Biology subjects

Jones, R. G.

Publications and source records attributed to Jones, R. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Exercise preconditioning confers skeletal muscle myometaplasticity

Previously exercise trained skeletal muscle is more growth-responsive to retraining. Using a murine training-detraining-retraining approach and multi-omics, we find that Pecam1 gene expression is lower, but capillarization is appreciably higher in previously trained (preconditioned) relative to naive trained control muscle. Greater capillarity could be permissive for accelerated hypertrophic adaptation. Exercise preconditioned myonuclei feature differential promoter CpG regulation in genes related to Wnt signaling. These epigenetic alterations with retraining align with our prior observations of methylation changes within the same pathway after a longer period of chronic training, suggesting a more rapid response due to preconditioning. Methylome-transcriptome integration and single myonucleus RNA-sequencing expose the polyamine metabolism enzyme Smox as a target that relates to heightened hypertrophic adaptability with retraining. Smox induction is sufficient to cause hypertrophy in aligned myotubes cultured on a stiffness-tuned substrate along with a growth-supportive transcriptional program. Integration of our multi-omics data suggests that Smox regulates repression of Ddit4/Redd1 (an inhibitor of mTORC1 signaling) after retraining. Smox may govern a favorable muscle fiber growth environment in previously trained muscle by sensitizing anabolic potential through polyamine metabolism. A lower adaptive threshold mediated by Smox could contribute to myometaplasticity, or a change to how subsequent muscle adaptations are made.

molecular biology↗

bioRxiv2026

Myonuclear loss, rather than senescent myonuclei, associates with fiber type-specific atrophy in aging human skeletal muscle

Age-related reductions in whole-muscle function are attributed, in part, to pronounced atrophy of muscle fibers expressing the fast myosin heavy chain (MyHC) II isoforms. Senescence, a state of irreversible cell cycle arrest that can be characterized by DNA damage ({gamma}H2AX) and chromatin remodeling (loss of nuclear HMGB1), may contribute to skeletal muscle aging. Muscle nuclei (myonuclei) maintain fiber size and function and could exhibit senescence-associated features; however, the prevalence of senescent myonuclei and whether they contribute to fast fiber atrophy in older adults remains unknown. Vastus lateralis biopsies from 20 young (19-34yr; 10 females) and 20 older (65-84yr; 10 females) adults were analyzed via immunohistochemistry for myonuclei positive for {gamma}H2AX ({gamma}H2AX+) and negative for HMGB1 (HMGB1-). MyHC II cross-sectional area (CSA) was [~]70% larger in young compared with old, whereas MyHC I CSA did not differ with age. The relative abundance of {gamma}H2AX+/HMGB1- myonuclei did not differ with age and was not associated with CSA in either fiber type. Single-nucleus RNA-sequencing corroborated no age-related difference in the prevalence of myonuclei with senescence-associated features. Myonuclear content of MyHC II fibers was [~]30% higher in young compared with old and was closely associated with CSA in both fiber types. Size-cluster analysis revealed a pronounced age-related leftward shift in MyHC II CSA that paralleled the reductions in myonuclear number, consistent with myonuclear loss. These data suggest that age-related fast fiber atrophy is not attributed to an increased prevalence of senescent myonuclei but instead occurs concomitantly with fiber type-specific myonuclear loss across the lifespan.

cell biology↗

bioRxiv2025

The Age-Dependent Resident Myonuclear Multi-Omic Response to a Skeletal Muscle Hypertrophic Stimulus

A detailed analysis of how muscle fiber nuclei (myonuclei) respond to a hypertrophic stimulus would provide a critical step toward understanding compromised skeletal muscle plasticity with age. We used recombination-independent doxycycline-inducible myonucleus-specific fluorescent labelling, tissue RNA-sequencing, myonuclear DNA methylation analysis, multi-omic integration, and single myonucleus RNA-sequencing to define the molecular characteristics of adult (6-8 month) and aged (24 month) murine skeletal muscle after acute mechanical overload (MOV). In adult and aged MOV muscles, we found that: 1) similarities in the transcriptional response to loading - specifically in metabolism genes - were partly explained by a post-transcriptional microRNA-mediated mechanism, which we corroborated using an inducible muscle fiber-specific miR-1 knockout model, 2) differences in age-dependent transcriptional responses were linked to the magnitude and location of differential DNA methylation in resident myonuclei, specifically around hypertrophy-associated genes such as Myc, Runx1, Mybph, Ankrd1, collagen genes, and minichromosome maintenance genes, 3) adult and aged resident myonuclear transcriptomes had differing enrichment for innervation-related transcripts as well as unique transcriptional profiles in an Atf3+ "sarcomere assembly" population after MOV, and 4) cellular deconvolution analysis supports a role for neuromuscular junction regulation in age-specific hypertrophic adaptation. These data are a roadmap for uncovering molecular targets to enhance aged muscle adaptability.

