bims-mepmim Biomed News
on Metabolites in pathological microenvironments and immunometabolism
Issue of 2023‒03‒19
28 papers selected by
Erika Mariana Palmieri
NIH/NCI Laboratory of Cancer ImmunoMetabolism

  1. Nat Commun. 2023 Mar 13. 14(1): 1362
      Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.
  2. Front Immunol. 2023 ;14 1101433
      Introduction: CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis.Methods: To dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate.
    Results: The lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma.
    Discussion: Our studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells.
    Keywords:  CD8+ T cells; lactate; metabolism; oxygen; transcriptome
  3. J Biol Chem. 2023 Mar 15. pii: S0021-9258(23)00253-3. [Epub ahead of print] 104611
      Adipose tissue (AT) plays a crucial role in maintaining metabolic homeostasis by storing lipids and glucose from circulation as intracellular fat. As peripheral tissues like AT become insulin resistant, decompensation of blood glucose levels occurs causing type 2 diabetes (T2D). Currently, modulating the glycocalyx, a layer of cell-surface glycans, is an underexplored pharmacological treatment strategy to improve glucose homeostasis in T2D patients. Here, we show a novel role for cell surface heparan sulfate (HS) in establishing glucose uptake capacity and metabolic utilization in differentiated adipocytes. Using a combination of chemical and genetic interventions, we identified that HS modulates this metabolic phenotype by attenuating levels of Wnt signaling during adipogenesis. By engineering the glycocalyx of preadipocytes with exogenous synthetic HS mimetics, we were able to enhance glucose clearance capacity after differentiation through modulation of Wnt ligand availability. These findings establish the cellular glycocalyx as a possible new target for therapeutic intervention in T2D patients by enhancing glucose clearance capacity independent of insulin secretion.
  4. J Clin Invest. 2023 Mar 16. pii: e167442. [Epub ahead of print]
      BACKGROUND: Hepatic de novo lipogenesis (DNL) and β-oxidation are tightly coordinated, and their dysregulation is thought to contribute to the pathogenesis of non-alcoholic fatty liver (NAFL). Fasting normally relaxes DNL-mediated inhibition of hepatic β-oxidation, dramatically increasing ketogenesis and decreasing reliance on the TCA cycle. Thus, we tested whether aberrant oxidative metabolism in fasting NAFL subjects is related to the inability to halt fasting DNL.METHODS: Forty consecutive non-diabetic individuals with and without a history of NAFL were recruited for this observational study. After phenotyping, subjects fasted for 24-hr, and hepatic metabolism was interrogated using a combination of 2H2O and 13C tracers, magnetic resonance spectroscopy, and high-resolution mass spectrometry.
    RESULTS: Within a subset of subjects, DNL was detectable after a 24-hr fast and was more prominent in those with NAFL, though it was poorly correlated with steatosis. However, fasting DNL negatively correlated with hepatic β-oxidation and ketogenesis and positively correlated with citrate synthesis. Subjects with NAFL but undetectable fasting DNL (25th percentile) were comparatively normal. However, those with the highest fasting DNL (75th percentile) were intransigent to the effects of fasting on the concentration of insulin, NEFA, and ketones. Additionally, they sustained glycogenolysis and spared the loss of oxaloacetate to gluconeogenesis in favor of citrate synthesis, which correlated with DNL and diminished ketogenesis.
    CONCLUSION: Metabolic flux analysis in fasted subjects indicates that shared metabolic mechanisms link the dysregulations of hepatic DNL, ketogenesis, and the TCA cycle in NAFL.
    TRIAL REGISTRATION: Data obtained during the enrollment/non-intervention phase of Effect of Vitamin E on Non-Alcoholic Fatty Liver Disease; NCT02690792.
