bims-mepmim Biomed News
on Metabolites in pathological microenvironments and immunometabolism
Issue of 2022‒08‒21
29 papers selected by
Erika Mariana Palmieri
NIH/NCI Laboratory of Cancer ImmunoMetabolism

  1. Cell Metab. 2022 Aug 11. pii: S1550-4131(22)00311-4. [Epub ahead of print]
      How environmental nutrient availability impacts T cell metabolism and function remains poorly understood. Here, we report that the presence of physiologic carbon sources (PCSs) in cell culture medium broadly impacts glucose utilization by CD8+ T cells, independent of transcriptional changes in metabolic reprogramming. The presence of PCSs reduced glucose contribution to the TCA cycle and increased effector function of CD8+ T cells, with lactate directly fueling the TCA cycle. In fact, CD8+ T cells responding to Listeria infection preferentially consumed lactate over glucose as a TCA cycle substrate in vitro, with lactate enhancing T cell bioenergetic and biosynthetic capacity. Inhibiting lactate-dependent metabolism in CD8+ T cells by silencing lactate dehydrogenase A (Ldha) impaired both T cell metabolic homeostasis and proliferative expansion in vivo. Together, our data indicate that carbon source availability shapes T cell glucose metabolism and identifies lactate as a bioenergetic and biosynthetic fuel for CD8+ effector T cells.
    Keywords:  (13)C tracing; T cells; TCA cycle; immunometabolism; lactate; metabolic programming; metabolomics
  2. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01015-4. [Epub ahead of print]40(7): 111198
      The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate.
    Keywords:  CP: Cell biology; CP: Metabolism; Drp1; Mfn1; Mfn2; Opa1; amino acid sensing; arginine; dynamics; fusion; glutamine; hyperfusion; leucine; mitochondria; stable isotope tracer
  3. Biochim Biophys Acta Mol Basis Dis. 2022 Aug 11. pii: S0925-4439(22)00190-9. [Epub ahead of print] 166519
      BACKGROUND: Community-acquired pneumonia (CAP) is responsible for a high morbidity and mortality worldwide. Monocytes are essential for pathogen recognition and the initiation of an innate immune response. Immune cells induce intracellular glycolysis upon activation to support several functions.OBJECTIVE: To obtain insight in the metabolic profile of blood monocytes during CAP, with a focus on glycolysis and branching metabolic pathways, and to determine a possible association between intracellular metabolite levels and monocyte function.
    METHODS: Monocytes were isolated from blood of patients with CAP within 24 h of hospital admission and from control subjects matched for age, sex and chronic comorbidities. Changes in glycolysis, oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle and the pentose phosphate pathway were investigated through RNA sequencing and metabolomics measurements. Monocytes were stimulated ex vivo with lipopolysaccharide (LPS) to determine their capacity to produce tumor necrosis factor (TNF), interleukin (IL)-1β and IL-10.
    RESULTS: 50 patients with CAP and 25 non-infectious control subjects were studied. When compared with control monocytes, monocytes from patients showed upregulation of many genes involved in glycolysis, including PKM, the gene encoding pyruvate kinase, the rate limiting enzyme for pyruvate production. Gene set enrichment analysis of OXPHOS, the TCA cycle and the pentose phosphate pathway did not reveal differences between monocytes from patients and controls. Patients' monocytes had elevated intracellular levels of pyruvate and the TCA cycle intermediate α-ketoglutarate. Monocytes from patients were less capable of producing cytokines upon LPS stimulation. Intracellular pyruvate (but not α-ketoglutarate) concentrations positively correlated with IL-1β and IL-10 levels released by patients' (but not control) monocytes upon exposure to LPS.
    CONCLUSION: These results suggest that elevated intracellular pyruvate levels may partially maintain cytokine production capacity of hyporesponsive monocytes from patients with CAP.
    Keywords:  Community-acquired pneumonia (CAP); Cytokine production; Monocytes; Pyruvate; Tolerance
  4. Mol Cell. 2022 Aug 12. pii: S1097-2765(22)00703-1. [Epub ahead of print]
      Proliferating cells exhibit a metabolic phenotype known as "aerobic glycolysis," which is characterized by an elevated rate of glucose fermentation to lactate irrespective of oxygen availability. Although several theories have been proposed, a rationalization for why proliferating cells seemingly waste glucose carbon by excreting it as lactate remains elusive. Using the NCI-60 cell lines, we determined that lactate excretion is strongly correlated with the activity of mitochondrial NADH shuttles, but not proliferation. Quantifying the fluxes of the malate-aspartate shuttle (MAS), the glycerol 3-phosphate shuttle (G3PS), and lactate dehydrogenase under various conditions demonstrated that proliferating cells primarily transform glucose to lactate when glycolysis outpaces the mitochondrial NADH shuttles. Increasing mitochondrial NADH shuttle fluxes decreased glucose fermentation but did not reduce the proliferation rate. Our results reveal that glucose fermentation, a hallmark of cancer, is a secondary consequence of MAS and G3PS saturation rather than a unique metabolic driver of cellular proliferation.
