Publications by authors named "Julie A Reisz"

105 Publications

Specificity of Human Sulfiredoxin for Reductant and Peroxiredoxin Oligomeric State.

Antioxidants (Basel) 2021 Jun 11;10(6). Epub 2021 Jun 11.

Center for Structural Biology, Department of Biochemistry, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA.

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., HO), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SOH) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (HS) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and HS in supporting Srx activity.
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http://dx.doi.org/10.3390/antiox10060946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230665PMC
June 2021

Time-Efficient Inspiratory Muscle Strength Training Lowers Blood Pressure and Improves Endothelial Function, NO Bioavailability, and Oxidative Stress in Midlife/Older Adults With Above-Normal Blood Pressure.

J Am Heart Assoc 2021 Jul 29;10(13):e020980. Epub 2021 Jun 29.

Department of Integrative Physiology University of Colorado Boulder Boulder CO.

Background High-resistance inspiratory muscle strength training (IMST) is a novel, time-efficient physical training modality. Methods and Results We performed a double-blind, randomized, sham-controlled trial to investigate whether 6 weeks of IMST (30 breaths/day, 6 days/week) improves blood pressure, endothelial function, and arterial stiffness in midlife/older adults (aged 50-79 years) with systolic blood pressure ≥120 mm Hg, while also investigating potential mechanisms and long-lasting effects. Thirty-six participants completed high-resistance IMST (75% maximal inspiratory pressure, n=18) or low-resistance sham training (15% maximal inspiratory pressure, n=18). IMST was safe, well tolerated, and had excellent adherence (≈95% of training sessions completed). Casual systolic blood pressure decreased from 135±2 mm Hg to 126±3 mm Hg (<0.01) with IMST, which was ≈75% sustained 6 weeks after IMST (<0.01), whereas IMST modestly decreased casual diastolic blood pressure (79±2 mm Hg to 77±2 mm Hg, =0.03); blood pressure was unaffected by sham training (all >0.05). Twenty-four hour systolic blood pressure was lower after IMST versus sham training (=0.01). Brachial artery flow-mediated dilation improved ≈45% with IMST (<0.01) but was unchanged with sham training (=0.73). Human umbilical vein endothelial cells cultured with subject serum sampled after versus before IMST exhibited increased NO bioavailability, greater endothelial NO synthase activation, and lower reactive oxygen species bioactivity (<0.05). IMST decreased C-reactive protein (=0.05) and altered select circulating metabolites (targeted plasma metabolomics) associated with cardiovascular function. Neither IMST nor sham training influenced arterial stiffness (>0.05). Conclusions High-resistance IMST is a safe, highly adherable lifestyle intervention for improving blood pressure and endothelial function in midlife/older adults with above-normal initial systolic blood pressure. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03266510.
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http://dx.doi.org/10.1161/JAHA.121.020980DOI Listing
July 2021

The AML microenvironment catalyzes a stepwise evolution to gilteritinib resistance.

Cancer Cell 2021 Jul 24;39(7):999-1014.e8. Epub 2021 Jun 24.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA; Department of Cell, Development, & Cancer Biology, Oregon Health & Science University, Portland, OR, USA.

Our study details the stepwise evolution of gilteritinib resistance in FLT3-mutated acute myeloid leukemia (AML). Early resistance is mediated by the bone marrow microenvironment, which protects residual leukemia cells. Over time, leukemia cells evolve intrinsic mechanisms of resistance, or late resistance. We mechanistically define both early and late resistance by integrating whole-exome sequencing, CRISPR-Cas9, metabolomics, proteomics, and pharmacologic approaches. Early resistant cells undergo metabolic reprogramming, grow more slowly, and are dependent upon Aurora kinase B (AURKB). Late resistant cells are characterized by expansion of pre-existing NRAS mutant subclones and continued metabolic reprogramming. Our model closely mirrors the timing and mutations of AML patients treated with gilteritinib. Pharmacological inhibition of AURKB resensitizes both early resistant cell cultures and primary leukemia cells from gilteritinib-treated AML patients. These findings support a combinatorial strategy to target early resistant AML cells with AURKB inhibitors and gilteritinib before the expansion of pre-existing resistance mutations occurs.
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http://dx.doi.org/10.1016/j.ccell.2021.06.003DOI Listing
July 2021

Accelerated aging of the brain transcriptome by the common chemotherapeutic doxorubicin.

Exp Gerontol 2021 Sep 18;152:111451. Epub 2021 Jun 18.

Department of Health and Exercise Science, Colorado State University, Fort Collins, CO, United States of America; Center for Healthy Aging, Colorado State University, Fort Collins, CO, United States of America. Electronic address:

Cancer is one of the most common age-related diseases, and over one-third of cancer patients will receive chemotherapy. One frequently reported side effect of chemotherapeutic agents like doxorubicin (Doxo) is impaired cognitive function, commonly known as "chemotherapy-induced cognitive impairment (CICI)", which may mimic accelerated brain aging. The biological mechanisms underlying the adverse effects of Doxo on the brain are unclear but could involve mitochondrial dysfunction. Here, we characterized brain (hippocampal) transcriptome and cognitive/behavioral changes in young mice treated with Doxo +/- the mitochondrial therapeutic MitoQ. We found that Doxo altered transcriptome/biological processes related to synaptic transmission and neurotransmitter function, neuronal health and behavior, and that these gene expression changes were: 1) similar to key differences observed in transcriptome data on brain aging; and 2) associated with related, aging-like behavioral differences, such as decreased exploration time and impaired novel object recognition test (NOR, an index of learning/memory) performance. Interestingly, MitoQ partially prevented Doxo-induced transcriptome changes in the brain, but it had no effect on behavior or cognitive function. Collectively, our findings are consistent with the idea that chemotherapeutic agents could induce neuronal/gene expression and behavioral changes similar to those that occur during brain aging. In this context, mitochondrial therapeutics may have potential as treatments for CICI at the biological level, but their effects on behavior/cognitive function require further investigation.
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http://dx.doi.org/10.1016/j.exger.2021.111451DOI Listing
September 2021

Erythrocyte adenosine A2B receptor prevents cognitive and auditory dysfunction by promoting hypoxic and metabolic reprogramming.

