Itaconate, the product of the decarboxylation of cis-aconitate, regulates numerous biological processes. We and others have revealed itaconate as a regulator of fatty acid β-oxidation, generation of ...mitochondrial reactive oxygen species and the metabolic interplay between resident macrophages and tumors. In the present study, we show that itaconic acid is upregulated in human non-alcoholic steatohepatitis and a mouse model of non-alcoholic fatty liver disease. Male mice deficient in the gene responsible for itaconate production (immunoresponsive gene (Irg)-1) have exacerbated lipid accumulation in the liver, glucose and insulin intolerance and mesenteric fat deposition. Treatment of mice with the itaconate derivative, 4-octyl itaconate, reverses dyslipidemia associated with high-fat diet feeding. Mechanistically, itaconate treatment of primary hepatocytes reduces lipid accumulation and increases their oxidative phosphorylation in a manner dependent upon fatty acid oxidation. We propose a model whereby macrophage-derived itaconate acts in trans upon hepatocytes to modulate the liver's ability to metabolize fatty acids.
ATP citrate lyase (ACLY) is the predominant nucleocytosolic source of acetyl-CoA and is aberrantly regulated in many diseases making it an attractive therapeutic target. Structural studies of ACLY ...reveal a central homotetrameric core citrate synthase homology (CSH) module flanked by acyl-CoA synthetase homology (ASH) domains, with ATP and citrate binding the ASH domain and CoA binding the ASH-CSH interface to produce acetyl-CoA and oxaloacetate products. The specific catalytic role of the CSH module and an essential D1026A residue contained within it has been a matter of debate. Here, we report biochemical and structural analysis of an ACLY-D1026A mutant demonstrating that this mutant traps a (3S)-citryl-CoA intermediate in the ASH domain in a configuration that is incompatible with the formation of acetyl-CoA, is able to convert acetyl-CoA and OAA to (3S)-citryl-CoA in the ASH domain, and can load CoA and unload acetyl-CoA in the CSH module. Together, this data support an allosteric role for the CSH module in ACLY catalysis.
Few therapies have produced significant improvement in cardiac structure and function after ischemic cardiac injury (ICI). Our possible explanation is activation of local inflammatory responses ...negatively impact the cardiac repair process following ischemic injury. Factors that can alter immune response, including significantly altered cytokine levels in plasma and polarization of macrophages and T cells towards a pro-reparative phenotype in the myocardium post-MI is a valid strategy for reducing infarct size and damage after myocardial injury.
Our previous studies showed that cortical bone stem cells (CBSCs) possess reparative effects after ICI. In our current study, we have identified that the beneficial effects of CBSCs appear to be mediated by miRNA in their extracellular vesicles (CBSC-EV). Our studies showed that CBSC-EV treated animals demonstrated reduced scar size, attenuated structural remodeling, and improved cardiac function versus saline treated animals. These effects were linked to the alteration of immune response, with significantly altered cytokine levels in plasma, and polarization of macrophages and T cells towards a pro-reparative phenotype in the myocardium post-MI. Our detailed in vitro studies demonstrated that CBSC-EV are enriched in miR-182/183 that mediates the pro-reparative polarization and metabolic reprogramming in macrophages, including enhanced OXPHOS rate and reduced ROS, via Ras p21 protein activator 1 (RASA1) axis under Lipopolysaccharides (LPS) stimulation. In summary, CBSC-EV deliver unique molecular cargoes, such as enriched miR-182/183, that modulate the immune response after ICI by regulating macrophage polarization and metabolic reprogramming to enhance repair.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Chronic, systemic inflammation is a pathophysiological manifestation of metabolic disorders. Inflammatory signaling leads to elevated glycolytic flux and a metabolic shift towards aerobic glycolysis ...and lactate generation. This rise in lactate corresponds with increased generation of lactoylLys modifications on histones, mediating transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Here, we report a regulatory role for LGSH in mediating histone lactoylation and inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH and histone lactoylation with a corresponding potentiation of the inflammatory response when exposed to lipopolysaccharides. An analysis of chromatin accessibility shows that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state; upon stimulation, however, regions of the genome associated with lactoylation become markedly more accessible. Lastly, we demonstrate a spontaneous S-to-S acyltransfer of lactate from LGSH to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGSH, and not intracellular lactate, is the primary driving factor facilitating histone lactoylation and a major contributor to inflammatory signaling.