    Keywords:  NADH shuttles; aerobic glycolysis; cancer metabolism; glycerol 3-phosphate shuttle; isotope-tracer analysis; malate-aspartate shuttle; metabolic flux; metabolomics; the Warburg effect
  5. Cell Stress Chaperones. 2022 Aug 17.
      The molecular chaperone TNF-receptor-associated protein-1 (TRAP1) controls mitochondrial respiration through regulation of Krebs cycle and electron transport chain activity. Post-translational modification (PTM) of TRAP1 regulates its activity, thereby controlling global metabolic flux. O-GlcNAcylation is one PTM that is known to impact mitochondrial metabolism, however the major effectors of this regulatory PTM remain inadequately resolved. Here we demonstrate that TRAP1-O-GlcNAcylation decreases TRAP1 ATPase activity, leading to increased mitochondrial metabolism. O-GlcNAcylation of TRAP1 occurs following mitochondrial import and provides critical regulatory feedback, as the impact of O-GlcNAcylation on mitochondrial metabolism shows TRAP1-dependence. Mechanistically, loss of TRAP1-O-GlcNAcylation decreased TRAP1 binding to ATP, and interaction with its client protein succinate dehydrogenase (SDHB). Taken together, TRAP1-O-GlcNAcylation serves to regulate mitochondrial metabolism by the reversible attenuation of TRAP1 chaperone activity.
    Keywords:  GlcNAcylation; Metabolism; Molecular chaperone; Post-translational modification; TRAP1
  6. Nat Commun. 2022 Aug 16. 13(1): 4812
      Production of high density lipoprotein (HDL) requires ATP-binding cassette transporter A1 (ABCA1) to drive phospholipid (PL) from the plasma membrane into extracellular apolipoprotein A-I. Here, we use simulations to show that domains of ABCA1 within the plasma membrane remove PL from the membrane's outer leaflet. In our simulations, after the lipid diffuses into the interior of ABCA1's outward-open cavity, PL extracted by the gateway passes through a ring-shaped domain, the annulus orifice, which forms the base of an elongated hydrophobic tunnel in the transporter's extracellular domain. Engineered mutations in the gateway and annulus strongly inhibit lipid export by ABCA1 without affecting cell-surface expression levels. Our finding that ABCA1 extracts lipid from the outer face of the plasma membrane and forces it through its gateway and annulus into an elongated hydrophobic tunnel contrasts with the alternating access model, which proposes that ABCA1 flops PL substrate from the inner leaflet to the outer leaflet of the membrane. Consistent with our model, ABCA1 lacks the charged amino acid residues in the transmembrane domain found in the floppase members of the ABC transporter family.
  7. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01010-5. [Epub ahead of print]40(7): 111193
      Succinate dehydrogenase (SDH) loss-of-function mutations drive succinate accumulation in tumor microenvironments, for example in the neuroendocrine tumors pheochromocytoma (PC) and paraganglioma (PG). Control of innate immune cell activity by succinate is described, but effects on T cells have not been interrogated. Here we report that exposure of human CD4+ and CD8+ T cells to tumor-associated succinate concentrations suppresses degranulation and cytokine secretion, including of the key anti-tumor cytokine interferon-γ (IFN-γ). Mechanistically, this is associated with succinate uptake-partly via the monocarboxylate transporter 1 (MCT1)-inhibition of succinyl coenzyme A synthetase activity and impaired glucose flux through the tricarboxylic acid cycle. Consistently, pharmacological and genetic interventions restoring glucose oxidation rescue T cell function. Tumor RNA-sequencing data from patients with PC and PG reveal profound suppression of IFN-γ-induced genes in SDH-deficient tumors compared with those with other mutations, supporting a role for succinate in modulating the anti-tumor immune response in vivo.
    Keywords:  CP: immunology; CP: metabolism; T cell; metabolism; metabolite; succinate; tumor microenvironment
  8. Blood. 2022 Aug 19. pii: blood.2022017575. [Epub ahead of print]
      Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, NMR-based metabolite quantifications and 13C-tracing, polysomal profiling, and ChIP-seq, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis and induced a cell cycle block. ROS levels were not increased following methionine depletion and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.