PLoS Biol 2021 Jun 17;19(6):e3001239. Epub 2021 Jun 17.

Department of Biochemistry and Molecular Biology, The University of Texas McGovern Medical School, Houston, Texas, United States of America.

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline.
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http://dx.doi.org/10.1371/journal.pbio.3001239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211187PMC
June 2021

Trisomy 21 results in modest impacts on mitochondrial function and central carbon metabolism.

Free Radic Biol Med 2021 Jun 12;172:201-212. Epub 2021 Jun 12.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, USA; Linda Crnic Institute for Down Syndrome, School of Medicine, University of Colorado, Aurora, CO, USA. Electronic address:

Down syndrome (DS) is the most common genetic cause of intellectual disability. Mechanistically, oxidative stress and mitochondrial dysfunction are reported to be etiological factors for many of the DS-related comorbidities and have previously been reported in a number of in vitro and in vivo models of DS. The purpose of this study was to test for the presence of mitochondrial dysfunction in fibroblast cells obtained via skin biopsy from individuals with DS, and to assess the impact of trisomy 21 on central carbon metabolism. Using extracellular flux assays in matched dermal fibroblasts from euploid and DS individuals, we found that basal mitochondrial dysfunction is quite mild. Stressing the cells with a cocktail of mitochondrial stressors revealed a significant mitochondrial deficit in DS cells compared to euploid controls. Evaluation of extracellular acidification rate did not reveal a baseline abnormality in glycolysis; however, metabolomic assessments utilizing isotopically labeled glucose and glutamine revealed altered central carbon metabolism in DS cells. Specifically, we observed greater glucose dependency, uptake and flux into the oxidative phase of the pentose phosphate pathway in DS fibroblasts. Furthermore, using induced pluripotent stem cells (iPSC) we found that mitochondrial function in DS iPSCs was similar to the previously published studies employing fetal cells. Together, these data indicate that aberrant central carbon metabolism is a candidate mechanism for stress-related mitochondrial dysfunction in DS.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.06.003DOI Listing
June 2021

SIRT5 IS A DRUGGABLE METABOLIC VULNERABILITY IN ACUTE MYELOID LEUKEMIA.

Blood Cancer Discov 2021 May 2;2(3):266-287. Epub 2019 Dec 2.

Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype-agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.
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http://dx.doi.org/10.1158/2643-3230.bcd-20-0168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133360PMC
May 2021

Lung metabolomics after ischemic acute kidney injury reveals increased oxidative stress, altered energy production, and ATP depletion.

Am J Physiol Lung Cell Mol Physiol 2021 Jul 5;321(1):L50-L64. Epub 2021 May 5.

Rocky Mountain Regional VA Medical Center, Denver, Colorado.

Acute kidney injury (AKI) is a complex disease associated with increased mortality that may be due to deleterious distant organ effects. AKI associated with respiratory complications, in particular, has a poor outcome. In murine models, AKI is characterized by increased circulating cytokines, lung chemokine upregulation, and neutrophilic infiltration, similar to other causes of indirect acute lung injury (ALI; e.g., sepsis). Many causes of lung inflammation are associated with a lung metabolic profile characterized by increased oxidative stress, a shift toward the use of other forms of energy production, and/or a depleted energy state. To our knowledge, there are no studies that have evaluated pulmonary energy production and metabolism after AKI. We hypothesized that based on the parallels between inflammatory acute lung injury and AKI-mediated lung injury, a similar metabolic profile would be observed. Lung metabolomics and ATP levels were assessed 4 h, 24 h, and 7 days after ischemic AKI in mice. Numerous novel findings regarding the effect of AKI on the lung were observed including ) increased oxidative stress, ) a shift toward alternate methods of energy production, and ) depleted levels of ATP. The findings in this report bring to light novel characteristics of AKI-mediated lung injury and provide new leads into the mechanisms by which AKI in patients predisposes to pulmonary complications.
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http://dx.doi.org/10.1152/ajplung.00042.2020DOI Listing
July 2021

Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension.

Front Immunol 2021 31;12:640718. Epub 2021 Mar 31.

Cardiovascular Pulmonary Research Laboratories, Departments of Pediatrics and Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted using a fibroblast-macrophage co-culture system and in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype , and are closely related to flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both and . In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.
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http://dx.doi.org/10.3389/fimmu.2021.640718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044406PMC
March 2021

Quantifying dynamic range in red blood cell energetics: Evidence of progressive energy failure during storage.

Transfusion 2021 05 8;61(5):1586-1599. Epub 2021 Apr 8.