•Ablation of the lactoylglutathione hydrolase, GLO2, exacerbates inflammatory signaling in RAW 264.7 macrophages.•Lactoyl-CoA is generated non-enzymatically from LGSH via an S-to-S acyl transfer.•Chromatin accessibility is regulated through histone lactoylation and a putative lactoylLysine reader.•Development of a mass spectrometry method to quantify site-specific lactoylation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract only Introduction: Myocardial Infarction (MI) triggers an inflammatory response associated with cardiac wound healing. T-regulatory cells (Tregs) play a crucial role in the MI heart during ...this inflammatory response via their regulation of cardiac repair and function. Stimulating and sustaining the pro-reparative T cell response after MI would aid in preserving cardiac structure and function. Cortical Bone derived Stem Cells (CBSCs) possess a diverse array of inflammatory signaling molecules that can influence the inflammatory microenvironment of the heart after MI. A paracrine factor, Osteoprotegerin (OPG), is abundantly present in CBSCs, but it is unknown if OPG can mediate Tregs in myocardial injury. Hypothesis: CBSCs-derived Osteoprotegerin can regulate cardiac wound healing post-MI by modulating T cell response. Methods and Results: T cells exposed to CBSC secretome underwent an upregulation of a Treg population (p≤0.0001) characterized by the expression of Tumor Necrosis Factor Receptor II (TNFRII). Further investigation into the CBSC secretome highlighted OPG as a potent signaling molecule responsible for inducing TNFRII+ Tregs (p≤0.0001) measured by FACS analysis. siRNA-mediated depletion of OPG studies showed compromised TNFRII+ Treg induction (p≤0.0001). In vivo studies, adult FoxP3-GFP (12-16 week) mice underwent left anterior descending artery ligation followed by intramyocardial injection of CBSCs, Mesenchymal Stem Cells (MSCs), or vehicle (saline) along the infarct border zone. Resident T-lymphocyte populations were assessed at 1- and 8-weeks post-MI via flow cytometry. CBSC-treated hearts yielded a greater prevalence of TNFRII+ Tregs at 1- and 8-weeks following MI compared to MSCs (p≤0.01; p≤0.0001) and vehicle (p≤0.0001; p≤0.05). CBSC induction following MI reduced infarct size compared to the vehicle (p≤0.05). Ablation of the Treg population in the ischemic zone following MI increased infarct size and solicited adverse cardiac remodeling (p≤0.05). Conclusion: Osteoprotegerin can promote cardiac wound healing by facilitating the expansion and preservation of pro-reparative TNFRII+ Tregs.
Cancer cells rewire metabolism to favour the generation of specialized metabolites that support tumour growth and reshape the tumour microenvironment
. Lysine functions as a biosynthetic molecule, ...energy source and antioxidant
, but little is known about its pathological role in cancer. Here we show that glioblastoma stem cells (GSCs) reprogram lysine catabolism through the upregulation of lysine transporter SLC7A2 and crotonyl-coenzyme A (crotonyl-CoA)-producing enzyme glutaryl-CoA dehydrogenase (GCDH) with downregulation of the crotonyl-CoA hydratase enoyl-CoA hydratase short chain 1 (ECHS1), leading to accumulation of intracellular crotonyl-CoA and histone H4 lysine crotonylation. A reduction in histone lysine crotonylation by either genetic manipulation or lysine restriction impaired tumour growth. In the nucleus, GCDH interacts with the crotonyltransferase CBP to promote histone lysine crotonylation. Loss of histone lysine crotonylation promotes immunogenic cytosolic double-stranded RNA (dsRNA) and dsDNA generation through enhanced H3K27ac, which stimulates the RNA sensor MDA5 and DNA sensor cyclic GMP-AMP synthase (cGAS) to boost type I interferon signalling, leading to compromised GSC tumorigenic potential and elevated CD8
T cell infiltration. A lysine-restricted diet synergized with MYC inhibition or anti-PD-1 therapy to slow tumour growth. Collectively, GSCs co-opt lysine uptake and degradation to shunt the production of crotonyl-CoA, remodelling the chromatin landscape to evade interferon-induced intrinsic effects on GSC maintenance and extrinsic effects on immune response.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Abstract
Itaconate, the product of the decarboxylation of cis-aconitate, regulates numerous biological processes. We and others have revealed itaconate as a regulator of fatty acid beta-oxidation, ...generation of mitochondrial reactive oxygen species and the metabolic interplay between resident macrophages and tumors. In the present study, we show that itaconic acid is upregulated in human non-alcoholic steatohepatitis and a mouse model of non-alcoholic fatty liver disease. Mice deficient in the gene responsible for itaconate production (Immunoresponsive gene /Irg-1) have exacerbated lipid accumulation in the liver, glucose and insulin intolerance and mesenteric fat deposition. Treatment of mice with the itaconate derivative, 4-OI, reverses dyslipidemia associated with high fat diet feeding. Mechanistically, itaconate treatment of primary hepatocytes reduces lipid accumulation and increases their oxidative phosphorylation in a manner dependent upon fatty acid oxidation. We propose a model whereby macrophage-derived itaconate acts in trans upon hepatocytes to modulate the liver’s ability to metabolize fatty acids.