cell biology↗

bioRxiv2024

An alternative route for β-hydroxybutyrate metabolism supports fatty acid synthesis in cancer cells

Cancer cells are exposed to diverse metabolites in the tumor microenvironment that are used to support the synthesis of nucleotides, amino acids, and lipids needed for rapid cell proliferation1-3. Recent work has shown that ketone bodies such as {beta}-hydroxybutyrate ({beta}-OHB), which are elevated in circulation under fasting conditions or low glycemic diets, can serve as an alternative fuel that is metabolized in the mitochondria to provide acetyl-CoA for the tricarboxylic acid (TCA) cycle in some tumors4-7. Here, we discover a non-canonical route for {beta}-OHB metabolism, in which {beta}-OHB can bypass the TCA cycle to generate cytosolic acetyl-CoA for de novo fatty acid synthesis in cancer cells. We show that {beta}-OHB-derived acetoacetate in the mitochondria can be shunted into the cytosol, where acetoacetyl-CoA synthetase (AACS) and thiolase convert it into acetyl-CoA for fatty acid synthesis. This alternative metabolic routing of {beta}-OHB allows it to avoid oxidation in the mitochondria and net contribute to anabolic biosynthetic processes. In cancer cells, {beta}-OHB is used for fatty acid synthesis to support cell proliferation under lipid-limited conditions in vitro and contributes to tumor growth under lipid-limited conditions induced by a calorie-restricted diet in vivo. Together, these data demonstrate that {beta}-OHB is preferentially used for fatty acid synthesis in cancer cells to support tumor growth.

cancer biology↗

bioRxiv2024

Glucose-dependent glycosphingolipid biosynthesis fuels CD8+ T cell function and tumor control

Glucose is essential for T cell proliferation and function, yet its specific metabolic roles in vivo remain poorly defined. Here, we identify glycosphingolipid (GSL) biosynthesis as a key pathway fueled by glucose that enables CD8+ T cell expansion and cytotoxic function in vivo. Using 13C-based stable isotope tracing, we demonstrate that CD8+ effector T cells use glucose to synthesize uridine diphosphate-glucose (UDP-Glc), a precursor for glycogen, glycan, and GSL biosynthesis. Inhibiting GSL production by targeting the enzymes UGP2 or UGCG impairs CD8+ T cell expansion and cytolytic activity without affecting glucose-dependent energy production. Mechanistically, we show that glucose-dependent GSL biosynthesis is required for plasma membrane lipid raft integrity and aggregation following TCR stimulation. Moreover, UGCG-deficient CD8+ T cells display reduced granzyme expression and tumor control in vivo. Together, our data establish GSL biosynthesis as a critical metabolic fate of glucose--independent of energy production--required for CD8+ T cell responses in vivo.

immunology↗

bioRxiv2024

NRF2-dependent regulation of the prostacyclin receptor PTGIR drives CD8 T cell exhaustion

The progressive decline of CD8 T cell effector function--also known as terminal exhaustion--is a major contributor to immune evasion in cancer. Yet, the molecular mechanisms that drive CD8 T cell dysfunction remain poorly understood. Here, we report that the Kelch-like ECH-associated protein 1 (KEAP1)-Nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis, which mediates cellular adaptations to oxidative stress, directly regulates CD8 T cell exhaustion. Transcriptional profiling of dysfunctional CD8 T cells from chronic infection and cancer reveals enrichment of NRF2 activity in terminally exhausted (Texterm) CD8 T cells. Increasing NRF2 activity in CD8 T cells (via conditional deletion of KEAP1) promotes increased glutathione production and antioxidant defense yet accelerates the development of terminally exhausted (PD-1+TIM-3+) CD8 T cells in response to chronic infection or tumor challenge. Mechanistically, we identify PTGIR, a receptor for the circulating eicosanoid prostacyclin, as an NRF2-regulated protein that promotes CD8 T cell dysfunction. Silencing PTGIR expression restores the anti-tumor function of KEAP1-deficient T cells. Moreover, lowering PTGIR expression in CD8 T cells both reduces terminal exhaustion and enhances T cell effector responses (i.e. IFN-{gamma} and granzyme production) to chronic infection and cancer. Together, these results establish the KEAP1-NRF2 axis as a metabolic sensor linking oxidative stress to CD8 T cell dysfunction and identify the prostacyclin receptor PTGIR as an NRF2-regulated immune checkpoint that regulates CD8 T cell fate decisions between effector and exhausted states. One Sentence SummaryThe KEAP1-NRF2 pathway is hyperactivated in terminally exhausted CD8 T cells and drives T cell dysfunction via transcriptional regulation of the prostacyclin receptor, Ptgir.

