria, which is reflected by elevated oxygen consumption. During dysregulated inflammation, high energy demand and low glucose availability can be boundary conditions for neuronal metabolic fitness as revealed by kinetic modeling of single neuron energetics. Collectively, these findings indicate that metabolic flexibility protects neuronal network function against alterations in local substrate availability during moderate neuroinflammation.
    Keywords:  Glycolysis; Immunometabolism; Lactate oxidation; Microglia; Neuronal oscillations
    DOI:  https://doi.org/10.1038/s41598-024-64872-1
  3. Sci Rep. 2024 06 26. 14(1): 14784
    Implications of mitochondrial membrane potential gradients on signaling and ATP production analyzed by correlative multi-parameter microscopy.
    Benjamin Gottschalk, Zhanat Koshenov, Roland Malli, Wolfgang F Graier.
      The complex architecture and biochemistry of the inner mitochondrial membrane generate ultra-structures with different phospholipid and protein compositions, shapes, characteristics, and functions. The crista junction (CJ) serves as an important barrier separating the cristae (CM) and inner boundary membranes (IBM). Thereby CJ regulates the movement of ions and ensures distinct electrical potentials across the cristae (ΔΨC) and inner boundary (ΔΨIBM) membranes. We have developed a robust and flexible approach to visualize the CJ permeability with super-resolution microscopy as a readout of local mitochondrial membrane potential (ΔΨmito) fluctuations. This method involves analyzing the distribution of TMRM fluorescence intensity in a model that is restricted to the mitochondrial geometry. We show that mitochondrial Ca2+ elevation hyperpolarizes the CM most likely caused by Ca2+ sensitive increase of mitochondrial tricarboxylic acid cycle (TCA) and subsequent oxidative phosphorylation (OXPHOS) activity in the cristae. Dynamic multi-parameter correlation measurements of spatial mitochondrial membrane potential gradients, ATP levels, and mitochondrial morphometrics revealed a CJ-based membrane potential overflow valve mechanism protecting the mitochondrial integrity during excessive cristae hyperpolarization.
    Keywords:  Correlative microscopy; Cristae junctions; Membrane potential gradient; Mitochondria; Mitochondrial membranes
    DOI:  https://doi.org/10.1038/s41598-024-65595-z
  4. Cell Death Dis. 2024 Jun 24. 15(6): 442
    Mitochondrial metabolic reprogramming in diabetic kidney disease.
    Xiaoting Fan, Meilin Yang, Yating Lang, Shangwei Lu, Zhijuan Kong, Ying Gao, Ning Shen, Dongdong Zhang, Zhimei Lv.
      Diabetic kidney disease, known as a glomerular disease, arises from a metabolic disorder impairing renal cell function. Mitochondria, crucial organelles, play a key role in substance metabolism via oxidative phosphorylation to generate ATP. Cells undergo metabolic reprogramming as a compensatory mechanism to fulfill energy needs for survival and growth, attracting scholarly attention in recent years. Studies indicate that mitochondrial metabolic reprogramming significantly influences the pathophysiological progression of DKD. Alterations in kidney metabolism lead to abnormal expression of signaling molecules and activation of pathways, inducing oxidative stress-related cellular damage, inflammatory responses, apoptosis, and autophagy irregularities, culminating in renal fibrosis and insufficiency. This review delves into the impact of mitochondrial metabolic reprogramming on DKD pathogenesis, emphasizing the regulation of metabolic regulators and downstream signaling pathways. Therapeutic interventions targeting renal metabolic reprogramming can potentially delay DKD progression. The findings underscore the importance of focusing on metabolic reprogramming to develop safer and more effective therapeutic approaches.
    DOI:  https://doi.org/10.1038/s41419-024-06833-0
  5. Anal Chem. 2024 Jun 26.
    Fluorescence Lifetime Super-Resolution Imaging Unveil the Dynamic Relationship between Mitochondrial Membrane Potential and Cristae Structure Using the Förster Resonance Energy Transfer Strategy.
    Fei Peng, Xiangnan Ai, Jing Sun, Xichuan Ge, Meiqi Li, Peng Xi, Baoxiang Gao.
      Mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) into the matrix, are the main site for the generation of ATP via oxidative phosphorylation, and mitochondrial membrane potential (MMP). Synchronous study of the dynamic relationship between cristae and MMP is very important for further understanding of mitochondrial function. Due to the lack of suitable IMM probes and imaging techniques, the dynamic relationship between MMP and cristae structure alterations remains poorly understood. We designed a pair of FRET-based molecular probes, with the donor (OR-LA) being rhodamine modified with mitochondrial coenzyme lipoic acid and the acceptor (SiR-BA) being silicon-rhodamine modified with a butyl chain, for simultaneous dynamic monitoring of mitochondrial cristae structure and MMP. The FRET process of the molecular pair in mitochondria is regulated by MMP, enabling more precise visualization of MMP through fluorescence intensity ratio and fluorescence lifetime. By combining FRET with FLIM super-resolution imaging technology, we achieved simultaneous dynamic monitoring of mitochondrial cristae structure and MMP, revealing that during the decline of MMP, there is a progression involving cristae dilation, fragmentation, mitochondrial vacuolization, and eventual rupture. Significantly, we successfully observed that the rapid decrease in MMP at the site of mitochondrial membrane rupture may be a critical factor in mitochondrial fragmentation. These data collectively reveal the dynamic relationship between cristae structural alterations and MMP decline, laying a foundation for further investigation into cellular energy regulation mechanisms and therapeutic strategies for mitochondria-related diseases.
    DOI:  https://doi.org/10.1021/acs.analchem.4c01905
  6. Int J Mol Sci. 2024 Jun 19. pii: 6735. [Epub ahead of print]25(12):
    Interplay between mTOR and Purine Metabolism Enzymes and Its Relevant Role in Cancer.
    Simone Allegrini, Marcella Camici, Mercedes Garcia-Gil, Rossana Pesi, Maria Grazia Tozzi.
      Tumor cells reprogram their metabolism to meet the increased demand for nucleotides and other molecules necessary for growth and proliferation. In fact, cancer cells are characterized by an increased "de novo" synthesis of purine nucleotides. Therefore, it is not surprising that specific enzymes of purine metabolism are the targets of drugs as antineoplastic agents, and a better knowledge of the mechanisms underlying their regulation would be of great help in finding new therapeutic approaches. The mammalian target of the rapamycin (mTOR) signaling pathway, which is often activated in cancer cells, promotes anabolic processes and is a major regulator of cell growth and division. Among the numerous effects exerted by mTOR, noteworthy is its empowerment of the "de novo" synthesis of nucleotides, accomplished by supporting the formation of purinosomes, and by increasing the availability of necessary precursors, such as one-carbon formyl group, bicarbonate and 5-phosphoribosyl-1-pyrophosphate. In this review, we highlight the connection between purine and mitochondrial metabolism, and the bidirectional relation between mTOR signaling and purine synthesis pathways.
    Keywords:  AKT; c-Myc; cancer; cell survival; mTOR; mitochondria; one-carbon metabolism; proliferation; purine metabolism; purinosome
    DOI:  https://doi.org/10.3390/ijms25126735
  7. bioRxiv. 2024 Jun 16. pii: 2024.06.15.599174. [Epub ahead of print]
    Endothelial Drp1 Couples VEGF-induced Redox Signaling with Glycolysis Through Cysteine Oxidation to Drive Angiogenesis.
    Sheela Nagarkoti, Young-Mee Kim, Archita Das, Dipankar Ash, Eric A Vitriol, Tracy-Ann Read, Varadarajan Sudhahar, Md Selim Hossain, Shikha Yadav, Malgorzata McMenamin, Stephanie Kelley, Rudolf Lucas, David Stepp, Eric J Belin de Chantemele, Ruth B Caldwell, David Jr Fulton, Tohru Fukai, Masuko Ushio-Fukai.