    Keywords:  Endocrinology; Fatty acid oxidation; Hepatology; Intermediary metabolism
  5. Immunol Rev. 2023 Mar 16.
      Neutrophil recruitment from circulation to sites of inflammation is guided by multiple chemoattractant cues emanating from tissue cells, immune cells, and platelets. Here, we focus on the function of one G-protein coupled receptor, GPR35, in neutrophil recruitment. GPR35 has been challenging to study due the description of multiple ligands and G-protein couplings. Recently, we found that GPR35-expressing hematopoietic cells respond to the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA). We discuss distinct response profiles of GPR35 to 5-HIAA compared to other ligands. To place the functions of 5-HIAA in context, we summarize the actions of serotonin in vascular biology and leukocyte recruitment. Important sources of serotonin and 5-HIAA are platelets and mast cells. We discuss the dynamics of cell migration into inflamed tissues and how multiple platelet and mast cell-derived mediators, including 5-HIAA, cooperate to promote neutrophil recruitment. Additional actions of GPR35 in tissue physiology are reviewed. Finally, we discuss how clinically approved drugs that modulate serotonin uptake and metabolism may influence 5-HIAA-GPR35 function, and we speculate about broader influences of the GPR35 ligand-receptor system in immunity and disease.
    Keywords:  GPCRs; chemotaxis; lipid mediators; mast cells; neutrophils; platelets
  6. Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00119-3. [Epub ahead of print]83(6): 877-889
      Mitochondria are membrane-enclosed organelles with endosymbiotic origins, harboring independent genomes and a unique biochemical reaction network. To perform their critical functions, mitochondria must maintain a distinct biochemical environment and coordinate with the cytosolic metabolic networks of the host cell. This coordination requires them to sense and control metabolites and respond to metabolic stresses. Indeed, mitochondria adopt feedback or feedforward control strategies to restrain metabolic toxicity, enable metabolic conservation, ensure stable levels of key metabolites, allow metabolic plasticity, and prevent futile cycles. A diverse panel of metabolic sensors mediates these regulatory circuits whose malfunctioning leads to inborn errors of metabolism with mild to severe clinical manifestations. In this review, we discuss the logic and molecular basis of metabolic sensing and control in mitochondria. The past research outlined recurring patterns in mitochondrial metabolic sensing and control and highlighted key knowledge gaps in this organelle that are potentially addressable with emerging technological breakthroughs.
  7. Am J Physiol Lung Cell Mol Physiol. 2023 Mar 15.
      In obesity, disturbed glutamine metabolism contributes to enhanced inflammation by inducing alterations in immune cells. As macrophages and innate lymphoid cells (ILCs) are known to be involved in the pathogenesis of obesity-related asthma, we tested our hypothesis that altered glutamine metabolism may link obesity to airway hyperresponsivenss (AHR), a cardinal feature of asthma, focusing on these innate immune cells. Four-week-old male C57BL/6 mice were fed a high-fat diet (HFD) for 13 weeks in the presence or absence of BPTES [Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a selective inhibitor of glutaminase 1 which converts glutamine to glutamate] and their blood, lung, and adipose tissues were analyzed. We then conducted in vitro experiments using bone marrow-derived macrophages (BMDMs) and mouse alveolar macrophage cell line. Furthermore, we investigated plasma glutamine and glutamate levels in obese and non-obese asthmatics. BPTES treatment prevented HFD-induced AHR and significantly decreased IL-1β+ classically activated macrophages (M1s) and type 3 ILC (ILC3s) which increased in the lungs of HFD-fed obese mice. In in vitro experiments, BPTES treatment or glutamine supplement significantly reduced the proportion of IL-1β+NLRP3+ M1s in lipopolysaccharide-stimulated BMDMs and mouse alveolar macrophage cell line. BPTES treatment also significantly reduced the IL-17 producing ILC3s differentiated from ILCs in naïve mouse lung. In addition, plasma glutamate/glutamine ratios were significantly higher in obese asthmatics compared to non-obese asthmatics. Inhibition of glutaminolysis reverses AHR in HFD-induced obese mice and decreases IL-1β+NLRP3+ M1s and IL-17 producing ILC3s, which suggests altered glutamine metabolism may have a role in the pathogenesis of obesity-related AHR.