  9. Immunity. 2022 Aug 10. pii: S1074-7613(22)00340-5. [Epub ahead of print]
      The risk of chronic diseases caused by aging is reduced by caloric restriction (CR)-induced immunometabolic adaptation. Here, we found that the matricellular protein, secreted protein acidic and rich in cysteine (SPARC), was inhibited by 2 years of 14% sustained CR in humans and elevated by obesity. SPARC converted anti-inflammatory macrophages into a pro-inflammatory phenotype with induction of interferon-stimulated gene (ISG) expression via the transcription factors IRF3/7. Mechanistically, SPARC-induced ISGs were dependent on toll-like receptor-4 (TLR4)-mediated TBK1, IRF3, IFN-β, and STAT1 signaling without engaging the Myd88 pathway. Metabolically, SPARC dampened mitochondrial respiration, and inhibition of glycolysis abrogated ISG induction by SPARC in macrophages. Furthermore, the N-terminal acidic domain of SPARC was required for ISG induction, while adipocyte-specific deletion of SPARC reduced inflammation and extended health span during aging. Collectively, SPARC, a CR-mimetic adipokine, is an immunometabolic checkpoint of inflammation and interferon response that may be targeted to delay age-related metabolic and functional decline.
    Keywords:  SPARC; TLR4; caloric restriction; inflammation; interferon-stimulated gene; macrophage; matricellular protein
  10. Biochem Biophys Rep. 2022 Sep;31 101317
      Macrophages play a major role in the immune defense against pathogenic factors; however, they can lead to tumor exacerbation and metastasis, as the tumor microenvironment (TME) polarizes tumor-associated macrophages (TAMs) into the M2 subtype. Lactate, a metabolite produced by carcinoma cells at high concentrations in the TME, induces an M2-polarization in macrophages, which ultimately leads to the secretion of factors, such as vascular endothelial growth factor (VEGF), and promotes tumor progression. However, the effect of TAM lactate import on tumor progression has not been fully elucidated. Aquaporin 9 (AQP9) is a transporter of water and glycerol expressed in macrophages. Here, we used a tumor allograft mouse model to show that AQP9 knockout (AQP9-/-) mice were more resistant against tumor cell growth and exhibited a suppressive M2-like polarization in tumor tissue than wild-type mice. Moreover, we discovered that the primary bone marrow-derived macrophages from AQP9-/- mice were less sensitive to lactate stimulation and exhibited reduced M2-like polarization as well as decreased VEGF production. To further investigate the role of AQP9 in macrophage polarization, we overexpressed AQP9 in Chinese hamster ovary cells and found that AQP9 functioned in lactate import. In contrast, primary AQP9-/- macrophages and AQP9 knockdown RAW264.7 cells exhibited a reduced lactate transport rate, suggesting the involvement of AQP9 in lactate transport in macrophages. Together, our results reveal the mechanism by which the TME modifies the polarization and function of tumor-infiltrating macrophages via AQP9 transport function.
    Keywords:  AQP9; Colon cancer; Lactate; Tumor microenvironment; Tumor-associated macrophages
  11. Cell Death Differ. 2022 Aug 16.
      Ferroptosis is a unique type of cell death that is hallmarked with the imbalanced redox homeostasis as triggered by iron-dependent lipid peroxidation. Cysteines often play critical roles in proteins to help maintain a healthy cellular environment by dynamically switching between their reduced and oxidized forms, however, how the global redox landscape of cysteinome is perturbed upon ferroptosis remains unknown to date. By using a quantitative chemical proteomic strategy, we systematically profiled the dynamic changes of cysteinome in ferroptotic cells and identified a list of candidate sites whose redox states are precisely regulated under ferroptosis-inducing and rescuing conditions. In particular, C106 of the protein/nucleic acid deglycase DJ-1 acts as an intriguing sensor switch for the ferroptotic condition, whose oxidation results in the disruption of its interaction with the 20S proteasome and leads to a marked activation in the proteasome system. Our chemoproteomic profiling and associated functional studies reveal a novel functional link between ferroptosis and the proteasome-mediated protein degradation. It also suggests proteasome as a promising target for developing treatment strategies for ferroptosis-related diseases.
  12. J Cell Physiol. 2022 Aug 20.
      Lipid droplets (LDs) are important cellular organelles due to their ability to accumulate and store lipids. LD dynamics are associated with various cellular and metabolic processes. Accurate monitoring of LD's size and shape is of prime importance as it indicates the metabolic status of the cells. Unintrusive continuous quantification techniques have a clear advantage in analyzing LDs as they measure and monitor the cells' metabolic function and droplets over time. Here, we present a novel machine-learning-based method for LDs analysis by segmentation of phase-contrast images of differentiated adipocytes (in vitro) and adipose tissue (in vivo). We developed a new workflow based on the ImageJ waikato environment for knowledge analysis segmentation plugin, which provides an accurate, label-free, live single-cell, and organelle quantification of LD-related parameters. By applying the new method on differentiating 3T3-L1 cells, the size of LDs was analyzed over time in differentiated adipocytes and their correlation with other morphological parameters. Moreover, we analyzed the LDs dynamics during catabolic changes such as lipolysis and lipophagy and demonstrated its ability to identify different cellular subpopulations based on their structural, numerical, and spatial variability. This analysis was also implemented on unstained ex vivo adipose tissues to measure adipocyte size, an important readout of the tissue's metabolism. The presented approach can be applied in different LD-related metabolic conditions to provide a better understanding of LD biogenesis and function in vivo and in vitro while serving as a new platform that enables rapid and accurate screening of data sets.