      Angiogenesis plays a vital role for postnatal development and tissue repair following ischemia. Reactive oxygen species (ROS) generated by NADPH oxidases (NOXes) and mitochondria act as signaling molecules that promote angiogenesis in endothelial cells (ECs) which mainly relies on aerobic glycolysis for ATP production. However, the connections linking redox signaling with glycolysis are not well understood. The GTPase Drp1 is a member of the dynamin superfamily that moves from cytosol to mitochondria through posttranslational modifications to induce mitochondrial fission. The role of Drp1 in ROS-dependent VEGF signaling and angiogenesis in ECs has not been previously described. Here, we identify an unexpected function of endothelial Drp1 as a redox sensor, transmitting VEGF-induced H 2 O 2 signals to enhance glycolysis and angiogenesis. Loss of Drp1 expression in ECs inhibited VEGF-induced angiogenic responses. Mechanistically, VEGF rapidly induced the NOX4-dependent sulfenylation (CysOH) of Drp1 on Cys 644 , promoting disulfide bond formation with the metabolic kinase AMPK and subsequent sulfenylation of AMPK at Cys 299 / 304 via the mitochondrial fission-mitoROS axis. This cysteine oxidation of AMPK, in turn, enhanced glycolysis and angiogenesis. In vivo , mice with EC-specific Drp1 deficiency or CRISPR/Cas9-engineered "redox-dead" (Cys to Ala) Drp1 knock-in mutations exhibited impaired retinal angiogenesis and post-ischemic neovascularization. Our findings uncover a novel role for endothelial Drp1 in linking VEGF-induced mitochondrial redox signaling to glycolysis through a cysteine oxidation-mediated Drp1-AMPK redox relay, driving both developmental and reparative angiogenesis.
    DOI:  https://doi.org/10.1101/2024.06.15.599174
  8. Int J Mol Sci. 2024 Jun 13. pii: 6516. [Epub ahead of print]25(12):
    Methionine Sulfoxide Speciation in Mouse Hippocampus Revealed by Global Proteomics Exhibits Age- and Alzheimer's Disease-Dependent Changes Targeted to Mitochondrial and Glycolytic Pathways.
    Filipa Blasco Tavares Pereira Lopes, Daniela Schlatzer, Mengzhen Li, Serhan Yilmaz, Rihua Wang, Xin Qi, Marzieh Ayati, Mehmet Koyutürk, Mark R Chance.
      Methionine oxidation to the sulfoxide form (MSox) is a poorly understood post-translational modification of proteins associated with non-specific chemical oxidation from reactive oxygen species (ROS), whose chemistries are linked to various disease pathologies, including neurodegeneration. Emerging evidence shows MSox site occupancy is, in some cases, under enzymatic regulatory control, mediating cellular signaling, including phosphorylation and/or calcium signaling, and raising questions as to the speciation and functional nature of MSox across the proteome. The 5XFAD lineage of the C57BL/6 mouse has well-defined Alzheimer's and aging states. Using this model, we analyzed age-, sex-, and disease-dependent MSox speciation in the mouse hippocampus. In addition, we explored the chemical stability and statistical variance of oxidized peptide signals to understand the needed power for MSox-based proteome studies. Our results identify mitochondrial and glycolytic pathway targets with increases in MSox with age as well as neuroinflammatory targets accumulating MSox with AD in proteome studies of the mouse hippocampus. Further, this paper establishes a foundation for reproducible and rigorous experimental MSox-omics appropriate for novel target identification in biological discovery and for biomarker analysis in ROS and other oxidation-linked diseases.
    Keywords:  5XFAD; Alzheimer’s disease; MSox; ROS; mass spectrometry; methionine oxidation; proteomics
    DOI:  https://doi.org/10.3390/ijms25126516
  9. J Clin Invest. 2024 Jun 17. pii: e164249. [Epub ahead of print]134(12):
    HIF-2α expression and metabolic signaling require ACSS2 in clear cell renal cell carcinoma.
    Zachary A Bacigalupa, Emily N Arner, Logan M Vlach, Melissa M Wolf, Whitney A Brown, Evan S Krystofiak, Xiang Ye, Rachel A Hongo, Madelyn Landis, Edith K Amason, Kathryn E Beckermann, W Kimryn Rathmell, Jeffrey C Rathmell.
      Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer driven by VHL loss and aberrant HIF-2α signaling. Identifying means to regulate HIF-2α thus has potential therapeutic benefit. Acetyl-CoA synthetase 2 (ACSS2) converts acetate to acetyl-CoA and is associated with poor patient prognosis in ccRCC. Here we tested the effects of ACSS2 on HIF-2α and cancer cell metabolism and growth in ccRCC models and clinical samples. ACSS2 inhibition reduced HIF-2α levels and suppressed ccRCC cell line growth in vitro, in vivo, and in cultures of primary ccRCC patient tumors. This treatment reduced glycolytic signaling, cholesterol metabolism, and mitochondrial integrity, all of which are consistent with loss of HIF-2α. Mechanistically, ACSS2 inhibition decreased chromatin accessibility and HIF-2α expression and stability. While HIF-2α protein levels are widely regulated through pVHL-dependent proteolytic degradation, we identify a potential pVHL-independent pathway of degradation via the E3 ligase MUL1. We show that MUL1 can directly interact with HIF-2α and that overexpression of MUL1 decreased HIF-2α levels in a manner partially dependent on ACSS2. These findings identify multiple mechanisms to regulate HIF-2α stability and ACSS2 inhibition as a strategy to complement HIF-2α-targeted therapies and deplete pathogenically stabilized HIF-2α.