immunology↗

bioRxiv2024

Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia

ObjectivesA high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. MethodsHere, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm [~]45, [~]75 and [~]90 days after EOC injection. ResultsPrimary ovarian tumour sizes were progressively larger at each time point while robust metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNF), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. ConclusionThis mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-metastatic weakness during EOC in addition to therapies targeting cachexia. HighlightsO_LIThis study reports the first orthotopic model of metastatic ovarian cancer cachexia that can be induced in adult immunocompetent mice C_LIO_LIDiaphragm and limb muscle weakness precedes metastasis and atrophy during ovarian cancer C_LIO_LISkeletal muscle mitochondrial oxidative and redox stress signatures occur during pre-metastatic stages of ovarian cancer C_LIO_LISpecific muscle force as well as mitochondrial pyruvate oxidation and creatine metabolism demonstrate compensation in later stages C_LIO_LIOvarian cancer has heterogeneous effects on distinct muscle types across time C_LI

physiology↗

bioRxiv2024

The 24-Hour Time Course of Integrated Molecular Responses to Resistance Exercise in Human Skeletal Muscle Implicates MYC as a Hypertrophic Regulator That is Sufficient for Growth

Molecular control of recovery after exercise in muscle is temporally dynamic. A time course of biopsies around resistance exercise (RE) combined with -omics is necessary to better comprehend the molecular contributions of skeletal muscle adaptation in humans. Vastus lateralis biopsies before and 30 minutes, 3-, 8-, and 24-hours after acute RE were collected. A time-point matched biopsy-only group was also included. RNA-sequencing defined the transcriptome while DNA methylomics and computational approaches complemented these data. The post-RE time course revealed: 1) DNA methylome responses at 30 minutes corresponded to upregulated genes at 3 hours, 2) a burst of translation- and transcription-initiation factor-coding transcripts occurred between 3 and 8 hours, 3) global gene expression peaked at 8 hours, 4) ribosome-related genes dominated the mRNA landscape between 8 and 24 hours, 5) methylation-regulated MYC was a highly influential transcription factor throughout the 24-hour recovery and played a primary role in ribosome-related mRNA levels between 8 and 24 hours. The influence of MYC in human muscle adaptation was strengthened by transcriptome information from acute MYC overexpression in mouse muscle. To test whether MYC was sufficient for hypertrophy, we generated a muscle fiber-specific doxycycline inducible model of pulsatile MYC induction. Periodic 48-hour pulses of MYC over 4 weeks resulted in higher muscle mass and fiber size in the soleus of adult female mice. Collectively, we present a temporally resolved resource for understanding molecular adaptations to RE in muscle and reveal MYC as a regulator of RE-induced mRNA levels and hypertrophy.

cell biology↗

bioRxiv2023

Quantitative Analysis of Autophagy in Single Cells: Differential Response to Amino Acid and Glucose Starvation

Autophagy is a highly conserved, intracellular recycling process by which cytoplasmic contents are degraded in the lysosome. This process occurs at a low level constitutively; however, it is induced robustly in response to stressors, in particular, starvation of critical nutrients such as amino acids and glucose. That said, the relative contribution of these inputs is ambiguous and many starvation medias are poorly defined or devoid of multiple nutrients. Here, we sought to generate a quantitative catalog of autophagy across multiple stages and in single, living cells under normal growth conditions as well as in media starved specifically of amino acids or glucose. We found that autophagy is induced by starvation of amino acids, but not glucose, in U2OS cells, and that MTORC1-mediated ULK1 regulation and autophagy are tightly linked to amino acid levels. While autophagy is engaged immediately during amino acid starvation, a heightened response occurs during a period marked by transcriptional upregulation of autophagy genes during sustained starvation. Finally, we demonstrated that cells immediately return to their initial, low-autophagy state when nutrients are restored, highlighting the dynamic relationship between autophagy and environmental conditions. In addition to sharing our findings here, we provide our data as a high-quality resource for others interested in mathematical modeling or otherwise exploring autophagy in individual cells across a population.