    Keywords:  adipocyte metabolism; adipose tissue; lipid droplets; lipolysis
  13. Nanoscale. 2022 Aug 16.
      Dense stroma and an immunosuppressive microenvironment severely hamper the antitumor therapeutic results of pancreatic cancer. Tumor-associated macrophages (TAMs) support the proliferation and invasion of tumor cells and contribute to the information of the immunosuppressive tumor microenvironment (TME). The repolarization of TAMs activates the antitumor immune response and sensitizes chemotherapy. Nevertheless, the difference in distributed mode between TAMs and tumor cells in tumor turns out to be an obstacle for dual targeting. To repolarize TAMs and elevate the chemoimmunotherapy outcome against pancreatic cancer, co-loading the TME responsive micellar system with gemcitabine (GEM) and PI3K inhibitor wortmannin (Wtmn) was used to dual target TAMs and tumor cells. GEM conjugated dendritic poly-lysine DGL (GD) nanoparticles were linked to polycaprolactone-polyethylene glycol micelles encapsulated with Wtmn (PP/Wtmn) via a cathepsin B (CTSB) substrate peptide to obtain raspberry-like GD@PP/Wtmn micelles. Upon arrival at the TME, GD was released in response to highly expressed CTSB, allowing deep penetration of the tumor and overcoming of the stromal barrier, while PP/Wtmn remained in the perivascular area where TAMs abundantly resided. By inhibiting the PI3K pathway, the M2-like TAMs were repolarized into M1-like TAMs and then activated antitumor immunity, further synergizing with GEM to suppress tumor growth. This tumor and TAMs dual targeting nanoplatform provides an alternative approach to sensitize chemoimmunotherapy against pancreatic cancer.
  14. J Agric Food Chem. 2022 Aug 15.
      Amino acid sensing plays an important role in regulating lipid metabolism by sensing amino acid nutrient disturbance. T1R1 (umami taste receptor, type 1, member 1) is a membrane G protein-coupled receptor that senses amino acids. Tas1r1-knockout (KO) mice were used to explore the function of umami receptors in lipid metabolism. Compared with wild-type (WT) mice, Tas1r1-KO mice showed decreased fat mass (P < 0.05) and adipocyte size, lower liver triglyceride (7.835 ± 0.809 vs 12.463 ± 0.916 mg/g WT, P = 0.013) and total cholesterol levels (0.542 ± 0.109 vs 1.472 ± 0.044 mmol/g WT, P < 0.001), and reduced lipogenesis gene expressions in adipose and liver tissues. Targeted liver amino acid metabolomics showed that the amino acid content of Tas1r1-KO mice was significantly decreased, which was consistent with the branched-chain ketoacid dehydrogenase protein levels. Proteomics analysis showed that the upregulated proteins were enriched in lipid and steroid metabolism pathways, and parallel reaction monitoring results illustrated that Tas1r1 ablation promoted lipid catabolism through oxysterol 7 α-hydroxylase and insulin-like growth factor binding protein 2. In summary, Tas1r1 disruption in mice could reduce lipid accumulation by reducing de novo lipid synthesis and improving lipid catabolism.
    Keywords:  Tas1r1; amino acid receptor; de novo lipid synthesis; lipid catabolism; liver
  15. BMC Med. 2022 Aug 18. 20(1): 255
      BACKGROUND: Periprostatic adipose tissue (PPAT) plays a role in prostate cancer (PCa) progression. PPAT lipidomic composition study may allow us to understand the tumor metabolic microenvironment and provide new stratification factors.METHODS: We used ultra-high-performance liquid chromatography-mass spectrometry-based non-targeted lipidomics to profile lipids in the PPAT of 40 patients with PCa (n = 20 with low-risk and n = 20 high-risk). Partial least squares-discriminant analysis (PLS-DA) and variable importance in projection (VIP) analysis were used to identify the most relevant features of PPAT between low- and high-risk PCa, and metabolite set enrichment analysis was used to detect disrupted metabolic pathways. Metabolic crosstalk between PPAT and PCa cell lines (PC-3 and LNCaP) was studied using ex vivo experiments. Lipid uptake and lipid accumulation were measured. Lipid metabolic-related genes (SREBP1, FASN, ACACA, LIPE, PPARG, CD36, PNPLA2, FABP4, CPT1A, FATP5, ADIPOQ), inflammatory markers (IL-6, IL-1B, TNFα), and tumor-related markers (ESRRA, MMP-9, TWIST1) were measured by RT-qPCR.