    Keywords:  Cancer; Cell biology; Hypoxia; Metabolism; Molecular biology
    DOI:  https://doi.org/10.1172/JCI164249
  10. J Neurosci. 2024 Jun 27. pii: e0003242024. [Epub ahead of print]
    Endoplasmic reticulum and mitochondrial calcium handling dynamically shape slow afterhyperpolarizations in vasopressin magnocellular neurons.
    Matthew K Kirchner, Ferdinand Althammer, Elba Campos-Lira, Juliana Montanez, Javier E Stern.
      Many neurons including vasopressin (VP) magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus (SON) generate afterhyperpolarizations (AHPs) during spiking to slow firing, a phenomenon known as spike frequency adaptation. The AHP is underlain by Ca2+-activated K+ currents, and while slow component (sAHP) features are well described, its mechanism remains poorly understood. Previous work demonstrated that Ca2+ influx through N-type Ca2+ channels is the primary source of sAHP activation in SON oxytocin neurons, but no obvious channel coupling was described for VP neurons. Given this, we tested the possibility of an intracellular source of sAHP activation, namely the Ca2+-handling organelles endoplasmic reticulum (ER) and mitochondria in male and female wistar rats. We demonstrate that ER Ca2+ depletion greatly inhibits sAHPs without a corresponding decrease in Ca2+ signal. Caffeine sensitized AHP activation by Ca2+ In contrast to ER, disabling mitochondria with CCCP or blocking mitochondria Ca2+ uniporter (MCU) enhanced sAHP amplitude and duration, implicating mitochondria as a vital buffer for sAHP-activating Ca2+ Block of mitochondria Na+-dependent Ca2+ release via triphenylphosphonium (TPP+) failed to affect sAHPs, indicating that mitochondria Ca2+ doesn't contribute to sAHP activation. Together, our results support that ER Ca2+-induced Ca2+ release activates sAHPs and mitochondria shape the spatiotemporal trajectory of the sAHP via Ca2+ buffering in VP neurons. Overall, this implicates organelle Ca2+, and specifically ER-mitochondria associated membrane contacts, as an important site of Ca2+ microdomain activity that regulates sAHP signaling pathways. Thus, this site plays a major role in influencing VP firing activity and systemic hormonal release.Significance Statement The slow afterhyperpolarization (sAHP) is mediated by a Ca2+-dependent K+ current. Despite its critical role in regulating neuronal spiking, the Ca2+-dependent mechanisms leading to its activation and spatiotemporal shape remains poorly understood. Here we show that in vasopressin (VP) neurons, dynamic interactions in Ca2+ handling between endoplasmic reticulum (ER) and mitochondria play a significant role in sAHP initiation (via ER Ca2+ release) and its spatiotemporal waveform (via mitochondrial Ca2+ uptake). Our results suggest that contact sites between ER and mitochondria represent Ca2+ microdomains critically involved in initiating the first steps of sAHP generation in VP neurons. Given that changes in the sAHP have been linked to abnormal firing activity in various diseases, our results have both wide-range physiological and pathological implications.
    DOI:  https://doi.org/10.1523/JNEUROSCI.0003-24.2024
  11. Nat Commun. 2024 Jun 25. 15(1): 5386
    Raman micro-spectroscopy reveals the spatial distribution of fumarate in cells and tissues.
    Marlous Kamp, Jakub Surmacki, Marc Segarra Mondejar, Tim Young, Karolina Chrabaszcz, Fadwa Joud, Vincent Zecchini, Alyson Speed, Christian Frezza, Sarah E Bohndiek.