This research was supported, in part, by the Intramural Research Program of the NIH, National Cancer Institute, CCR, CIL. RNA sequencing and initial data analysis were conducted at the Frederick National Laboratory for Cancer Research at the CCR Sequencing Facility, NCI Frederick. Human specimens were provided by the Clinical Biospecimen Repository and Processing Core of the Pittsburgh Liver Research Center (supported by National Institutes of Health grant 1P30DK120531). Acyl-CoA analysis was supported by R01GM132261 to NWS.
Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been ...reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The ability of cells to store and rapidly mobilize energy reserves in response to nutrient availability is essential for survival. Breakdown of carbon stores produces acetyl-CoA (AcCoA), which fuels ...essential metabolic pathways and is also the acyl donor for protein lysine acetylation. Histones are abundant and highly acetylated proteins, accounting for 40% to 75% of cellular protein acetylation. Notably, histone acetylation is sensitive to AcCoA availability, and nutrient replete conditions induce a substantial accumulation of acetylation on histones. Deacetylation releases acetate, which can be recycled to AcCoA, suggesting that deacetylation could be mobilized as an AcCoA source to feed downstream metabolic processes under nutrient depletion. While the notion of histones as a metabolic reservoir has been frequently proposed, experimental evidence has been lacking. Therefore, to test this concept directly, we used acetate-dependent, ATP citrate lyase–deficient mouse embryonic fibroblasts (Acly−/− MEFs), and designed a pulse-chase experimental system to trace deacetylation-derived acetate and its incorporation into AcCoA. We found that dynamic protein deacetylation in Acly−/− MEFs contributed carbons to AcCoA and proximal downstream metabolites. However, deacetylation had no significant effect on acyl-CoA pool sizes, and even at maximal acetylation, deacetylation transiently supplied less than 10% of cellular AcCoA. Together, our data reveal that although histone acetylation is dynamic and nutrient-sensitive, its potential for maintaining cellular AcCoA-dependent metabolic pathways is limited compared to cellular demand.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract only
Introduction:
Few therapies have produced significant improvement in cardiac function after ischemic cardiac injury (ICI). The activation of local inflammatory responses is critical to ...cardiac repair after ICI. Our previous studies showed that cortical bone derived stem cells (CBSCs) possess can enhance repair after ICI. Beneficial effects of CBSCs appear to be mediated by paracrine factors including extracellular vesicles (EVs). This study explored if and how these EVs enhance cardiac repair by modulating the ICI immune response.
Hypothesis:
CBSCs derived extracellular vesicles (CBSC-EV) modulate immune response after ICI.
Methods and Results:
Both CBSCs and CBSC-EV were transplanted into mice after myocardial infarction (MI). CBSCs and CBSC-EV treated animals had better ICI repair compared with Saline, with reduced scar size, attenuated structural remodeling, improved cardiac function, and reduced cell apoptosis. These effects were linked to alteration of immune response. Plasma analysis of CBSCs and CBSC-EV treated animals showed significantly lower level of pro-inflammatory cytokines such as TNFα 24 hours after MI. CBSCs and CBSC-EV treatment induced significant polarization from CD86+ M1 macrophages towards CD206+ M2 macrophages phenotype 5 days post-MI, with subsequent reduction of CD8+ T cells and increase of CD4+ T cells, especially the FoxP3+ Treg population, from 7 days to 14 days post-MI. RNA sequencing analysis revealed that CBSC-EV contained a distinct transcriptome compared with endothelial progenitor cells and cardiosphere-derived cells. Gene Ontology analysis suggested the differentially expressed genes in CBSC-EV significantly enriched in immune cell receptor binding. MiR-182 and miR-183, which ranked top of the most significantly upregulated genes in CBSC-EV, attenuated M1 macrophage polarization after LPS treatment and promote CD25+ FoxP3+ Treg differentiation in vitro.
Conclusions:
CBSC-EV enhance cardiac repair by modulating the immune response after injury and highlight the molecular bases of into the beneficial effects of cell-free therapy after ICI.