    Keywords:  asthma; glutamine; innate lymphoid cells; macrophages; obesity
  8. Redox Biol. 2023 Mar 08. pii: S2213-2317(23)00062-9. [Epub ahead of print]62 102661
      There is growing appreciation that hematopoietic alterations underpin the ubiquitous detrimental effects of metabolic disorders. The susceptibility of bone marrow (BM) hematopoiesis to perturbations of cholesterol metabolism is well documented, while the underlying cellular and molecular mechanisms remain poorly understood. Here we reveal a distinct and heterogeneous cholesterol metabolic signature within BM hematopoietic stem cells (HSCs). We further show that cholesterol directly regulates maintenance and lineage differentiation of long-term HSCs (LT-HSCs), with high levels of intracellular cholesterol favoring maintenance and myeloid bias of LT-HSCs. During irradiation-induced myelosuppression, cholesterol also safeguards LT-HSC maintenance and myeloid regeneration. Mechanistically, we unravel that cholesterol directly and distinctively enhances ferroptosis resistance and boosts myeloid but dampens lymphoid lineage differentiation of LT-HSCs. Molecularly, we identify that SLC38A9-mTOR axis mediates cholesterol sensing and signal transduction to instruct lineage differentiation of LT-HSCs as well as to dictate ferroptosis sensitivity of LT-HSCs through orchestrating SLC7A11/GPX4 expression and ferritinophagy. Consequently, myeloid-biased HSCs are endowed with a survival advantage under both hypercholesterolemia and irradiation conditions. Importantly, a mTOR inhibitor rapamycin and a ferroptosis inducer imidazole ketone erastin prevent excess cholesterol-induced HSC expansion and myeloid bias. These findings unveil an unrecognized fundamental role of cholesterol metabolism in HSC survival and fate decisions with valuable clinical implications.
    Keywords:  Cholesterol; Ferroptosis; Hematopoietic stem cell; Ionizing radiation; Myeloid bias; Myelosuppression
  9. Front Physiol. 2023 ;14 1122895
      Numerous cellular proteins are post-translationally modified by addition of a lipid group to their structure, which dynamically influences the proteome by increasing hydrophobicity of proteins often impacting protein conformation, localization, stability, and binding affinity. These lipid modifications include myristoylation and palmitoylation. Palmitoylation involves a 16-carbon saturated fatty acyl chain being covalently linked to a cysteine thiol through a thioester bond. Palmitoylation is unique within this group of modifications, as the addition of the palmitoyl group is reversible and enzyme driven, rapidly affecting protein targeting, stability and subcellular trafficking. The palmitoylation reaction is catalyzed by a large family of Asp-His-His-Cys (DHHCs) motif-containing palmitoyl acyltransferases, while the reverse reaction is catalyzed by acyl-protein thioesterases (APTs), that remove the acyl chain. Palmitoyl-CoA serves an important dual purpose as it is not only a key metabolite fueling energy metabolism, but is also a substrate for this PTM. In this review, we discuss protein palmitoylation in regulating substrate metabolism, focusing on membrane transport proteins and kinases that participate in substrate uptake into the cell. We then explore the palmitoylation of mitochondrial proteins and the palmitoylation regulatory enzymes, a less explored field for potential lipid metabolic regulation.
    Keywords:  fatty acid signalling; membrane transporter; metabolism; mitochondria; palmitoylation
  10. EMBO J. 2023 Mar 14. e111901
      Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Here, using cellular models representing a wide variety of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced fragmentation upon knockdown or knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing. Remarkably, the genetic induction of fragmentation phenocopies distinct cell type-specific biological functions of enhanced FAO. These include stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet β-cells exposed to fatty acids, and survival of FAO-dependent lymphoma subtypes. We find that fragmentation increases long-chain but not short-chain FAO, identifying carnitine O-palmitoyltransferase 1 (CPT1) as the downstream effector of mitochondrial morphology in regulation of FAO. Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increases CPT1 sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined.
    Keywords:  CPT1; fatty acid oxidation; fission; fusion; mitochondrial dynamics
  11. EMBO J. 2023 Mar 15. e113490
      Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact-dependent but uptake-independent manner. We use time-lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX-1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface-exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX-1-mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake-independent killing of macrophages and that the 50-kDa isoform of the ESX-1-secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX-1 inhibitor reduces uptake-independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti-phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung.