      Aberrantly accumulated metabolites elicit intra- and inter-cellular pro-oncogenic cascades, yet current measurement methods require sample perturbation/disruption and lack spatio-temporal resolution, limiting our ability to fully characterize their function and distribution. Here, we show that Raman spectroscopy (RS) can directly detect fumarate in living cells in vivo and animal tissues ex vivo, and that RS can distinguish between Fumarate hydratase (Fh1)-deficient and Fh1-proficient cells based on fumarate concentration. Moreover, RS reveals the spatial compartmentalization of fumarate within cellular organelles in Fh1-deficient cells: consistent with disruptive methods, we observe the highest fumarate concentration (37 ± 19 mM) in mitochondria, where the TCA cycle operates, followed by the cytoplasm (24 ± 13 mM) and then the nucleus (9 ± 6 mM). Finally, we apply RS to tissues from an inducible mouse model of FH loss in the kidney, demonstrating RS can classify FH status. These results suggest RS could be adopted as a valuable tool for small molecule metabolic imaging, enabling in situ non-destructive evaluation of fumarate compartmentalization.
    DOI:  https://doi.org/10.1038/s41467-024-49403-w
  12. Nat Aging. 2024 Jun 26.
    Ferroptotic stress drives liver aging and metabolic dysfunction.
      
    DOI:  https://doi.org/10.1038/s43587-024-00654-8
  13. Sci Adv. 2024 Jun 28. 10(26): eadn4508
    Lactate transported by MCT1 plays an active role in promoting mitochondrial biogenesis and enhancing TCA flux in skeletal muscle.
    Lingling Zhang, Chenhao Xin, Shuo Wang, Shixuan Zhuo, Jing Zhu, Zi Li, Yuyi Liu, Lifeng Yang, Yan Chen.
      Once considered as a "metabolic waste," lactate is now recognized as a major fuel for tricarboxylic acid (TCA) cycle. Our metabolic flux analysis reveals that skeletal muscle mainly uses lactate to fuel TCA cycle. Lactate is transported through the cell membrane via monocarboxylate transporters (MCTs) in which MCT1 is highly expressed in the muscle. We analyzed how MCT1 affects muscle functions using mice with specific deletion of MCT1 in skeletal muscle. MCT1 deletion enhances running performance, increases oxidative fibers while decreasing glycolytic fibers, and enhances flux of glucose to TCA cycle. MCT1 deficiency increases the expression of mitochondrial proteins, augments cell respiration rate, and elevates mitochondrial activity in the muscle. Mechanistically, the protein level of PGC-1α, a master regulator of mitochondrial biogenesis, is elevated upon loss of MCT1 via increases in cellular NAD+ level and SIRT1 activity. Collectively, these results demonstrate that MCT1-mediated lactate shuttle plays a key role in regulating muscle functions by modulating mitochondrial biogenesis and TCA flux.
    DOI:  https://doi.org/10.1126/sciadv.adn4508
  14. JCI Insight. 2024 Jun 27. pii: e178725. [Epub ahead of print]
    A metabolic redox relay supports ER proinsulin export in pancreatic islet β-cells.
    Kristen E Rohli, Nicole J Stubbe, Emily M Walker, Gemma L Pearson, Scott A Soleimanpour, Samuel B Stephens.
      Endoplasmic reticulum (ER) stress and proinsulin misfolding are heralded as contributing factors to β-cell dysfunction in Type 2 diabetes (T2D), yet how ER function becomes compromised is not well understood. Recent data identifies altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple β-cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that β-cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas Txnip suppression restored ER redox and proinsulin trafficking. Taken together, we propose that β-cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.
    Keywords:  Beta cells; Cell biology; Endocrinology; Insulin; Protein traffic
    DOI:  https://doi.org/10.1172/jci.insight.178725
  15. bioRxiv. 2024 Jun 10. pii: 2024.06.09.598152. [Epub ahead of print]
    ATP Citrate Lyase Supports Cardiac Function and NAD+/NADH Balance And Is Depressed in Human Heart Failure.
    Mariam Meddeb, Navid Koleini, Seungho Jun, Mohammad Keykhaei, Farnaz Farshidfar, Liang Zhao, Seoyoung Kwon, Brian Lin, Gizem Keceli, Nazareno Paolocci, Virginia Hahn, Kavita Sharma, Erika L Pearce, David A Kass.