    RESULTS: Significant differences in the content of 67 lipid species were identified in PPAT samples between high- and low-risk PCa. PLS-DA and VIP analyses revealed a discriminating lipidomic panel between low- and high-risk PCa, suggesting the occurrence of disordered lipid metabolism in patients related to PCa aggressiveness. Functional analysis revealed that alterations in fatty acid biosynthesis, linoleic acid metabolism, and β-oxidation of very long-chain fatty acids had the greatest impact in the PPAT lipidome. Gene analyses of PPAT samples demonstrated that the expression of genes associated with de novo fatty acid synthesis such as FASN and ACACA were significantly lower in PPAT from high-risk PCa than in low-risk counterparts. This was accompanied by the overexpression of inflammatory markers (IL-6, IL-1B, and TNFα). Co-culture of PPAT explants with PCa cell lines revealed a reduced gene expression of lipid metabolic-related genes (CD36, FASN, PPARG, and CPT1A), contrary to that observed in co-cultured PCa cell lines. This was followed by an increase in lipid uptake and lipid accumulation in PCa cells. Tumor-related genes were increased in co-cultured PCa cell lines.
    CONCLUSIONS: Disturbances in PPAT lipid metabolism of patients with high-risk PCa are associated with tumor cell metabolic changes.
    Keywords:  De novo fatty acid synthesis; Lipid metabolism; Lipidomic; Periprostatic adipose tissue; Prostate cancer
  16. Immunometabolism (Cobham). 2022 Jul;4(3): e00002
      The awareness that polyamines play a critical role in immune system regulation and function is coming into focus as the biological systems and analytical tools necessary to evaluate their roles have become available. Puleston et al have recently demonstrated that polyamine metabolism plays a central role in helper T-cell lineage determination through the production of the translational cofactor hypusinated eIF5A and faithful epigenetic regulation through proper histone acetylation. Their findings add to the rapidly growing body of data implicating properly controlled polyamine metabolism as essential for a normally functioning immune system.
    Keywords:  T cells; histone acetylation; hypusine; immune; polyamines; spermidine; spermine
  17. Nitric Oxide. 2022 Aug 13. pii: S1089-8603(22)00083-0. [Epub ahead of print]128 12-24
      Epigallocatechin gallate (EGCG) is the main bioactive component of green tea. Through screening of a small library of natural compounds, we discovered that EGCG inhibits cystathionine β-synthase (CBS), a major H2S-generating enzyme. Here we characterize EGCG's mechanism of action in the context of CBS-derived H2S production. In the current project, biochemical, pharmacological and cell biology approaches were used to characterize the effect of EGCG on CBS in cellular models of cancer and Down syndrome (DS). The results show that EGCG binds to CBS and inhibits H2S-producing CBS activity almost 30-times more efficiently than the canonical cystathionine formation (IC50 0.12 versus 3.3 μM). Through screening structural analogs and building blocks, we identified that gallate moiety of EGCG represents the pharmacophore responsible for CBS inhibition. EGCG is a mixed-mode, CBS-specific inhibitor with no effect on the other two major enzymatic sources of H2S, CSE and 3-MST. Unlike the prototypical CBS inhibitor aminooxyacetate, EGCG does not bind the catalytic cofactor of CBS pyridoxal-5'-phosphate. Molecular modeling suggests that EGCG blocks a substrate access channel to pyridoxal-5'-phosphate. EGCG inhibits cellular H2S production in HCT-116 colon cancer cells and in DS fibroblasts. It also exerts effects that are consistent with the functional role of CBS in these cells: in HCT-116 cells it decreases, while in DS cells it improves viability and proliferation. In conclusion, EGCG is a potent inhibitor of CBS-derived H2S production. This effect may contribute to its pharmacological effects in various pathophysiological conditions.
    Keywords:  Cancer; Down syndrome; Green tea; Hydrogen sulfide; Medicinal chemistry; Pyridoxal phosphate
  18. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01035-X. [Epub ahead of print]40(7): 111218
      Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.