Department of Pediatrics, Divisions of Critical Care Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Background: During storage, red blood cells (RBCs) undergo significant biochemical and morphologic changes, referred to collectively as the "storage lesion". It was hypothesized that these defects may arise from disrupted oxygen-based regulation of RBC energy metabolism, with resultant depowering of intrinsic antioxidant systems.

Study Design And Methods: As a function of storage duration, the dynamic range in RBC metabolic response to three models of biochemical oxidant stress (methylene blue, hypoxanthine/xanthine oxidase, and diamide) was assessed, comparing glycolytic flux by NMR and UHPLC-MS methodologies. Blood was processed/stored under standard conditions (AS-1 additive solution) with leukoreduction. Over a 6-week period, RBC metabolic and antioxidant status were assessed at baseline and following exposure to the three biochemical oxidant models. Comparison was made of glycolytic flux ( H-NMR tracking of [2- C]-glucose and metabolomic phenotyping with [1,2,3- C ] glucose), reducing equivalent (NADPH/NADP ) recycling, and thiol-based (GSH/GSSG) antioxidant status.

Results: As a function of storage duration, we observed the following: (1) a reduction in baseline hexose monophosphate pathway (HMP) flux, the sole pathway responsible for the regeneration of the essential reducing equivalent NADPH; with (2) diminished stress-based dynamic range in both overall glycolytic as well as proportional HMP flux. In addition, progressive with storage duration, RBCs showed (3) constraint in reducing equivalent (NADPH) recycling capacity, (4) loss of thiol based (GSH) recycling capacity, and (5) dysregulation of metabolon assembly at the cytoplasmic domain of Band 3 membrane protein (cdB3).

Conclusion: Blood storage disturbs normal RBC metabolic control, depowering antioxidant capacity and enhancing vulnerability to oxidative injury.
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http://dx.doi.org/10.1111/trf.16395DOI Listing
May 2021

Glutathionylation chemistry promotes interleukin-1 beta-mediated glycolytic reprogramming and pro-inflammatory signaling in lung epithelial cells.

FASEB J 2021 05;35(5):e21525

Department of Pathology and Laboratory Medicine, University of Vermont College of Medicine, Burlington, VT, USA.

Glycolysis is a well-known process by which metabolically active cells, such as tumor or immune cells meet their high metabolic demands. Previously, our laboratory has demonstrated that in airway epithelial cells, the pleiotropic cytokine, interleukin-1 beta (IL1B) induces glycolysis and that this contributes to allergic airway inflammation and remodeling. Activation of glycolysis is known to increase NADPH reducing equivalents generated from the pentose phosphate pathway, linking metabolic reprogramming with redox homeostasis. In addition, numerous glycolytic enzymes are known to be redox regulated. However, whether and how redox chemistry regulates metabolic reprogramming more generally remains unclear. In this study, we employed a multi-omics approach in primary mouse airway basal cells to evaluate the role of protein redox biochemistry, specifically protein glutathionylation, in mediating metabolic reprogramming. Our findings demonstrate that IL1B induces glutathionylation of multiple proteins involved in metabolic regulation, notably in the glycolysis pathway. Cells lacking Glutaredoxin-1 (Glrx), the enzyme responsible for reversing glutathionylation, show modulation of multiple metabolic pathways including an enhanced IL1B-induced glycolytic response. This was accompanied by increased secretion of thymic stromal lymphopoietin (TSLP), a cytokine important in asthma pathogenesis. Targeted inhibition of glycolysis prevented TSLP release, confirming the functional relevance of enhanced glycolysis in cells stimulated with IL1B. Collectively, data herein point to an intriguing link between glutathionylation chemistry and glycolytic reprogramming in epithelial cells and suggest that glutathionylation chemistry may represent a therapeutic target in pulmonary pathologies with perturbations in the glycolysis pathway.
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http://dx.doi.org/10.1096/fj.202002687RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073242PMC
May 2021

The COVIDome Explorer Researcher Portal.

medRxiv 2021 Mar 8. Epub 2021 Mar 8.

COVID-19 pathology involves dysregulation of diverse molecular, cellular, and physiological processes. In order to expedite integrated and collaborative COVID-19 research, we completed multi-omics analysis of hospitalized COVID-19 patients including matched analysis of the whole blood transcriptome, plasma proteomics with two complementary platforms, cytokine profiling, plasma and red blood cell metabolomics, deep immune cell phenotyping by mass cytometry, and clinical data annotation. We refer to this multidimensional dataset as the COVIDome. We then created the COVIDome Explorer, an online researcher portal where the data can be analyzed and visualized in real time. We illustrate here the use of the COVIDome dataset through a multi-omics analysis of biosignatures associated with C-reactive protein (CRP), an established marker of poor prognosis in COVID-19, revealing associations between CRP levels and damage-associated molecular patterns, depletion of protective serpins, and mitochondrial metabolism dysregulation. We expect that the COVIDome Explorer will rapidly accelerate data sharing, hypothesis testing, and discoveries worldwide.
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http://dx.doi.org/10.1101/2021.03.04.21252945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987038PMC
March 2021

Multi 'Omics Analysis of Intestinal Tissue in Ankylosing Spondylitis Identifies Alterations in the Tryptophan Metabolism Pathway.

Front Immunol 2021 3;12:587119. Epub 2021 Mar 3.

Division of Rheumatology, Department of Medicine, University of Colorado, Aurora, CO, United States.