cell biology↗

bioRxiv2023

Acod1 Expression in Cancer Cells Promotes Immune Evasion through the Generation of Inhibitory Peptides

Targeting PD-1 is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment (TME). Here, we find that PD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to PD-1, as decreasing Acod1 levels in PD-1 resistant cancer cells can sensitize tumors to PD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naive CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on secretion of ITA, but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.

cancer biology↗

bioRxiv2023

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8+ T cells

Infusion of 13C-labeled metabolites provides a gold-standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, acetate) in Listeria monocytogenes (Lm)-infected mice, we demonstrate that CD8+ T effector (Teff) cells utilize metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily towards nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support ATP and de novo pyrimidine synthesis. Additionally, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)--which regulates de novo aspartate synthesis--for effector cell expansion in vivo. Importantly, Teff cells change fuel preference over the course of infection, switching from glutamine-to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with Teff cell function in vivo. TeaserInterrogating dynamics of fuel utilization by CD8+ T cells in vivo reveals new metabolic checkpoints for immune function in vivo.

immunology↗

bioRxiv2023

A rapid microglial metabolic response controls metabolism and improves memory

Chronic high-fat feeding triggers chronic metabolic dysfunction including obesity, insulin resistance, and diabetes. How high-fat intake first triggers these pathophysiological states remains unknown. Here, we identify an acute microglial metabolic response that rapidly translates intake of high-fat diet (HFD) to a surprisingly beneficial effect on metabolism and spatial / learning memory. High-fat intake rapidly increases palmitate levels in cerebrospinal fluid and triggers a wave of microglial metabolic activation characterized by mitochondrial membrane activation and fission as well as metabolic skewing towards aerobic glycolysis. These effects are detectable throughout the brain and can be detected within as little as 12 hours of HFD exposure. In vivo, microglial ablation and conditional DRP1 deletion show that the microglial metabolic response is necessary for the acute effects of HFD. 13C-tracing experiments reveal that in addition to processing via {beta}-oxidation, microglia shunt a substantial fraction of palmitate towards anaplerosis and re-release of bioenergetic carbons into the extracellular milieu in the form of lactate, glutamate, succinate, and intriguingly, the neuro-protective metabolite itaconate. Together, these data identify microglia as a critical nutrient regulatory node in the brain, metabolizing away harmful fatty acids and releasing the same carbons as alternate bioenergetic and protective substrates for surrounding cells. The data identify a surprisingly beneficial effect of short-term HFD on learning and memory. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/535373v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1b9699corg.highwire.dtl.DTLVardef@1c48efcorg.highwire.dtl.DTLVardef@18757d0org.highwire.dtl.DTLVardef@962cf6_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

The Time-Course of Cancer Cachexia Onset Reveals Biphasic Transcriptional Disruptions in Female Skeletal Muscle Distinct from Males