    Keywords:  AMPK; CP: Cancer; CP: Metabolism; Cdk5; PRKAG2; SDHB; Warburg effect; cancer bioenergetics; neuroendocrine tumor; p53; pheochromocytoma; senescence
  19. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01018-X. [Epub ahead of print]40(7): 111201
      Stimulatory type 1 conventional dendritic cells (cDC1s) engage in productive interactions with CD8+ effectors along tumor-stroma boundaries. The paradoxical accumulation of "poised" cDC1s within stromal sheets is unlikely to simply reflect passive exclusion from tumor cores. Drawing parallels with embryonic morphogenesis, we hypothesized that invasive margin stromal remodeling generates developmentally conserved cell fate cues that regulate cDC1 behavior. We find that, in human T cell-inflamed tumors, CD8+ T cells penetrate tumor nests, whereas cDC1s are confined within adjacent stroma that recurrently displays site-specific proteolysis of the matrix proteoglycan versican (VCAN), an essential organ-sculpting modification in development. VCAN is necessary, and its proteolytic fragment (matrikine) versikine is sufficient for cDC1 accumulation. Versikine does not influence tumor-seeding pre-DC differentiation; rather, it orchestrates a distinctive cDC1 activation program conferring exquisite sensitivity to DNA sensing, supported by atypical innate lymphoid cells. Thus, peritumoral stroma mimicking embryonic provisional matrix remodeling regulates cDC1 abundance and activity to elicit T cell-inflamed tumor microenvironments.
    Keywords:  CD40; CP: Cancer; CP: Immunology; cDC1; checkpoint inhibitors; dendritic cells; immunotherapy; proteoglycans; tumor matrix; tumor stroma; versican; versikine
  20. Antioxid Redox Signal. 2022 Aug 18.
      Aims: Oxidative modifications of cysteine thiols regulate various physiological processes including inflammatory responses. The thioredoxin system plays a key role in thiol redox control. The aim of this study was to characterize the dynamic cysteine proteome of human macrophages upon activation by the prototypical pro-inflammatory agent, bacterial lipopolysaccharide (LPS), and/or perturbation of the thioredoxin system. Results: Herein, we profiled the cellular and redox proteome of human THP-1-derived macrophages during the early phase of LPS activation and/or inhibition of thioredoxin system activity by auranofin, by employing a peptide-centric, resin-assisted capture, redox proteomic workflow. Among 4200 identified cysteines, the oxidation of nearly 10% was selectively affected by LPS or auranofin treatments. Notably, the proteomic analysis uncovered a subset of ~100 thiols, mapped to proteins involved in diverse processes, whose oxidation is antagonistically regulated by LPS and thioredoxin. Compared to the redox proteome, the cellular proteome was largely unchanged, highlighting the importance of redox modifications as a mechanism that allows for rapid modulation of macrophage activities in response to a pro-inflammatory or a pro-oxidant insult. Structural-functional analyses provided mechanistic insights into redox regulation of selected proteins, including the glutathione synthesizing enzyme, glutamate cysteine ligase and the autophagy adaptor SQSTM1/p62, suggesting mechanisms by which macrophages adapt and fine-tune their responses according to a changing inflammatory and redox environment. Innovation: This study provides a rich resource for further characterization of redox mechanisms that regulate macrophage inflammatory activities. Conclusion: The dynamic thiol redox proteome allows macrophages to efficiently respond and adapt to redox and inflammatory challenges.
  21. Biochem Biophys Res Commun. 2022 Jul 03. pii: S0006-291X(22)00888-9. [Epub ahead of print]626 8-14
      Colorectal cancer (CRC) is a lethal malignant tumor and 25-30% of CRC patients develop liver metastasis (LM) with a worse prognosis, but the metastasis mechanism is yet elucidated. To identify the potential immune regulatory mechanism of CRC liver metastasis, single-cell sequencing and multiplex immunohistochemistry were applied to identify key cell populations of the tumor microenvironment (TME) in the CRC and LM sites. We found memory CD8+ T cells, B cells, and CTSB + macrophages were enriched in the LM site, forming the memory immune hub, which was important for the anti-tumor response against LM. Therefore, our results revealed that memory immune responses were called in the LM sites and probably meditated by CTSB + macrophages.
    Keywords:  CTSB+ macrophage; Colorectal cancer; Liver metastasis; Multi-omics analysis; Suppressive immune hub
  22. Dis Model Mech. 2022 Aug 01. pii: dmm049550. [Epub ahead of print]15(8):
      An integrated evaluation of the tissue distribution and pharmacodynamic properties of a therapeutic is essential for successful translation to the clinic. To date, however, cost-effective methods to measure these parameters at the systems level in model organisms are lacking. Here, we introduce a multidimensional workflow to evaluate drug activity that combines mass spectrometry-based imaging, absolute drug quantitation across different biological matrices, in vivo isotope tracing and global metabolome analysis in the adult zebrafish. As a proof of concept, we quantitatively determined the whole-body distribution of the anti-rheumatic agent hydroxychloroquine sulfate (HCQ) and measured the systemic metabolic impacts of drug treatment. We found that HCQ distributed to most organs in the adult zebrafish 24 h after addition of the drug to water, with the highest accumulation of both the drug and its metabolites being in the liver, intestine and kidney. Interestingly, HCQ treatment induced organ-specific alterations in metabolism. In the brain, for example, HCQ uniquely elevated pyruvate carboxylase activity to support increased synthesis of the neuronal metabolite, N-acetylaspartate. Taken together, this work validates a multidimensional metabolomics platform for evaluating the mode of action of a drug and its potential off-target effects in the adult zebrafish. This article has an associated First Person interview with the first author of the paper.