Intestinal microbial dysbiosis, intestinal inflammation, and Th17 immunity are all linked to the pathophysiology of spondyloarthritis (SpA); however, the mechanisms linking them remain unknown. One potential hypothesis suggests that the dysbiotic gut microbiome as a whole produces metabolites that influence human immune cells. To identify potential disease-relevant, microbiome-produced metabolites, we performed metabolomics screening and shotgun metagenomics on paired colon biopsies and fecal samples, respectively, from subjects with axial SpA (axSpA, N=21), Crohn's disease (CD, N=27), and Crohn's-axSpA overlap (CD-axSpA, N=12), as well as controls (HC, N=24). Using LC-MS based metabolomics of 4 non-inflamed pinch biopsies of the distal colon from subjects, we identified significant alterations in tryptophan pathway metabolites, including an expansion of indole-3-acetate (IAA) in axSpA and CD-axSpA compared to HC and CD and indole-3-acetaldehyde (I3Ald) in axSpA and CD-axSpA but not CD compared to HC, suggesting possible specificity to the development of axSpA. We then performed shotgun metagenomics of fecal samples to characterize gut microbial dysbiosis across these disease states. In spite of no significant differences in alpha-diversity among the 4 groups, our results confirmed differences in gene abundances of numerous enzymes involved in tryptophan metabolism. Specifically, gene abundance of indolepyruvate decarboxylase, which generates IAA and I3Ald, was significantly elevated in individuals with axSpA while gene abundances in HC demonstrated a propensity towards tryptophan synthesis. Such genetic changes were not observed in CD, again suggesting disease specificity for axSpA. Given the emerging role of tryptophan and its metabolites in immune function, altogether these data indicate that tryptophan metabolism into I3Ald and then IAA is one mechanism by which the gut microbiome potentially influences the development of axSpA.
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http://dx.doi.org/10.3389/fimmu.2021.587119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966505PMC
July 2021

Metabolic Characterization of Plasma and Cyst Fluid from Cystic Precursors to Pancreatic Cancer Patients Reveal Metabolic Signatures of Bacterial Infection.

J Proteome Res 2021 05 15;20(5):2725-2738. Epub 2021 Mar 15.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora 80045, Colorado, United States.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, with a 5 year survival rate as low as 9%. One factor complicating the management of pancreatic cancer is the lack of reliable tools for early diagnosis. While up to 50% of the adult population has been shown to develop precancerous pancreatic cysts, limited and insufficient approaches are currently available to determine whether a cyst is going to progress into pancreatic cancer. Recently, we used metabolomics approaches to identify candidate markers of disease progression in patients diagnosed with intraductal papillary mucinous neoplasms (IPMNs) undergoing pancreatic resection. Here, we enrolled an independent cohort to verify the candidate markers from our previous study with orthogonal quantitative methods in plasma and cyst fluid from serous cystic neoplasm and IPMN (either low- or high-grade dysplasia or pancreatic ductal adenocarcinoma). We thus validated these markers with absolute quantitative methods through the auxilium of stable isotope-labeled internal standards in a new independent cohort. Finally, we identified novel markers of IPMN status and disease progression-including amino acids, carboxylic acids, conjugated bile acids, free and carnitine-conjugated fatty acids, purine oxidation products, and trimethylamine-oxide. We show that the levels of these metabolites of potential bacterial origin correlated with the degree of bacterial enrichment in the cyst, as determined by 16S RNA. Overall, our findings are interesting per se, owing to the validation of previous markers and identification of novel small molecule signatures of IPMN and disease progression. In addition, our findings further fuel the provoking debate as to whether bacterial infections may represent an etiological contributor to the development and severity of the disease in pancreatic cancer, in like fashion to other cancers (e.g., and gastric cancer).
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http://dx.doi.org/10.1021/acs.jproteome.1c00018DOI Listing
May 2021

Maturation of Pluripotent Stem Cell-Derived Cardiomyocytes Enables Modeling of Human Hypertrophic Cardiomyopathy.

Stem Cell Reports 2021 03 25;16(3):519-533. Epub 2021 Feb 25.

Division of Cardiology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; The Consortium for Fibrosis Research & Translation, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. Electronic address:

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for biomedical research. However, they are immature, which is a barrier to modeling adult-onset cardiovascular disease. Here, we sought to develop a simple method that could drive cultured hiPSC-CMs toward maturity across a number of phenotypes, with the aim of utilizing mature hiPSC-CMs to model human cardiovascular disease. hiPSC-CMs were cultured in fatty acid-based medium and plated on micropatterned surfaces. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, sarcomeric maturity, and increased myofibril contractile force. In addition, mature hiPSC-CMs develop pathological hypertrophy, with associated myofibril relaxation defects, in response to either a pro-hypertrophic agent or genetic mutations. The more mature hiPSC-CMs produced by these methods could serve as a useful in vitro platform for characterizing cardiovascular disease.
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http://dx.doi.org/10.1016/j.stemcr.2021.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940251PMC
March 2021

Mechanisms of stearoyl CoA desaturase inhibitor sensitivity and acquired resistance in cancer.

Sci Adv 2021 Feb 10;7(7). Epub 2021 Feb 10.

