Biology subjects

Sheldon, R. D.

Publications and source records attributed to Sheldon, R. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Reducing background ion burden in tributylamine ion-pairing LC-MS improves signal intensity and feature coverage in metabolomics

Background chemical ions are a pervasive but often underappreciated limitation in LC-MS metabolomics, where they can suppress analyte signal, obscure endogenous metabolites, increase spectral complexity, and consume MS/MS acquisition events. Tributylamine (TBA) ion-pairing reversed-phase LC-MS provides stable retention and broad coverage of polar anionic metabolites, including central carbon intermediates, nucleotides, cofactors, and bile acids, but the back-ground burden introduced by the ion-pairing reagent itself has not been systematically addressed. Here, we identify commercial TBA as a major source of nonbiological contaminant ions and develop a practical strategy to reduce background burden while preserving metabolite coverage. Serial solid-phase extraction of TBA using orthogonal reversed-phase, strong anion-exchange, and strong cation-exchange sorbents removed chemically diverse contaminants, including isobaric background ions that interfered with endogenous hydroxybutyrate isomers. We further optimized the workflow by reducing medronic acid concentration, restricting medronic acid to the organic mobile phase, replacing phosphoric-acid column conditioning with metal-passivated column hardware, and adding EDTA to the sample reconstitution solvent to improve citrate detection. In mouse liver extracts, the optimized method increased signal intensity for most annotated metabolites and improved the fraction of full-scan ion current attributable to target analytes. Method optimization also altered compound-specific retention behavior, resolving some co-elution-based interferences while introducing new suppression relationships for selected analytes. Across mouse liver, human B lymphocytes, and NIST SRM 1950 plasma, the optimized workflow increased total feature detection by 45%, 72%, and 42%, respectively, and improved the number of low-variance features, precursors with data-dependent MS/MS spectra, and MS/MS library matches. These findings establish background-ion mitigation as a central design principle for LC-MS method development. More broadly, this work provides a generalizable framework for identifying, reducing, and validating reagent- and additive-derived background to improve targeted and untargeted LC-MS data quality.

biochemistry↗

bioRxiv2026

Nanoflow ion-pairing LC-MS for ultra-low-input polar metabolomics and isotope tracing

Low-input and single-cell metabolomics remain constrained by the poor retention of polar metabolites in conventional reversed-phase nanoflow LC-MS workflows. Here, we establish nanoflow tributylamine (TBA) ion-pairing LC-MS as a platform for ultra-low-input polar metabolomics and stable isotope tracing. By adapting an analytical-flow TBA ion-pairing method to the nanoflow scale, this workflow extends the sensitivity and inline concentration advantages of nanoflow chromatography to charged metabolites involved in central carbon metabolism. Using mouse liver metabolite extracts, we show that the nanoflow method preserves chromatographic retention and separation of chemically diverse metabolite classes, including adenine nucleotides, nucleotide cofactors, TCA cycle intermediates, acyl-CoAs, and bile acid isomers. Despite loading 20-fold less tissue-equivalent material on column, nanoflow LC-MS produced higher signal intensity than the analytical-flow method for many metabolites. Across representative compounds, the nanoflow workflow reduced the biomass required for detection by approximately 20- to >600-fold, with pronounced gains for low-abundance metabolites such as NADPH and acetyl-CoA. TBA ion-pairing also enabled trap-and-elute nanoflow analysis of retained polar metabolites from single-cell-equivalent inputs. ATP was detected from one cell equivalent using both full-scan and targeted parallel reaction monitoring acquisition, with targeted acquisition further increasing signal over blank. Finally, we applied the workflow to stable isotope tracing in uniformly labeled 13C-glucose-treated cells. 13C-labeled ATP isotopologues were detectable from single-cell-equivalent input, and targeted acquisition improved isotopologue measurement near the detection limit. Together, these results demonstrate that nanoflow TBA ion-pairing LC-MS enables retained, high-sensitivity analysis of polar metabolites from ultra-low inputs and provides a foundation for extending central carbon metabolite analysis and isotope tracing toward single-cell-scale applications.