BackgroundCancer-cachexia (CC) is experienced by 80% of cancer patients, representing 40% of cancer-related deaths. Evidence suggests biological sex dimorphism is associated with CC. Assessments of the female transcriptome in CC are lacking and direct comparisons between biological sex are scarce. The purpose of this study was to define the time course of LLC-induced CC in females using transcriptomics, while directly comparing the effects of biological sex. MethodsEight-week-old female mice were injected with LLC cells (1x106) or sterile PBS to the hind flank. Tumors developed for 1, 2, 3 or 4-weeks. Due to dimorphism between tumor weight in 3- and 4-weeks of development, these were reorganized as low-tumor weight (LT, tumor-weight [≤]1.2g), or high-tumor weight (HT, tumor-weight [≥]2g). Gastrocnemius muscle was collected for RNA-sequencing (RNA-seq). Differentially expressed genes (DEGs) were defined as FDR<0.05. Data were further compared to RNA-seq of male mice from a previous study. ResultsGlobal gene expression of female gastrocnemius muscle reveals consistent DEGs at all timepoints, all associated with type-II interferon signaling (FDR<0.05). Early transcriptomic upregulation of extracellular-matrix pathways was noted at 1wk (p<0.05), JAK-STAT pathway was upregulated in 2wk, LT, and HT. Type II interferon signaling was downregulated in 1wk, LT, and HT (p<0.05). A second major transcriptomic downregulation in oxidative phosphorylation, electron transport chain and TCA cycle were noted in cachectic (HT) muscle only (p<0.05). Male-female comparison of cachectic groups revealed 69% of DEGs were distinct between sex (FDR<0.05). Comparison of the top 10-up and down DEGs revealed downregulation of type-II Interferon genes was unique to female, while males show upregulation of interferon-signaling pathways. ConclusionWe demonstrate biphasic disruptions in transcriptome of female LLC tumor-bearing mice: an early phase associated with ECM remodeling and a late phase, accompanied by onset of systemic cachexia, affecting overall skeletal muscle energy metabolism. Comparison of cachectic female-male mice reveals ~2/3 of DEGs are biological sex specific, providing evidence of dimorphic mechanisms of cachexia between sexes. Alterations to Type-II Interferon signaling appears specific to CC development in females, suggesting a new biological sex-specific marker of CC. Our data support biological sex dimorphisms in development of CC. HighlightsO_LIWhile males show impairments in skeletal muscle energy metabolism in early stages of CC, early transcriptomic alterations impact ECM remodeling that precedes impairments in skeletal muscle energy metabolism in female tumor-bearing mice. C_LIO_LI2/3 of differently expressed genes in skeletal muscle undergoing cachexia are biological sex specific. C_LIO_LIDownregulation of Type-II Interferon genes is unique to female mice, which displayed preserved gastrocnemius mass despite systemic cachexia, representing a potential therapeutic target for muscle mass maintenance in cancer-induced atrophy. C_LIO_LIMechanisms of LLC-induced cachexia appear to be biological sex specific which needs to be considered in further study of mechanisms and therapeutic modalities. C_LI

bioinformatics↗

bioRxiv2022

Reprogramming of Iron Metabolism Confers Ferroptosis Resistance in ECM-Detached Cells

Cancer cells often acquire resistance to cell death programs induced by loss of integrin-mediated attachment to extracellular matrix (ECM). Given that adaptation to ECM-detached conditions can facilitate tumor progression and metastasis, there is significant interest in effective elimination of ECM-detached cancer cells. Here, we find that ECM-detached cells are remarkably resistant to the induction of ferroptosis. While alterations in membrane lipid content are observed during ECM-detachment, it is instead fundamental changes in iron metabolism that underlie resistance of ECM-detached cells to ferroptosis. More specifically, our data demonstrate that levels of free iron are low during ECM-detachment due to changes in both iron uptake and iron storage. In addition, we establish that lowering the levels of iron storage proteins sensitizes ECM-detached cells to death by ferroptosis. Taken together, our data suggest that therapeutics designed to kill cancer cells by ferroptosis may be hindered by lack of efficacy towards ECM-detached cells.

cell biology↗

bioRxiv2022

Combined single-sample metabolomics and RNAseq reveals a hepatic pyrimidine metabolic response to acute viral infection

ObjectiveMetabolomics and RNA sequencing (RNAseq) each provide powerful readouts of phenotype, and integration of these data can provide information greater than the sum of their parts. The ability to conduct such analysis on a single sample has many practical advantages, especially when dealing with rare or difficult-to-obtain samples. While methods exist to isolate multiple biomolecular subclasses from the same sample, in-depth analysis of the suitability of these approaches for multi- omics readouts is lacking. MethodsMice were injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh) control and liver tissue was harvested 2.5-days later. RNA was isolated from aliquots of pulverized liver tissue either following metabolite extraction using 80% methanol (MetRNA) or directly from frozen tissue (RNA). RNA sequencing data was evaluated by differential expression analysis via edgeR and dispersion using Ginis mean differences. Differential metabolite abundance was assessed using LIMMA. Pathway enrichment analysis was conducted on metabolomics and RNAseq data using MetaboAnalysts joint-integration tools. ResultsPrior metabolite extraction had no deleterious effects on quality or quantity of isolated RNA. RNA and MetRNA generated from the same sample clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Of the 2,169 genes that were differentially expressed between LCMV and Veh, the vast majority (n=1,848) were shared between extraction method, with the remainder evenly divided between RNA (n=165) and MetRNA (n=156). These differentially expressed genes unique to extraction method were attributed to randomness around the false discovery rate (FDR) = 0.05 cutoff and stochastic changes in variance estimation. Gini analysis further revealed that extraction method had no effect on the dispersion of detected transcripts across the entire dataset. To demonstrate the power of multi-omics integration on interrogated metabolic phenotypes, we next performed integrated pathway enrichment analysis on RNAseq data and metabolomics data. Our analysis revealed pyrimidine metabolism as the most impacted pathway by LCMV infection. Plotting up- and down-regulated genes and metabolites on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pyrimidine pathway exposed a pattern enzymatic degradation of pyrimidine nucleotides to generate the nucleobase uracil. Further, uracil was among the most differentially abundant metabolite in serum of LCMV infected mice, suggesting a novel mechanism of hepatic uracil export in acute infection response. ConclusionsWe demonstrate that prior metabolite extraction does not have a deleterious effect on RNAseq quality, which enables investigators to confidently perform metabolomics and RNAseq on the same sample. Implementation of this approach revealed a novel involvement of the hepatic pyrimidine metabolism during acute viral infection.