      BACKGROUND: ATP-citrate lyase (ACLY) converts citrate into acetyl-CoA and oxaloacetate in the cytosol. It plays a prominent role in lipogenesis and fat accumulation coupled to excess glucose, and its inhibition is approved for treating hyperlipidemia. In RNAseq analysis of human failing myocardium, we found ACLY gene expression is reduced; however the impact this might have on cardiac function and/or metabolism has not been previously studied. As new ACLY inhibitors are in development for cancer and other disorders, such understanding has added importance.METHODS: Cardiomyocytes, ex-vivo beating hearts, and in vivo hearts with ACLY inhibited by selective pharmacologic (BMS303141, ACLYi) or genetic suppression, were studied. Regulation of ACLY gene/protein expression, and effects of ACLYi on function, cytotoxicity, tricarboxylic acid (TCA)-cycle metabolism, and redox and NAD+/NADH balance were assessed. Mice with cardiac ACLY knockdown induced by AAV9-acly-shRNA or cardiomyocyte tamoxifen-inducible Acly knockdown were studied.
    RESULTS: Acly gene expression was reduced more in obese patients with heart failure and preserved EF (HFpEF) than HF with reduced EF. In vivo pressure-overload and in vitro hormonal stress increased ACLY protein expression, whereas it declined upon fatty-acid exposure. Acute ACLYi (1-hr) dose-dependently induced cytotoxicity in adult and neonatal cardiomyocytes, and caused substantial reduction of systolic and diastolic function in myocytes and ex-vivo beating hearts. In the latter, ATP/ADP ratio also fell and lactate increased. U13C-glucose tracing revealed an ACLYdependent TCA-bypass circuit in myocytes, where citrate generated in mitochondria is transported to the cytosol, metabolized by ACLY and then converted to malate to re-enter mitochondria,bypassing several NADH-generating steps. ACLYi lowered NAD+/NADH ratio and restoring this balance ameliorated cardiomyocyte toxicity. Oxidative stress was undetected with ACLYi. Adult hearts following 8-weeks of reduced cardiac and/or cardiomyocyte ACLY downregulation exhibited ventricular dilation and reduced function that was prevented by NAD augmentation. Cardiac dysfunction from ACLY knockdown was worse in hearts subjected to sustained pressureoverload, supporting a role in stress responses.
    CONCLUSIONS: ACLY supports normal cardiac function through maintenance of the NAD+/NADH balance and is upregulated by hemodynamic and hormonal stress, but depressed by lipid excess. ACLY levels are most reduced in human HFpEF with obesity potentially worsening cardio-metabolic reserve.
    DOI:  https://doi.org/10.1101/2024.06.09.598152
  16. EMBO Mol Med. 2024 Jun 26.
    FADS1/2 control lipid metabolism and ferroptosis susceptibility in triple-negative breast cancer.
    Nicla Lorito, Angela Subbiani, Alfredo Smiriglia, Marina Bacci, Francesca Bonechi, Laura Tronci, Elisabetta Romano, Alessia Corrado, Dario Livio Longo, Marta Iozzo, Luigi Ippolito, Giuseppina Comito, Elisa Giannoni, Icro Meattini, Alexandra Avgustinova, Paola Chiarugi, Angela Bachi, Andrea Morandi.
      Triple-negative breast cancer (TNBC) has limited therapeutic options, is highly metastatic and characterized by early recurrence. Lipid metabolism is generally deregulated in TNBC and might reveal vulnerabilities to be targeted or used as biomarkers with clinical value. Ferroptosis is a type of cell death caused by iron-dependent lipid peroxidation which is facilitated by the presence of polyunsaturated fatty acids (PUFA). Here we identify fatty acid desaturases 1 and 2 (FADS1/2), which are responsible for PUFA biosynthesis, to be highly expressed in a subset of TNBC with a poorer prognosis. Lipidomic analysis, coupled with functional metabolic assays, showed that FADS1/2 high-expressing TNBC are susceptible to ferroptosis-inducing agents and that targeting FADS1/2 by both genetic interference and pharmacological approach renders those tumors ferroptosis-resistant while unbalancing PUFA/MUFA ratio by the supplementation of exogenous PUFA sensitizes resistant tumors to ferroptosis induction. Last, inhibiting lipid droplet (LD) formation and turnover suppresses the buffering capacity of LD and potentiates iron-dependent cell death. These findings have been validated in vitro and in vivo in mouse- and human-derived clinically relevant models and in a retrospective cohort of TNBC patients.