Division of Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

The lipogenic enzyme stearoyl CoA desaturase (SCD) plays a key role in tumor lipid metabolism and membrane architecture. SCD is often up-regulated and a therapeutic target in cancer. Here, we report the unexpected finding that median expression of SCD is low in glioblastoma relative to normal brain due to hypermethylation and unintentional monoallelic co-deletion with phosphatase and tensin homolog (PTEN) in a subset of patients. Cell lines from this subset expressed undetectable SCD, yet retained residual SCD enzymatic activity. Unexpectedly, these lines evolved to survive independent of SCD through unknown mechanisms. Cell lines that escaped such genetic and epigenetic alterations expressed higher levels of SCD and were highly dependent on SCD for survival. Last, we identify that SCD-dependent lines acquire resistance through a previously unknown FBJ murine osteosarcoma viral oncogene homolog B (FOSB)-mediated mechanism. Accordingly, FOSB inhibition blunted acquired resistance and extended survival of tumor-bearing mice treated with SCD inhibitor.
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http://dx.doi.org/10.1126/sciadv.abd7459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875532PMC
February 2021

Chaperone-mediated autophagy sustains haematopoietic stem-cell function.

Nature 2021 03 13;591(7848):117-123. Epub 2021 Jan 13.

Department of Development and Molecular Biology, Albert Einstein College of Medicine, New York, NY, USA.

The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA), a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
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http://dx.doi.org/10.1038/s41586-020-03129-zDOI Listing
March 2021

Nitrogen recycling buffers against ammonia toxicity from skeletal muscle breakdown in hibernating arctic ground squirrels.

Nat Metab 2020 12 7;2(12):1459-1471. Epub 2020 Dec 7.

Department of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, AK, USA.

Hibernation is a state of extraordinary metabolic plasticity. The pathways of amino acid metabolism as they relate to nitrogen homeostasis in hibernating mammals in vivo are unknown. Here we show, using pulse isotopic tracing, evidence of increased myofibrillar (skeletal muscle) protein breakdown and suppressed whole-body production of metabolites in vivo throughout deep torpor. As whole-body production of metabolites is suppressed, amino acids with nitrogenous side chains accumulate during torpor, while urea cycle intermediates do not. Using N stable isotope methodology in arctic ground squirrels (Urocitellus parryii), we provide evidence that free nitrogen is buffered and recycled into essential amino acids, non-essential amino acids and the gamma-glutamyl system during the inter-bout arousal period of hibernation. In the absence of nutrient intake or physical activity, our data illustrate the orchestration of metabolic pathways that sustain the provision of essential and non-essential amino acids and prevent ammonia toxicity during hibernation.
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http://dx.doi.org/10.1038/s42255-020-00312-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744440PMC
December 2020

Maneb alters central carbon metabolism and thiol redox status in a toxicant model of Parkinson's disease.

Free Radic Biol Med 2021 01 3;162:65-76. Epub 2020 Dec 3.

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO, 80045, USA. Electronic address:

The dithiocarbamate fungicide maneb (MB) has attracted interest due to increasing concern of the negative health effects of pesticides, as well as its association with Parkinson's disease (PD). Our laboratory has previously reported distinct phenotypic changes of neuroblastoma cells exposed to acute, sub-toxic levels of MB, including decreased mitochondrial respiration, altered lactate dynamics, and metabolic stress. In this study, we aimed to further define the specific molecular mechanisms of MB toxicity through the comparison of several thiol-containing compounds and their effects on cellular energy metabolism and thiol redox nodes. Extracellular flux analyses and stable isotope labeled tracer metabolomics were employed to evaluate alterations in energy metabolism of SK-N-AS human neuroblastoma cells after acute exposure of an array of compounds, including dithiocarbamates (maneb, nabam, zineb) and other thiol-containing small molecules (glutathione, N-acetylcysteine). These studies revealed MB and its methylated form (MeDTC) as unique toxicants with significant alterations to mitochondrial respiration, proliferation, and glycolysis. We observed MB to significantly impact cellular thiol redox status by oxidizing cellular glutathione and altering the thiol redox status of peroxiredoxin 3 (Prx3, mitochondrial) after acute exposure. Redox Western blotting revealed a MB-specific modification of cellular Prx3, strengthening the argument that MB can preferentially target mitochondrial enzymes containing reactive cysteine thiols. Further, stable isotope tracer metabolomics confirmed our energetics assessments, and demonstrated that MB exposure results in acute derangement of central carbon metabolism. Specifically, we observed shunting of cellular glucose into the pentose-phosphate pathway and reduction of TCA intermediates derived from glucose and glutamine. Also, we report novel lactate utilization for TCA enrichment and glutathione synthesis after MB exposure. In summary, our results further confirm that MB exerts its toxic effects via thiol modification, and significantly transforms central carbon metabolism.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889696PMC
January 2021

Metabolic abnormalities in G6PC3-deficient human neutrophils result in severe functional defects.

Blood Adv 2020 12;4(23):5888-5901

Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO.

Severe congenital neutropenia type 4 (SCN-4) is an autosomal recessive condition in which mutations in the G6PC3 gene encoding for the catalytic 3 subunit of glucose-6-phosphatase-β result in neutropenia, neutrophil dysfunction, and other syndromic features. We report a child with SCN-4 caused by compound heterozygous mutations in G6PC3, a previously identified missense mutation in exon 6 (c.758G>A[p.R235H]), and a novel missense mutation in exon 2 (c.325G>A[p.G109S]). The patient had recurrent bacterial infections, inflammatory bowel disease, neutropenia, and intermittent thrombocytopenia. Administration of granulocyte colony-stimulating factor (G-CSF) resolved the neutropenia and allowed for detailed evaluation of human neutrophil function. Random and directed migration by the patient's neutrophils was severely diminished. Associated with this were defects in CD11b expression and F-actin assembly. Bactericidal activity at bacteria/neutrophil ratios >1:1 was also diminished and was associated with attenuated ingestion. Superoxide anion generation was <25% of control values, but phox proteins appeared quantitatively normal. Extensive metabolomics analysis at steady state and upon incubation with stable isotope-labeled tracers (U-13C-glucose, 13C,15N-glutamine, and U-13C-fructose) demonstrated dramatic impairments in early glycolysis (hexose phosphate levels), hexosemonophosphate shunt (required for the generation of the NADPH), and the total adenylate pool, which could explain the dramatic cell dysfunction displayed by the patient's neutrophils. Preliminary experiments with fructose supplementation to bypass the enzyme block demonstrated that the metabolic profile could be reversed, but was not sustained long enough for functional improvement. In human deficiency of G6PC3, metabolic defects resulting from the enzyme deficiency account for diverse neutrophil functional defects and present a major risk of infection.
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http://dx.doi.org/10.1182/bloodadvances.2020002225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724913PMC
December 2020