molecular biology↗

bioRxiv2022

Ketolysis is a metabolic driver of CD8+ T cell effector function through histone acetylation

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. However, the metabolic pathways critical for optimal T cell responses remain poorly understood. Here, we identify ketone bodies (KBs) - including {beta}-hydroxybutyrate ({beta}OHB) and acetoacetate (AcAc) - as essential fuels supporting CD8+ T cell metabolism and effector function. Ketolysis is an intrinsic feature of highly functional CD8+ T effector (Teff) cells and {beta}OHB directly increases CD8+ Teff cell IFN-{gamma} production and cytolytic activity. Using metabolic tracers, we establish that CD8+ Teff cells preferentially use KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boost the respiratory capacity of CD8+ T cells and TCA cycle-dependent metabolic pathways that fuel T cell growth. Mechanistically, we find that {beta}OHB is a major substrate for acetyl-CoA production in CD8+ T cells and regulates effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses. One Sentence summaryKetone bodies promote CD8+ T cell metabolism and effector function through regulation of epigenetic programming

immunology↗

bioRxiv2021

A non-canonical role for glutamate decarboxylase 1 in cancer cell amino acid homeostasis, independent of the GABA shunt.

Glutamate decarboxylase 1 (GAD1) is best known for its role in producing the neurotransmitter {gamma}-amino butyric acid (GABA) as part of the "GABA shunt" metabolic pathway, an alternative mechanism of glutamine anaplerosis for TCA cycle metabolism (Yogeeswari et al., 2005). However, understanding of the metabolic function of GAD1 in non-neuronal tissues has remained limited. Here, we show that GAD1 supports cancer cell proliferation independent of the GABA shunt. Despite its elevated expression in lung cancer tissue, GAD1 is not engaged in the GABA shunt in proliferating non-small cell lung cancer (NSCLC) cells, but rather is required for regulating amino acid homeostasis. Silencing GAD1 promotes a broad deficiency in amino acid uptake, leading to reduced glutamine-dependent TCA cycle metabolism and defects in serum- and amino acid-stimulated mTORC1 activation. Mechanistically, GAD1 regulates amino acid uptake through ATF4-dependent amino acid transporter expression including SLC7A5 (LAT1), an amino acid transporter required for branched chain amino acid (BCAA) uptake. Overexpression of LAT1 rescues the proliferative and mTORC1 signalling defects of GAD1-deficient tumor cells. Our results, therefore, define a non-canonical role for GAD1, independent of its characterised role in GABA metabolism, whereby GAD1 regulates amino acid homeostasis to maintain tumor cell proliferation.

cancer biology↗

bioRxiv2020

In vivo metabolite tracing of T cells

T cells are integral players in the adaptive immune system that readily adapt their metabolism to meet their energetic and biosynthetic needs. A major hurdle to understand physiologic T cell metabolism has been the differences between in vitro cell culture conditions and the complex in vivo milieu. To address this, we have developed a protocol that merges traditional immunology infection models with whole-body metabolite infusion and mass spectrometry (MS)-based metabolomic profiling to assess T cell metabolism in vivo. In this protocol, pathogen-infected mice are infused via the tail vein with an isotopically labeled metabolite, followed by rapid magnetic bead isolation to purify T cell populations and then stable isotope labeling (SIL) analysis conducted by MS. This procedure enables researchers to evaluate metabolic substrate utilization by specific T cell subpopulations in the context of physiological immune responses in vivo.

molecular biology↗