    Keywords:  EspB; calcium flux; plasma membrane damage; pyroptosis; time-lapse fluorescence microscopy
  12. Immunity. 2023 Mar 07. pii: S1074-7613(23)00092-4. [Epub ahead of print]
      Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.
    Keywords:  Citrobacter rodentium; ROS signals; TH17 cells; commensal bacterial; epithelial endoplasmic reticulum stress; inflammatory bowel disease; purine metabolism
  13. STAR Protoc. 2023 Mar 17. pii: S2666-1667(23)00133-8. [Epub ahead of print]4(2): 102175
      Regulation of bioenergetics and cell death are pivotal mitochondrial functions determining the responses of macrophages to infection. Here, we provide a protocol to investigate mitochondrial functions during infection of macrophages by intracellular bacteria. We describe steps for quantifying mitochondrial polarization, cell death, and bacterial infection in infected, living, human primary macrophages at the single-cell level. We also detail the use of the pathogen Legionella pneumophila as model. This protocol can be adapted to investigate mitochondrial functions in other settings. For complete details on the use and execution of this protocol, please refer to Escoll et al. (2021).1.
    Keywords:  Cell Biology; Cell-based Assays; High-throughput Screening; Immunology; Metabolism; Microbiology; Microscopy; Single Cell
  14. Elife. 2023 Mar 15. pii: e85103. [Epub ahead of print]12
      Chronically high blood glucose (hyperglycemia) leads to diabetes and fatty liver disease. Obesity is a major risk factor for hyperglycemia, but the underlying mechanism is unknown. Here, we show that a high-fat diet (HFD) in mice causes early loss of expression of the glycolytic enzyme Hexokinase 2 (HK2) specifically in adipose tissue. Adipose-specific knockout of Hk2 reduced glucose disposal and lipogenesis and enhanced fatty acid release in adipose tissue. In a non-cell-autonomous manner, Hk2 knockout also promoted glucose production in liver. Furthermore, we observed reduced hexokinase activity in adipose tissue of obese and diabetic patients, and identified a loss-of-function mutation in the hk2 gene of naturally hyperglycemic Mexican cavefish. Mechanistically, HFD in mice led to loss of HK2 by inhibiting translation of Hk2 mRNA. Our findings identify adipose HK2 as a critical mediator of local and systemic glucose homeostasis, and suggest that obesity-induced loss of adipose HK2 is an evolutionarily conserved mechanism for the development of selective insulin resistance and thereby hyperglycemia.
    Keywords:  adipose tissue; astyanax mexicanus; cell biology; diabetes; glucose; human; lipid metabolism; mouse; obesity; selective insulin resistance
  15. Nat Commun. 2023 Mar 13. 14(1): 1368
      Inhibition of AMPK is tightly associated with metabolic perturbations upon over nutrition, yet the molecular mechanisms underlying are not clear. Here, we demonstrate the serine/threonine-protein phosphatase 6 regulatory subunit 3, SAPS3, is a negative regulator of AMPK. SAPS3 is induced under high fat diet (HFD) and recruits the PP6 catalytic subunit to deactivate phosphorylated-AMPK, thereby inhibiting AMPK-controlled metabolic pathways. Either whole-body or liver-specific deletion of SAPS3 protects male mice against HFD-induced detrimental consequences and reverses HFD-induced metabolic and transcriptional alterations while loss of SAPS3 has no effects on mice under balanced diets. Furthermore, genetic inhibition of AMPK is sufficient to block the protective phenotype in SAPS3 knockout mice under HFD. Together, our results reveal that SAPS3 is a negative regulator of AMPK and suppression of SAPS3 functions as a guardian when metabolism is perturbed and represents a potential therapeutic strategy to treat metabolic syndromes.