Succinate Activation of SUCNR1 Predisposes Severely Injured Patients to Neutrophil-Mediated ARDS.

Ann Surg 2020 Nov 18. Epub 2020 Nov 18.

Department of Surgery, School of Medicine University of Colorado, Aurora, CO.

Objectives: Identify the metabolites that are increased in the plasma of severely injured patients that developed ARDS versus severely injured patients that did not, and assay if these increased metabolites prime PMNs and induce pulmonary sequestration in an animal model of ARDS. We hypothesize that metabolic derangement due to advanced shock in critically injured patients leads to the pulmonary sequestration of neutrophils (PMNs), which serves as the first event in the acute respiratory distress syndrome (ARDS).

Summary Background Data: Intracellular metabolites accumulate in the plasma of severely injured patients.

Methods: Untargeted metabolomics profiling of 67 critically injured patients was completed to establish a metabolic signature associated with ARDS development. Metabolites that signficantly increased were assayed for PMN priming activity in vitro. The metabolites that primed PMNs were tested in a two-event animal model of ARDS to identify a molecular link between circulating metabolites and clinical risk for ARDS.

Results: After controlling for confounders, four metabolites significantly increased: creatine, dehydroascorbate, fumarate, and succinate in trauma patients who developed ARDS (p<0.05). Succinate alone primed the PMN oxidase in vitro at physiologically relevant levels. Intravenous (IV) succinate-induced PMN sequestration in the lung, a first event, and followed by IV lipopolysaccharide, a second event, resulted in ARDS in vivo requiring PMNs. Succinate receptor (SUCNR1) inhibition abrogated PMN priming, PMN sequestration, and ARDS.

Conclusion: Significant increases in plasma succinate post-injury may serve as the first event in ARDS. Targeted inhibition of the SUCNR1 may decrease ARDS development from other disease states to prevent ARDS globally.
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http://dx.doi.org/10.1097/SLA.0000000000004644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128932PMC
November 2020

Metabolic characterization of plasma and cyst fluid from cystic precursors to pancreatic cancer patients reveal metabolic signatures of bacterial infection.

medRxiv 2020 Nov 4. Epub 2020 Nov 4.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, with a 5-year survival rate as low as 9%. One factor complicating the management of pancreatic cancer is the lack of reliable tools for early diagnosis. While up to 50% of the adult population has been shown to develop precancerous pancreatic cysts, limited and insufficient approaches are currently available to determine whether a cyst is going to progress into pancreatic cancer. Recently, we used metabolomics approaches to identify candidate markers of disease progression in patients diagnosed with intraductal papillary mucinous neoplasms (IPMNs) undergoing pancreatic resection. Here we enrolled an independent cohort to verify the candidate markers from our previous study with orthogonal quantitative methods in plasma and cyst fluid from serous cystic neoplasm and IPMN (either low- or high-grade dysplasia or pancreatic ductal adenocarcinoma). We thus validated these markers with absolute quantitative methods through the auxilium of stable isotope-labelled internal standards in a new independent cohort. Finally, we identified novel markers of IPMN status and disease progression - including amino acids, carboxylic acids, conjugated bile acids, free and carnitine-conjugated fatty acids, purine oxidation products and TMAO. We show that the levels of these metabolites of potential bacterial origin correlated with the degree of bacterial enrichment in the cyst, as determined by 16S RNA. Overall, our findings are interesting per se, owing to the validation of previous markers and identification of novel small molecule signatures of IPMN and disease progression. In addition, our findings further fuel the provoking debate as to whether bacterial infections may represent an etiological contributor to the development and severity of the disease in pancreatic cancer, in like fashion to other cancers (e.g., and gastric cancer).

Key Points: We identified and quantified novel markers of IPMN cyst status and pancreatic cancer disease progression - including amino acids, carboxylic acids, conjugated bile acids, free and carnitine-conjugated fatty acids, purine oxidation products and TMAO.We show that the levels of these metabolites of potential bacterial origin correlated with the degree of bacterial enrichment in the cyst, as determined by 16S RNA.
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http://dx.doi.org/10.1101/2020.11.03.20225524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654893PMC
November 2020

Doxorubicin-Induced Oxidative Stress and Endothelial Dysfunction in Conduit Arteries Is Prevented by Mitochondrial-Specific Antioxidant Treatment.

JACC CardioOncol 2020 Sep 15;2(3):475-488. Epub 2020 Sep 15.

Department of Integrative Physiology, University of Colorado Boulder, Boulder, Colorado.