    Keywords:  Desaturases; Ferroptosis; Lipid Droplets; Lipid Metabolism; Polyunsaturated Fatty Acids
    DOI:  https://doi.org/10.1038/s44321-024-00090-6
  17. Anal Chem. 2024 Jun 28.
    Visualizing Metabolism in Biotechnologically Important Yeasts with dDNP NMR Reveals Evolutionary Strategies and Glycolytic Logic.
    Sebastian Meier, Ke-Chuan Wang, Francesca Sannelli, Jakob Blæsbjerg Hoof, Jürgen Wendland, Pernille Rose Jensen.
      Saccharomyces cerevisiae has long been a pillar of biotechnological production and basic research. More recently, strides to exploit the functional repertoire of nonconventional yeasts for biotechnological production have been made. Genomes and genetic tools for these yeasts are not always available, and yeast genomics alone may be insufficient to determine the functional features in yeast metabolism. Hence, functional assays of metabolism, ideally in the living cell, are best suited to characterize the cellular biochemistry of such yeasts. Advanced in cell NMR methods can allow the direct observation of carbohydrate influx into central metabolism on a seconds time scale: dDNP NMR spectroscopy temporarily enhances the nuclear spin polarization of substrates by more than 4 orders of magnitude prior to functional assays probing central metabolism. We use various dDNP enhanced carbohydrates for in-cell NMR to compare the metabolism of S. cerevisiae and nonconventional yeasts, with an emphasis on the wine yeast Hanseniaspora uvarum. In-cell observations indicated more rapid exhaustion of free cytosolic NAD+ in H. uvarum and alternative routes for pyruvate conversion, in particular, rapid amination to alanine. In-cell observations indicated that S. cerevisiae outcompetes other biotechnologically relevant yeasts by rapid ethanol formation due to the efficient adaptation of cofactor pools and the removal of competing reactions from the cytosol. By contrast, other yeasts were better poised to use redox neutral processes that avoided CO2-emission. Beyond visualizing the different cellular strategies for arriving at redox neutral end points, in-cell dDNP NMR probing showed that glycolytic logic is more conserved: nontoxic precursors of cellular building blocks formed high-population intermediates in the influx of glucose into the central metabolism of eight different biotechnologically important yeasts. Unsupervised clustering validated that the observation of rapid intracellular chemistry is a viable means to functionally classify biotechnologically important organisms.
    DOI:  https://doi.org/10.1021/acs.analchem.4c00809
  18. bioRxiv. 2024 Jun 12. pii: 2024.06.10.598354. [Epub ahead of print]
    Intra-organ cell specific mitochondrial quantitative interactomics.
    A A Bakhtina, M D Campbell, B D Sibley, M Sanchez-Contreras, M T Sweetwyne, J E Bruce.
      Almost every organ consists of many cell types, each with its unique functions. Proteomes of these cell types are thus unique too. But it is reasonable to assume that interactome (inter and intra molecular interactions of proteins) are also distinct since protein interactions are what ultimately carry out the function. Podocytes and tubules are two cell types within kidney with vastly different functions: podocytes envelop the blood vessels in the glomerulus and act as filters while tubules are located downstream of the glomerulus and are responsible for reabsorption of important nutrients. It has been long known that for tubules mitochondria plays an important role as they require a lot of energy to carry out their functions. In podocytes, however, it has been assumed that mitochondria might not matter as much in both normal physiology and pathology 1 . Here we have applied quantitative cross-linking mass spectrometry to compare mitochondrial interactomes of tubules and podocytes using a transgenic mitochondrial tagging strategy to immunoprecipitate cell-specific mitochondria directly from whole kidney. We have uncovered that mitochondrial proteomes of these cell types are quite similar, although still showing unique features that correspond to known functions, such as high energy production through TCA cycle in tubules and requirements for detoxification in podocytes which are on the frontline of filtration where they encounter toxic compounds and therefore, as a non-renewing cell type they must have ways to protect themselves from cellular toxicity. But we gained much deeper insight with the interactomics data. We were able to find pathways differentially regulated in podocytes and tubules based on changing cross-link levels and not just protein levels. Among these pathways are betaine metabolism, lysine degradation, and many others. We have also demonstrated how quantitative interactomics could be used to detect different activity levels of an enzyme even when protein abundances of it are the same between cell types. We have validated this finding with an orthogonal activity assay. Overall, this work presents a new view of mitochondrial biology for two important, but functionally distinct, cell types within the mouse kidney showing both similarities and unique features. This data can continue to be explored to find new aspects of mitochondrial biology, especially in podocytes, where mitochondria has been understudied. In the future this methodology can also be applied to other organs to uncover differences in the function of cell types.