  16. Nat Metab. 2023 Mar 13.
      Our understanding of how global changes in cellular metabolism contribute to human kidney disease remains incompletely understood. Here we show that nicotinamide adenine dinucleotide (NAD+) deficiency drives mitochondrial dysfunction causing inflammation and kidney disease development. Using unbiased global metabolomics in healthy and diseased human kidneys, we identify NAD+ deficiency as a disease signature. Furthermore using models of cisplatin- or ischaemia-reperfusion induced kidney injury in male mice we observed NAD+ depletion Supplemental nicotinamide riboside or nicotinamide mononucleotide restores NAD+ levels and improved kidney function. We find that cisplatin exposure causes cytosolic leakage of mitochondrial RNA (mtRNA) and activation of the cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I), both of which can be ameliorated by restoring NAD+. Male mice with RIG-I knock-out (KO) are protected from cisplatin-induced kidney disease. In summary, we demonstrate that the cytosolic release of mtRNA and RIG-I activation is an NAD+-sensitive mechanism contributing to kidney disease.
  17. EMBO J. 2023 Mar 15. e111494
      Tumor growth is influenced by a complex network of interactions between multiple cell types in the tumor microenvironment (TME). These constrained conditions trigger the endoplasmic reticulum (ER) stress response, which extensively reprograms mRNA translation. When uncontrolled over time, chronic ER stress impairs the antitumor effector function of CD8 T lymphocytes. How cells promote adaptation to chronic stress in the TME without the detrimental effects of the terminal unfolded protein response (UPR) is unknown. Here, we find that, in effector CD8 T lymphocytes, RNA-binding protein CPEB4 constitutes a new branch of the UPR that allows cells to adapt to sustained ER stress, yet remains decoupled from the terminal UPR. ER stress, induced during CD8 T-cell activation and effector function, triggers CPEB4 expression. CPEB4 then mediates chronic stress adaptation to maintain cellular fitness, allowing effector molecule production and cytotoxic activity. Accordingly, this branch of the UPR is required for the antitumor effector function of T lymphocytes, and its disruption in these cells exacerbates tumor growth.
    Keywords:  CPEB; T lymphocytes; endoplasmic reticulum stress; mRNA translation; tumor microenvironment
  18. Nat Commun. 2023 Mar 15. 14(1): 1439
      A growing body of evidence demonstrates that fetal-derived tissue-resident macrophages have developmental functions. It has been proposed that macrophages promote testicular functions, but which macrophage populations are involved is unclear. Previous studies showed that macrophages play critical roles in fetal testis morphogenesis and described two adult testicular macrophage populations, interstitial and peritubular. There has been debate regarding the hematopoietic origins of testicular macrophages and whether distinct macrophage populations promote specific testicular functions. Here our hematopoietic lineage-tracing studies in mice show that yolk-sac-derived macrophages comprise the earliest testicular macrophages, while fetal hematopoietic stem cells (HSCs) generate monocytes that colonize the gonad during a narrow time window in a Sertoli-cell-dependent manner and differentiate into adult testicular macrophages. Finally, we show that yolk-sac-derived versus HSC-derived macrophages have distinct functions during testis morphogenesis, while interstitial macrophages specifically promote adult Leydig cell steroidogenesis. Our findings provide insight into testicular macrophage origins and their tissue-specific roles.
  19. Blood Adv. 2023 Mar 17. pii: bloodadvances.2022008735. [Epub ahead of print]
      Platelets utilize signal transduction pathways facilitated by Class I phosphatidylinositol transfer proteins (PITPs). The two mammalian Class I PITPs, PITPα and PITPβ, are single PITP domain soluble proteins that are encoded by different genes and have 77% sequence identity, though their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominant expressed murine PITP isoform, had no effect on hemostasis, but had impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we find that mice lacking the lesser expressed PITPβ in their platelets exhibit a similar phenotype. However, in contrast to PITPα-null platelet lysates that have impaired lipid transfer activity, PITPβ-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs leads to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our studies also demonstrate that PITP isoforms are required for maintaining endogenous phosphoinositide PI(4,5)P2 levels and agonist stimulated second messenger formation. The data shown here demonstrate that both class I PITP isoforms contribute to phosphoinositide signaling in platelets, likely through distinct biochemical mechanisms or in different subcellular domains. They are functionally overlapping and either single isoform is able to maintain the homeostasis of platelets.