    Keywords:  Drug discovery; Mass spectrometry imaging; Metabolomics; Pharmacodynamics; Zebrafish
  23. J Exp Clin Cancer Res. 2022 Aug 19. 41(1): 253
      BACKGROUND: Tumor-associated macrophages (TAMs), which form a large part of the tumor microenvironment, are normally regulated by metabolic reprogramming. However, the potential mechanisms of the immune-metabolism interaction between hepatocellular carcinoma (HCC) cells and TAMs remain unclear.METHODS: The candidate long non-coding RNAs (lncRNAs) were screened by Smart-seq based scRNA-seq method and then validated by qPCR. Immunostaining analysis was done to examine the levels of markers for TAMs and glycolysis. Exosomes from primary TAMs of human HCC tissues were isolated by centrifugation, and their internalization with lncRNAs was confirmed by immunofluorescence. The underlying mechanism of TAMs-derived exosomal lncRNA to HCC was confirmed by luciferase reporter assay and RNA immunoprecipitation. Metabolism regulation was evaluated through glucose consumption, lactate productions and extracellular acidification rates (ECARs). Mouse xenograft models were used to elucidate the in vivo effect of candidate lncRNAs on tumor growth.
    RESULTS: TAMs augment the aerobic glycolysis in HCC cells and their proliferation by the extracellular exosome transmission of a myeloid-derived lncRNA, M2 macrophage polarization associated lncRNA (lncMMPA). Mechanistically, lncMMPA not only could polarize M2 macrophage, but also could act as an microRNA sponge to interact with miR-548 s and increase the mRNA level of ALDH1A3, then further promote glucose metabolism and cell proliferation in HCC. Moreover, lncMMPA increased HCC cell multiplication through interacting with miR-548 s in vivo. Clinically, lncMMPA expression associates with glycolysis in TAMs and reduced survival of HCC patients.
    CONCLUSION: LncMMPA plays an important role in regulating HCC malignancy and metabolic reprogramming of miR-548 s/ALDH1A3 pathway.
    Keywords:  Hepatocellular carcinoma; LncMMPA; Metabolic reprogramming; Tumor associated macrophage
  24. Oncogene. 2022 Aug 13.
      Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.
  25. Physiol Rep. 2022 Aug;10(16): e15422
      Ketone bodies (KB) serve as the food for mitochondrial biogenetics. Interestingly, probiotics are known to promote KB formation in the gut (especially those that belong to the Lactobacillus genus). Furthermore, Lactobacillus helps produce folate that lowers the levels of homocysteine (Hcy); a hallmark non-proteinogenic amino acid that defines the importance of epigenetics, and its landscape. In this study, we decided to test whether hydrogen sulfide (H2 S), another Hcy lowering agent regulates the epigenetic gene writer DNA methyltransferase (DNMT), eraser FTO and TET2, and thus mitigates the skeletal muscle remodeling. We treated hyperhomocysteinemic (HHcy, cystathionine beta-synthase heterozygote knockout; CBS+/- ) mice with NaHS (the H2 S donor). The results suggested multi-organ damage by HHcy in the CBS+/- mouse strain compared with WT control mice (CBS+/+ ). H2 S treatment abrogated most of the HHcy-induced damage. The levels of gene writer (DNMT2) and H3K9 (methylation) were higher in the CBS+/- mice, and the H2 S treatment normalized their levels. More importantly, the levels of eraser FTO, TET, and associated GADD45, and MMP-13 were decreased in the CBS+/- mice; however, H2 S treatment mitigated their respective decrease. These events were associated with mitochondrial fission, i.e., an increase in DRP1, and mitophagy. Although the MMP-2 level was lower in CBS+/- compared to WT but H2 S could further lower it in the CBS+/- mice. The MMPs levels were associated with an increase in interstitial fibrosis in the CBS+/- skeletal muscle. Due to fibrosis, the femoral artery blood flow was reduced in the CBS+/- mice, and that was normalized by H2 S. The bone and muscle strengths were found to be decreased in the CBS+/- mice but the H2 S treatment normalized skeletal muscle strength in the CBS+/- mice. Our findings suggest that H2 S mitigates the mitophagy-led skeletal muscle remodeling via epigenetic regulation of the gene writer and eraser function.