    DOI:  https://doi.org/10.1101/2024.06.10.598354
  19. Curr Protoc. 2024 Jun;4(6): e1060
    Using Fluorescent GAP Indicators to Monitor ER Ca2.
    Jonathan Rojo-Ruiz, Cinthia Sánchez-Rabadán, Belen Calvo, Javier García-Sancho, Maria Teresa Alonso.
      The endoplasmic reticulum (ER) is the main reservoir of Ca2+ of the cell. Accurate and quantitative measuring of Ca2+ dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca2+ indicators have been developed, including a family of fluorescent Ca2+ indicators, dubbed GFP-Aequorin Proteins (GAPs). They are based on the fusion of two jellyfish proteins, the green fluorescent protein (GFP) and the Ca2+-binding protein aequorin. GAP Ca2+ indicators exhibit a combination of several features: they are excitation ratiometric indicators, with reciprocal changes in the fluorescence excited at 405 and 470 nm, which is advantageous for imaging experiments; they exhibit a Hill coefficient of 1, which facilitates the calibration of the fluorescent signal into Ca2+ concentrations; they are insensible to variations in the Mg2+ concentrations or pH variations (in the 6.5-8.5 range); and, due to the lack of mammalian homologues, these proteins have a favorable expression in transgenic animals. A low Ca2+ affinity version of GAP, GAP3 (KD ≅ 489 µM), has been engineered to conform with the estimated [Ca2+] in the ER. GAP3 targeted to the lumen of the ER (erGAP3) can be utilized for imaging intraluminal Ca2+. The ratiometric measurements provide a quantitative method to assess accurate [Ca2+]ER, both dynamically and at rest. In addition, erGAP3 can be combined with synthetic cytosolic Ca2+ indicators to simultaneously monitor ER and cytosolic Ca2+. Here, we provide detailed methods to assess erGAP3 expression and to perform Ca2+ imaging, either restricted to the ER lumen, or simultaneously in the ER and the cytosol. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Detection of erGAP3 in the ER by immunofluorescence Basic Protocol 2: Monitoring ER Ca2+ Basic Protocol 3: Monitoring ER- and cytosolic-Ca2+ Support Protocol: Generation of a stable cell line expressing erGAP3.
    Keywords:  Ca2+ calibration; GFP; aequorin; fluorescence; imaging
    DOI:  https://doi.org/10.1002/cpz1.1060
  20. Aging Cell. 2024 Jun 26. e14199
    Integrated multi-omic analyses uncover the effects of aging on cell-type regulation in glucose-responsive tissues.
    Peng Xu, Yimeng Kong, Nicholette D Palmer, Maggie C Y Ng, Bin Zhang, Swapan K Das.
      Aging significantly influences cellular activity and metabolism in glucose-responsive tissues, yet a comprehensive evaluation of the impacts of aging and associated cell-type responses has been lacking. This study integrates transcriptomic, methylomic, single-cell RNA sequencing, and metabolomic data to investigate aging-related regulations in adipose and muscle tissues. Through coexpression network analysis of the adipose tissue, we identified aging-associated network modules specific to certain cell types, including adipocytes and immune cells. Aging upregulates the metabolic functions of lysosomes and downregulates the branched-chain amino acids (BCAAs) degradation pathway. Additionally, aging-associated changes in cell proportions, methylation profiles, and single-cell expressions were observed in the adipose. In the muscle tissue, aging was found to repress the metabolic processes of glycolysis and oxidative phosphorylation, along with reduced gene activity of fast-twitch type II muscle fibers. Metabolomic profiling linked aging-related alterations in plasma metabolites to gene expression in glucose-responsive tissues, particularly in tRNA modifications, BCAA metabolism, and sex hormone signaling. Together, our multi-omic analyses provide a comprehensive understanding of the impacts of aging on glucose-responsive tissues and identify potential plasma biomarkers for these effects.
    Keywords:  adipose; aging; cell‐type; metabolite; multi‐omic; muscle; network
    DOI:  https://doi.org/10.1111/acel.14199
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