  20. FEBS J. 2023 Mar 12.
      Ferroptosis, featuring an iron-dependent peroxidation of lipids, is a novel form of programmed cell death that may hold great potential in cancer therapy. Our study found that palmitic acid (PA) inhibited colon cancer cell viability in vitro and in vivo, in conjunction with an accumulation of reactive oxygen species (ROS) and lipid peroxidation. The ferroptosis inhibitor Ferrostatin-1 but not Z-VAD-FMK (a pan-caspase inhibitor), Necrostatin-1 (a potent necroptosis inhibitor), or CQ (a potent inhibitor of autophagy), rescued the cell death phenotype induced by PA. Subsequently, we verified that PA induces ferroptotic cell death through excess iron as cell death was inhibited by iron chelator deferiprone (DFP), while it was exacerbated by a supplement of ferric ammonium citrate (FAC). Mechanistically, PA affects intracellular iron content by inducing endoplasmic reticulum (ER) stress leading to ER calcium release and regulating transferrin transport through increasing cytosolic calcium levels. Furthermore, we observed that cells with high expression of CD36 were more vulnerable to PA-induced ferroptosis. Altogether, our findings reveal that PA engages in anticancer properties by activating ER stress/ER calcium release/transferrin-dependent ferroptosis, and PA might serve as a compound to activate ferroptosis in colon cancer cells with high CD36 expression.
    Keywords:  colon cancer,palmitic acid ,ferroptosis,CD36,ER stress
  21. Nat Cell Biol. 2023 Mar 16.
      The pancreatic islets are composed of discrete hormone-producing cells that orchestrate systemic glucose homeostasis. Here we identify subsets of beta cells using a single-cell transcriptomic approach. One subset of beta cells marked by high CD63 expression is enriched for the expression of mitochondrial metabolism genes and exhibits higher mitochondrial respiration compared with CD63lo beta cells. Human and murine pseudo-islets derived from CD63hi beta cells demonstrate enhanced glucose-stimulated insulin secretion compared with pseudo-islets from CD63lo beta cells. We show that CD63hi beta cells are diminished in mouse models of and in humans with type 2 diabetes. Finally, transplantation of pseudo-islets generated from CD63hi but not CD63lo beta cells into diabetic mice restores glucose homeostasis. These findings suggest that loss of a specific subset of beta cells may lead to diabetes. Strategies to reconstitute or maintain CD63hi beta cells may represent a potential anti-diabetic therapy.
  22. Redox Biol. 2023 Mar 04. pii: S2213-2317(23)00058-7. [Epub ahead of print]62 102657
      Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE) activity. eNOS function is suppressed in hyperglycaemic conditions, resulting in reduced NO bioavailability, which is paralleled by reduced levels of hydrogen sulfide (H2S). Here we have addressed the molecular basis of the interplay between the eNOS and CSE pathways. We tested the impact of H2S replacement by using the mitochondrial-targeted H2S donor AP123 in isolated vessels and cultured endothelial cells in high glucose (HG) environment, at concentrations not causing any vasoactive effect per se. Aorta exposed to HG displayed a marked reduction of acetylcholine (Ach)-induced vasorelaxation that was restored by the addition of AP123 (10 nM). In HG condition, bovine aortic endothelial cells (BAEC) showed reduced NO levels, downregulation of eNOS expression, and suppression of CREB activation (p-CREB). Similar results were obtained by treating BAEC with propargylglycine (PAG), an inhibitor of CSE. AP123 treatment rescued eNOS expression, as well as NO levels, and restored p-CREB expression in both the HG environment and the presence of PAG. This effect was mediated by a PI3K-dependent activity since wortmannin (PI3K inhibitor) blunted the rescuing effects operated by the H2S donor. Experiments performed in the aorta of CSE-/- mice confirmed that reduced levels of H2S not only negatively affect the CREB pathway but also impair Ach-induced vasodilation, significantly ameliorated by AP123. We have demonstrated that the endothelial dysfunction due to HG involves H2S/PI3K/CREB/eNOS route, thus highlighting a novel aspect of the H2S/NO interplay in the vasoactive response.
