Metabolic reprogramming has emerged as a crucial regulator of immune cell activation, but how systemic metabolism influences immune cell metabolism and function remains to be investigated. To ...investigate the effect of dyslipidemia on immune cell metabolism, we performed in-depth transcriptional, metabolic, and functional characterization of macrophages isolated from hypercholesterolemic mice. Systemic metabolic changes in such mice alter cellular macrophage metabolism and attenuate inflammatory macrophage responses. In addition to diminished maximal mitochondrial respiration, hypercholesterolemia reduces the LPS-mediated induction of the pentose phosphate pathway (PPP) and the Nrf2-mediated oxidative stress response. Our observation that suppression of the PPP diminishes LPS-induced cytokine secretion supports the notion that this pathway contributes to inflammatory macrophage responses. Overall, this study reveals that systemic and cellular metabolism are strongly interconnected, together dictating macrophage phenotype and function.
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•Systemic metabolism affects immune cell metabolism•Hypercholesterolemia suppresses the PPP and Nrf2 pathway in macrophages•PPP inhibition and hypercholesterolemia deactivate inflammatory macrophage responses•The Nrf2 pathway regulates the PPP in an LXR-independent manner
The link between systemic and cellular metabolism is a neglected aspect in immunometabolism. Baardman et al. show that hypercholesterolemia alters macrophage metabolism and phenotype. The suppressed pentose phosphate pathway (PPP) in those “foam cell” macrophages attenuates inflammatory responses, signifying that systemic and cellular metabolism together regulate macrophage function.
Activation of brown adipose tissue (BAT) contributes to total body energy expenditure through energy dissipation as heat. Activated BAT increases the clearance of lipids and glucose from the ...circulation, but how BAT accommodates large influx of multiple substrates is not well defined. The purpose of this work was to assess the metabolic fluxes in brown adipocytes during β3-adrenergic receptor (β3-AR) activation.T37i murine preadipocytes were differentiated into brown adipocytes and we used Seahorse respirometry employing a set of specific substrate inhibitors in the presence or absence of β3-AR agonist CL316,243. The main substrate used by these brown adipocytes were fatty acids, which were oxidized equally during activation as well as during resting condition. U-
C-glucose tracer-based metabolomics revealed that the flux through the TCA cycle was enhanced and regulated by pyruvate dehydrogenase (PDH) activity. Based on
C-tracer incorporation in lipids, it appeared that most glucose was oxidized via TCA cycle activity, while some was utilized for glycerol-3-phosphate synthesis to replenish the triglyceride pool. Collectively, we show that while fatty acids are the main substrates for oxidation, glucose is also oxidized to meet the increased energy demand during short term β3-AR activation. PDH plays an important role in directing glucose carbons towards oxidation.
Barth syndrome (BTHS) is a rare disorder caused by mutations in the TAFAZZIN gene. Previous studies from both patients and model systems have established metabolic dysregulation as a core component ...of BTHS pathology. In particular, features such as lactic acidosis, pyruvate dehydrogenase (PDH) deficiency, and aberrant fatty acid and glucose oxidation have been identified. However, the lack of a mechanistic understanding of what causes these conditions in the context of BTHS remains a significant knowledge gap, and this has hindered the development of effective therapeutic strategies for treating the associated metabolic problems. In the current study, we utilized tafazzin-knockout C2C12 mouse myoblasts (TAZ-KO) and cardiac and skeletal muscle tissue from tafazzin-knockout mice to identify an upstream mechanism underlying impaired PDH activity in BTHS. This mechanism centers around robust upregulation of pyruvate dehydrogenase kinase 4 (PDK4), resulting from hyperactivation of AMP-activated protein kinase (AMPK) and subsequent transcriptional upregulation by forkhead box protein O1 (FOXO1). Upregulation of PDK4 in tafazzin-deficient cells causes direct phospho-inhibition of PDH activity accompanied by increased glucose uptake and elevated intracellular glucose concentration. Collectively, our findings provide a novel mechanistic framework whereby impaired tafazzin function ultimately results in robust PDK4 upregulation, leading to impaired PDH activity and likely linked to dysregulated metabolic substrate utilization. This mechanism may underlie previously reported findings of BTHS-associated metabolic dysregulation.
Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin ...(TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome.
•Barth syndrome patient cells were analyzed by proteomic and metabolic profiling.•Complexome profiling revealed effects on 200 different mitochondrial proteins.•Cristae organization and cardiolipin trafficking complexes were prominently changed.•2-Oxoglutarate and branched-chain ketoacid dehydrogenase were destabilized.•Metabolic flux distribution was not reduced.
Carnitine derivatives of disease‐specific acyl‐CoAs are the diagnostic hallmark for long‐chain fatty acid β‐oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very‐long‐chain ...acyl‐CoA dehydrogenase deficiency (VLCADD), long‐chain 3‐hydroxyacyl‐CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO‐intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO‐intermediates and to explore the presence of disease‐specific markers, we used tracer‐based lipidomics with deuterium‐labeled oleic acid (D9‐C18:1) in lcFAOD patient‐derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease‐specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1‐carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S‐(3‐hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3‐hydroxyacyl‐CoAs. S‐(3‐hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease‐specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.
Tracer‐based lipidomics enables the discovery of disease‐specific candidate biomarkers in mitochondrial β‐oxidation disorders.