Background: Doxorubicin (DOXO) chemotherapy increases risk for cardiovascular disease in part by inducing endothelial dysfunction in conduit arteries. However, the mechanisms mediating DOXO-associated endothelial dysfunction in (intact) arteries and treatment strategies are not established.

Objectives: We tested the hypothesis that DOXO impairs endothelial function in conduit arteries via excessive mitochondrial reactive oxygen species (ROS) and that these effects could be prevented by treatment with a mitochondrial-targeted antioxidant (MitoQ).

Methods: Endothelial function (endothelium-dependent dilation [EDD] to acetylcholine) and vascular mitochondrial ROS were assessed 4 weeks following administration (10 mg/kg intraperitoneal injection) of DOXO. A separate cohort of mice received chronic (4 weeks) oral supplementation with MitoQ (drinking water) for 4 weeks following DOXO.

Results: EDD in isolated pressurized carotid arteries was 55% lower 4 weeks following DOXO (peak EDD, DOXO: 42 ± 7% vs. sham: 94 ± 3%; p = 0.006). Vascular mitochondrial ROS was 52% higher and manganese (mitochondrial) superoxide dismutase was 70% lower after DOXO versus sham (p = 0.0008). Endothelial function was rescued by administration of the mitochondrial-targeted antioxidant, MitoQ, to the perfusate. Exposure to plasma from DOXO-treated mice increased mitochondrial ROS in cultured endothelial cells. Analyses of plasma showed differences in oxidative stress-related metabolites and a marked reduction in vascular endothelial growth factor A in DOXO mice, and restoring vascular endothelial growth factor A to sham levels normalized mitochondrial ROS in endothelial cells incubated with plasma from DOXO mice. Oral MitoQ supplementation following DOXO prevented the reduction in EDD (97 ± 1%; p = 0.002 vs. DOXO alone) by ameliorating mitochondrial ROS suppression of EDD.

Conclusions: DOXO-induced endothelial dysfunction in conduit arteries is mediated by excessive mitochondrial ROS and ameliorated by mitochondrial-specific antioxidant treatment. Mitochondrial ROS is a viable therapeutic target for mitigating arterial dysfunction with DOXO. (J Am Coll Cardiol CardioOnc 2020;2:475-88) © 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.
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http://dx.doi.org/10.1016/j.jaccao.2020.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561020PMC
September 2020

Oxidized Low-Density Lipoprotein Drives Dysfunction of the Liver Lymphatic System.

Cell Mol Gastroenterol Hepatol 2021 19;11(2):573-595. Epub 2020 Sep 19.

Division of Gastroenterology and Hepatology, University of Colorado Anschutz Medical Campus, Aurora, Colorado; RNA Biosciences Initiative, University of Colorado Anschutz Medical Campus, Aurora, Colorado; Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado. Electronic address:

Background And Aims: As the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease.

Methods: To answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo.

Results: Confirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries.

Conclusions: We identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease.
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http://dx.doi.org/10.1016/j.jcmgh.2020.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803659PMC
September 2020

Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells.

Cell Stem Cell 2020 11 20;27(5):748-764.e4. Epub 2020 Aug 20.

Division of Hematology, University of Colorado Denver, Aurora, CO 80045, USA. Electronic address:

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.
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http://dx.doi.org/10.1016/j.stem.2020.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655603PMC
November 2020

COVID-19 infection alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status.

JCI Insight 2020 07 23;5(14). Epub 2020 Jul 23.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado, USA.

BACKGROUNDReprogramming of host metabolism supports viral pathogenesis by fueling viral proliferation, by providing, for example, free amino acids and fatty acids as building blocks.METHODSTo investigate metabolic effects of SARS-CoV-2 infection, we evaluated serum metabolites of patients with COVID-19 (n = 33; diagnosed by nucleic acid testing), as compared with COVID-19-negative controls (n = 16).RESULTSTargeted and untargeted metabolomics analyses identified altered tryptophan metabolism into the kynurenine pathway, which regulates inflammation and immunity. Indeed, these changes in tryptophan metabolism correlated with interleukin-6 (IL-6) levels. Widespread dysregulation of nitrogen metabolism was also seen in infected patients, with altered levels of most amino acids, along with increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and renal dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis. Interestingly, metabolite levels in these pathways correlated with clinical laboratory markers of inflammation (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen).CONCLUSIONIn conclusion, this initial observational study identified amino acid and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.FUNDINGBoettcher Foundation Webb-Waring Biomedical Research Award; National Institute of General and Medical Sciences, NIH; and National Heart, Lung, and Blood Institute, NIH.
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http://dx.doi.org/10.1172/jci.insight.140327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453907PMC
July 2020

COVID-19 infection results in alterations of the kynurenine pathway and fatty acid metabolism that correlate with IL-6 levels and renal status.

medRxiv 2020 May 16. Epub 2020 May 16.