    Keywords:  Endothelium; Gasotransmitters; H(2)S donors; Hyperglycemia; Vascular function
  23. Proc Natl Acad Sci U S A. 2023 Mar 21. 120(12): e2220816120
      Methanol is an ideal feedstock for chemical and biological manufacturing. Constructing an efficient cell factory is essential for producing complex compounds through methanol biotransformation, in which coordinating methanol use and product synthesis is often necessary. In methylotrophic yeast, methanol utilization mainly occurs in peroxisomes, which creates challenges in driving the metabolic flux toward product biosynthesis. Here, we observed that constructing the cytosolic biosynthesis pathway resulted in compromised fatty alcohol production in the methylotrophic yeast Ogataea polymorpha. Alternatively, peroxisomal coupling of fatty alcohol biosynthesis and methanol utilization significantly improved fatty alcohol production by 3.9-fold. Enhancing the supply of precursor fatty acyl-CoA and cofactor NADPH in the peroxisomes by global metabolic rewiring further improved fatty alcohol production by 2.5-fold and produced 3.6 g/L fatty alcohols from methanol under fed-batch fermentation. We demonstrated that peroxisome compartmentalization is helpful for coupling methanol utilization and product synthesis, and with this approach, constructing efficient microbial cell factories for methanol biotransformation is feasible.
    Keywords:  cell factory; metabolic engineering; methanol metabolism; peroxisome; synthetic biology
  24. Nature. 2023 Mar 15.
      Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.
  25. Nat Immunol. 2023 Mar 13.
      Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LATG135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LATG135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LATG135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LATG135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.
  26. JCI Insight. 2023 Mar 14. pii: e164883. [Epub ahead of print]
      Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose importer, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and post-natal maintenance of the disc. Surprisingly, primary NP cells treated with two well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We show in vitro that NP cells mitigate GLUT1 loss by rewiring glucose import through GLUT3. Noteworthy, we demonstrate that substrates, such as glutamine and palmitate, do not compensate for glucose restriction resulting from dual inhibition of GLUT1/3 and inhibition compromises long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated two NP-specific knockout mice: Krt19CreERT; Glut1f/f and Foxa2Cre; Glut1f/f. Noteworthy, there were no apparent defects in post-natal disc health or development and maturation in mutant mice. Microarray analysis confirmed that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells.
    Keywords:  Bone Biology; Cartilage; Glucose metabolism; Hypoxia; Metabolism
  27. Nat Commun. 2023 Mar 13. 14(1): 1379
      Obesity, one of the most serious public health issues, is caused by the imbalance of energy intake and energy expenditure. N(6)-methyladenosine (m6A) RNA modification has been recently identified as a key regulator of obesity, while the detailed mechanism is elusive. Here, we find that YTH RNA binding protein 1 (YTHDF1), an m6A reader, acts as an essential regulator of white adipose tissue metabolism. The expression of YTHDF1 decreases in adipose tissue of male mice fed a high-fat diet. Adipocyte-specific Ythdf1 deficiency exacerbates obesity-induced metabolic defects and inhibits beiging of inguinal white adipose tissue (iWAT) in male mice. By contrast, male mice with WAT-specific YTHDF1 overexpression are resistant to obesity and shows promotion of beiging. Mechanistically, YTHDF1 regulates the translation of diverse m6A-modified mRNAs. In particular, YTHDF1 facilitates the translation of bone morphogenetic protein 8b (Bmp8b) in an m6A-dependent manner to induce the beiging process. Here, we show that YTHDF1 may be an potential therapeutic target for the management of obesity-associated diseases.
  28. Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00028-X. [Epub ahead of print]83(6): 1012-1012.e1
      Mitochondria have emerged as signaling organelles with roles beyond their well-established function in generating ATP and metabolites for macromolecule synthesis. Healthy mitochondria integrate various physiologic inputs and communicate signals that control cell function or fate as well as adaptation to stress. Dysregulation of these mitochondrial signaling networks are linked to pathology. Here we outline a few modes of signaling between the mitochondrion and the cytoplasm. To view this SnapShot, open or download the PDF.