    Keywords:  1-carbon metabolism; cystathionine β synthase; homocysteine; mitochondria
  26. J Biol Chem. 2022 Aug 10. pii: S0021-9258(22)00806-7. [Epub ahead of print] 102363
      Inositol is an essential metabolite that serves as a precursor for structural and signaling molecules. Although perturbation of inositol homeostasis has been implicated in numerous human disorders, surprisingly little is known about how inositol levels are regulated in mammalian cells. A recent study in mouse embryonic fibroblasts (MEFs) demonstrated that nuclear translocation of inositol hexakisphosphate kinase 1 (IP6K1) mediates repression of myo-inositol-3-P synthase (MIPS), the rate-limiting inositol biosynthetic enzyme. Binding of IP6K1 to phosphatidic acid (PA) is required for this repression. Here, we elucidate the role of PA in IP6K1 repression. Our results indicate that increasing PA levels through pharmacological stimulation of phospholipase D (PLD) or direct supplementation of 18:1 PA induces nuclear translocation of IP6K1 and represses expression of the MIPS protein. We found that this effect was specific to PA synthesized in the plasma membrane, as ER-derived PA did not induce IP6K1 translocation. Furthermore, we determined that PLD-mediated PA synthesis can be stimulated by the master metabolic regulator 5' AMP-activated protein kinase (AMPK). We show that activation of AMPK by glucose deprivation or by treatment with the mood-stabilizing drugs valproate (VPA) or lithium recapitulated IP6K1 nuclear translocation and decreased MIPS expression. This study demonstrates for the first time that modulation of PA levels through the AMPK-PLD pathway regulates IP6K1-mediated repression of MIPS.
    Keywords:  AMPK; Glucose; IP6K1; Inositol; Inositol phosphate; Lithium; MIPS; Phosphatidic acid; Phospholipase D; Valproate
  27. Mol Cell Endocrinol. 2022 Aug 15. pii: S0303-7207(22)00201-5. [Epub ahead of print] 111753
      Accumulation of excess lipids in non-adipose tissues, such as the hypothalamus, is termed lipotoxicity and causative of free fatty acid-mediated pathology in metabolic disease. This study aimed to elucidate the molecular mechanisms behind oleate (OA)- and palmitate (PA)-mediated changes in hypothalamic neurons. Using the well-characterized hypothalamic neuronal cell model, mHypoE-46, we assessed gene changes through qRT-PCR, cell death with quantitative imaging, PA metabolism using stable isotope labeling, and cellular mechanisms using pharmacological modulation of lipid metabolism and autophagic flux. Palmitate (PA) disrupts gene expression, including Npy, Grp78, and Il-6 mRNA in mHypoE-46 hypothalamic neurons. Blocking PA metabolism using triacsin-C prevented the increase of these genes, implying that these changes depend on PA intracellular metabolism. Co-incubation with oleate (OA) is also potently protective and prevents cell death induced by increasing concentrations of PA. However, OA does not decrease 13C-PA incorporation into diacylglycerol and phospholipids. Remarkably, OA can reverse PA toxicity even after significant PA metabolism and cellular impairment. OA can restore PA-mediated impairment of autophagy to prevent or reverse the accumulation of PA metabolites through lysosomal degradation, and not through other reported mechanisms. The autophagic flux inhibitor chloroquine mimics PA toxicity by upregulating autophagy-related genes, Npy, Grp78, and Il-6, an effect partially reversed by OA. Chloroquine also prevented the OA defense against PA toxicity, whereas the autophagy inducer rapamycin provided some protection. Thus, PA impairment of autophagic flux significantly contributes to its lipotoxicity, and OA-mediated protection requires functional autophagy. Overall, our results suggest that impairment of autophagy contributes to hypothalamic lipotoxicity.
    Keywords:  Autophagy; Hypothalamus; Lipid metabolism; Lipotoxicity; Oleate; Palmitate
  28. Immunometabolism (Cobham). 2022 Jul;4(3): e00007
      The endoplasmic reticulum (ER) is a specialized organelle that participates in multiple cellular functions including protein folding, maturation, trafficking, and degradation to maintain homeostasis. However, hostile conditions in the tumor microenvironment (TME) disturb ER homeostasis. To overcome these conditions, cells activate ER stress response pathways, which are shown to augment the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process remain elusive. Here, we discuss a recent study by Raines et al, that suggests the role of the helper T-cell 2 (TH2) cytokine interleukin-4 (IL-4), and the TME in facilitating a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages, which promotes immunosuppressive M2 macrophage activation and proliferation. Further, the authors showed that PERK signaling promotes both mitochondrial respirations to fulfill cellular energy requirements and signaling through ATF4, which regulate phosphoserine aminotransferase 1 (PSAT1) activity to mediate the serine biosynthesis pathway. These results highlight a previously uncharacterized role for PERK in cellular metabolism and epigenetic modification in M2 macrophages, and thus offers a new therapeutic strategy for overcoming the immunosuppressive effects in the TME.
    Keywords:  ER stress; M2 macrophage; endoplasmic reticulum; myeloid cell-derived suppressor cells; protein kinase RNA-like ER kinase; tumor-associated macrophages