Previous studies suggest a role for systemic reprogramming of host metabolism during viral pathogenesis to fuel rapidly expanding viral proliferation, for example by providing free amino acids and fatty acids as building blocks. In addition, general alterations in metabolism can provide key understanding of pathogenesis. However, little is known about the specific metabolic effects of SARS-COV-2 infection. The present study evaluated the serum metabolism of COVID-19 patients (n=33), identified by a positive nucleic acid test of a nasopharyngeal swab, as compared to COVID-19-negative control patients (n=16). Targeted and untargeted metabolomics analyses specifically identified alterations in the metabolism of tryptophan into the kynurenine pathway, which is well-known to be involved in regulating inflammation and immunity. Indeed, the observed changes in tryptophan metabolism correlated with serum interleukin-6 (IL-6) levels. Metabolomics analysis also confirmed widespread dysregulation of nitrogen metabolism in infected patients, with decreased circulating levels of most amino acids, except for tryptophan metabolites in the kynurenine pathway, and increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and kidney dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis in COVID-19 patients. Metabolite levels in these pathways correlated with clinical laboratory markers of inflammation and disease severity (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen). In conclusion, this initial observational study of the metabolic consequences of COVID-19 infection in a clinical cohort identified amino acid metabolism (especially kynurenine and cysteine/taurine) and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.
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http://dx.doi.org/10.1101/2020.05.14.20102491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274252PMC
May 2020

CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean Variant.

Hypertension 2020 08 8;76(2):523-532. Epub 2020 Jun 8.

From the Department of Pharmacology, New York Medical College, Valhalla (A.K., I.K., C.J., K.M., P.R., S.A.G.).

Epidemiological studies suggest that individuals in the Mediterranean region with a loss-of-function, nonsynonymous single nucleotide polymorphism (S188F), in glucose-6-phosphate dehydrogenase () are less susceptible to vascular diseases. However, this association has not yet been experimentally proven. Here, we set out to determine whether the Mediterranean mutation confers protection from vascular diseases and to discover the underlying protective mechanism. We generated a rat model with the Mediterranean single nucleotide polymorphism (G6PD) using CRISPR-Cas9 genome editing. In rats carrying the mutation, G6PD activity, but not expression, was reduced to 20% of wild-type (WT) littermates. Additionally, unbiased metabolomics analysis revealed that the pentose phosphate pathway and other ancillary metabolic pathways connected to the pentose phosphate pathway were reduced (<0.05) in the arteries of G6PD versus WT rats. Intriguingly, G6PD mutants, as compared with WT rats, developed less large arterial stiffness and hypertension evoked by high-fat diet and nitric oxide synthase inhibition with L-N-nitroarginine methyl ester. Intravenous injection of a voltage-gated L-type Ca channel agonist (methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate; Bay K8644) acutely increased blood pressure in WT but not in G6PD rats. Finally, our results suggested that (1) lower resting membrane potential of smooth muscle caused by increased expression of K channel proteins and (2) decreased voltage-gated Ca channel activity in smooth muscle contributed to reduced hypertension and arterial stiffness evoked by L-N-nitroarginine methyl ester and high-fat diet to G6PD mutants as compared with WT rats. In summary, a mutation resulting in the replacement of a single amino acid (S188F) in G6PD, the rate-limiting enzyme in the pentose phosphate pathway, ascribed properties to the vascular smooth muscle that shields the organism from risk factors associated with vascular diseases.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.14772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726709PMC
August 2020

Metabolic Reprogramming of Mouse Bone Marrow Derived Macrophages Following Erythrophagocytosis.

Front Physiol 2020 30;11:396. Epub 2020 Apr 30.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, United States.

Reticuloendothelial macrophages engulf ∼0.2 trillion senescent erythrocytes daily in a process called erythrophagocytosis (EP). This critical mechanism preserves systemic heme-iron homeostasis by regulating red blood cell (RBC) catabolism and iron recycling. Although extensive work has demonstrated the various effects on macrophage metabolic reprogramming by stimulation with proinflammatory cytokines, little is known about the impact of EP on the macrophage metabolome and proteome. Thus, we performed mass spectrometry-based metabolomics and proteomics analyses of mouse bone marrow-derived macrophages (BMDMs) before and after EP of IgG-coated RBCs. Further, metabolomics was performed on BMDMs incubated with free IgG to ensure that changes to macrophage metabolism were due to opsonized RBCs and not to free IgG binding. Uniformly labeled tracing experiments were conducted on BMDMs in the presence and absence of IgG-coated RBCs to assess the flux of glucose through the pentose phosphate pathway (PPP). In this study, we demonstrate that EP significantly alters amino acid and fatty acid metabolism, the Krebs cycle, OXPHOS, and arachidonate-linoleate metabolism. Increases in levels of amino acids, lipids and oxylipins, heme products, and RBC-derived proteins are noted in BMDMs following EP. Tracing experiments with U-C glucose indicated a slower flux through glycolysis and enhanced PPP activation. Notably, we show that it is fueled by glucose derived from the macrophages themselves or from the extracellular media prior to EP, but not from opsonized RBCs. The PPP-derived NADPH can then fuel the oxidative burst, leading to the generation of reactive oxygen species necessary to promote digestion of phagocytosed RBC proteins via radical attack. Results were confirmed by redox proteomics experiments, demonstrating the oxidation of Cys152 and Cys94 of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hemoglobin-β, respectively. Significant increases in early Krebs cycle and C-branched dibasic acid metabolites (α-ketoglutarate and 2-hydroxyglutarate, respectively) indicate that EP promotes the dysregulation of mitochondrial metabolism. Lastly, EP stimulated aminolevulinic acid (ALA) synthase and arginase activity as indicated by significant accumulations of ALA and ornithine after IgG-mediated RBC ingestion. Importantly, EP-mediated metabolic reprogramming of BMDMs does not occur following exposure to IgG alone. In conclusion, we show that EP reprograms macrophage metabolism and modifies macrophage polarization.
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http://dx.doi.org/10.3389/fphys.2020.00396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204509PMC
April 2020
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