Transcriptome‐based drug screening is emerging as a powerful tool to identify geroprotective compounds to intervene in age‐related disease. We hypothesized that, by mimicking the transcriptional ...signature of the highly conserved longevity intervention of FOXO3 (daf‐16 in worms) overexpression, we could identify and repurpose compounds with similar downstream effects to increase longevity. Our in silico screen, utilizing the LINCS transcriptome database of genetic and compound interventions, identified several FDA‐approved compounds that activate FOXO downstream targets in mammalian cells. These included the neuromuscular blocker atracurium, which also robustly extends both lifespan and healthspan in Caenorhabditis elegans. This longevity is dependent on both daf‐16 signaling and inhibition of the neuromuscular acetylcholine receptor subunit unc‐38. We found unc‐38 RNAi to improve healthspan, lifespan, and stimulate DAF‐16 nuclear localization, similar to atracurium treatment. Finally, using RNA‐seq transcriptomics, we identify atracurium activation of DAF‐16 downstream effectors. Together, these data demonstrate the capacity to mimic genetic lifespan interventions with drugs, and in doing so, reveal that the neuromuscular acetylcholine receptor regulates the highly conserved FOXO/DAF‐16 longevity pathway.
Transcriptome‐based drug screening is a powerful tool to identify geroprotective compounds. By mimicking the transcriptional signature of FOXO3 (daf‐16 in worms) overexpression, we identified the neuromuscular blocker atracurium, which extends both lifespan and healthspan in Caenorhabditis elegans. Here we demonstrate the capacity to mimic genetic lifespan interventions with drugs, and reveal that the neuromuscular acetylcholine receptor regulates the highly conserved DAF‐16 longevity pathway.
Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss ...leading to blindness. OAT is a pyridoxal-5′-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of 14C-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro, which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT, but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care.
•We developed an in vitro pyridoxine responsiveness assay in fibroblasts of patients with GACR.•We identified a prevalent variant within the Dutch GACR cohort, p.(Gly353Asp).•We developed a new diagnostic laboratory pipeline to extend diagnostic practices in GACR.
Background
Proton magnetic resonance spectroscopy (1H‐MRS) of the human heart is deemed to be a quantitative method to investigate myocardial metabolite content, but thorough validations of in vivo ...measurements against invasive techniques are lacking.
Purpose
To determine measurement precision and accuracy for quantifications of myocardial total creatine and triglyceride content with localized 1H‐MRS.
Study type
Test–retest repeatability and measurement validation study.
Subjects
Sixteen volunteers and 22 patients scheduled for open‐heart aortic valve replacement or septal myectomy.
Field Strength/Sequence
Prospectively ECG‐triggered respiratory‐gated free‐breathing single‐voxel point‐resolved spectroscopy (PRESS) sequence at 3 T.
Assessment
Myocardial total creatine and triglyceride content were quantified relative to the total water content by fitting the 1H‐MR spectra. Precision was assessed with measurement repeatability. Accuracy was assessed by validating in vivo 1H‐MRS measurements against biochemical assays in myocardial tissue from the same subjects.
Statistical Tests
Intrasession and intersession repeatability was assessed using Bland–Altman analyses. Agreement between 1H‐MRS measurements and biochemical assay was tested with regression analyses.
Results
The intersession repeatability coefficient for myocardial total creatine content was 41.8% with a mean value of 0.083% ± 0.020% of the total water signal, and 36.7% for myocardial triglyceride content with a mean value of 0.35% ± 0.13% of the total water signal. Ex vivo myocardial total creatine concentrations in tissue samples correlated with the in vivo myocardial total creatine content measured with 1H‐MRS: n = 22, r = 0.44; P < 0.05. Likewise, ex vivo myocardial triglyceride concentrations correlated with the in vivo myocardial triglyceride content: n = 20, r = 0.50; P < 0.05.
Data Conclusion
We validated the use of localized 1H‐MRS of the human heart at 3 T for quantitative assessments of in vivo myocardial tissue metabolite content by estimating the measurement precision and accuracy.
Level of Evidence
2
Technical Efficacy Stage
2
Cellular metabolism is important for determining cell function and shaping immune responses. Studies have shown a crucial role for stromal cells in steering proper immune responses in the lymph node ...microenvironment. These lymph node stromal cells (LNSCs) tightly regulate immune tolerance. We hypothesize that malfunctioning LNSCs create a microenvironment in which normal immune responses are not properly controlled, possibly leading to the development of autoimmune diseases such as rheumatoid arthritis (RA). Therefore, we set out to determine their metabolic profile during health and systemic autoimmunity. We included autoantibody positive individuals at risk of developing RA (RA-risk individuals), RA patients and healthy volunteers. All study subjects underwent lymph node biopsy sampling. Mitochondrial function in cultured LNSCs was assessed by quantitative PCR, flow cytometry, Seahorse and oleate oxidation assays. Overall, mitochondrial respiration was lower in RA(-risk) LNSCs compared with healthy LNSCs, while metabolic potential was only lower in RA LNSCs. To maintain basal mitochondrial respiration, all LNSCs were mostly dependent on fatty acid oxidation. However, RA(-risk) LNSCs were also dependent on glutamine oxidation. Finally, we showed that RA LNSCs have impaired metabolic flexibility. Our results show that the metabolic landscape of LNSCs is not only altered during established disease, but partly already in individuals at risk of developing RA. Future studies are needed to investigate the impact of restoring metabolic capacity in LNSC-mediated immunomodulation and disease progression.
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