biochemistry↗

bioRxiv2026

Functional partitioning of lipoic acid decouples cellular abundance from mitochondrial utilization

-Lipoic acid (LA) is widely included in "mitochondrial cocktails" recommended to patients with primary mitochondrial disorders, yet its mechanism of action remains unclear. Here, we define the intracellular availability and functional utilization of LA in mammalian cells. We show that LA exists in two functionally distinct cellular pools: a low-abundance free pool and a protein-bound pool generated through mitochondrial fatty acid synthesis (mtFAS). Disruption of the mtFAS pathway abolishes protein lipoylation and impairs oxidative phosphorylation without altering free LA levels. Conversely, supplementation with exogenous LA markedly increases free intracellular LA without restoring protein lipoylation, mitochondrial respiration, or cell proliferation. Instead, the cellular effects of LA supplementation resemble those of the antioxidant N-acetylcysteine. These findings clarify the mechanism of action of a widely used mitochondrial supplement and identify a fundamental disconnect between cellular LA abundance and mitochondrial utilization, challenging the rationale for using LA supplementation to restore mitochondrial function.

biochemistry↗

bioRxiv2026

Cells Engage Endogenous Malonate Synthesis to Drive Mitochondrial Metabolism

Malonate is often described as an endogenous inhibitor of complex II of the electron transport chain. However, the cellular source of malonate is unclear, and current knowledge concerning its metabolism is limited to the action of a single enzyme, Acyl-CoA Synthetase Family Member 3 (ACSF3), which converts malonate to malonyl-CoA in the mitochondrial matrix. One potential route of malonate metabolism downstream of ACSF3 is its consumption by the mitochondrial fatty acid synthesis (mtFAS) pathway. However, studies examining the link between ACSF3 and mtFAS have yielded conflicting results. We developed a novel mass spectrometry approach to perform stable isotope tracing into products of mtFAS, and found that while malonate is in fact a carbon source for mtFAS, ACSF3 is not required for malonate incorporation into mtFAS products. Using this method to trace other nutrients into mtFAS, we also found evidence of acetyl-CoA carboxylase 1 (ACC1)-dependent malonate synthesis from glucose. We further show that ACC1 is required for optimal mtFAS activity, with downstream effects on oxidative phosphorylation. Together these findings establish the malonate as a regulated endogenous intermediate that supports mtFAS activity and mitochondrial oxidative function.

biochemistry↗

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

Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking

Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic acid and adrenic acid. These PUFAs then accumulate in phospholipids, particularly ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools dictates their sensitivity to ferroptosis.

cancer biology↗

bioRxiv2024

Single-embryo metabolomics reveals developmental metabolism in the early Drosophila embryo

Early embryonic development is characterized by the transition from maternal factor reliance to zygotic control. These processes set the stage for the embryos basic structure and cellular differentiation. While relatively detailed knowledge exists of the transcriptional events during early development, little is known about the concurrent metabolic processes. Understanding these processes, however, is important since they are linked to cell fate determination and organ and tissue formation. The primary reasons for the limited progress in the field are technical limitations due to the small amount of material available during early embryonic time windows. Here, we introduce a novel single-embryo methodology that places us in an exciting position to analyze the early embryos metabolome and transcriptome in an integrated manner and at high temporal resolution. The resulting data allow us to map concomitant metabolic and transcriptional programs in early Drosophila embryonic development. Our results reveal that a substantial number of metabolites exhibit dynamic patterns with some changing even before the onset of zygotic transcription. dNTPs for example show a temporal pattern that correlates with cell division patterns in the early embryo. In summary, here we present an operationally simple single-embryo metabolomics methodology and provide a detailed picture of early developmental metabolic processes at unprecedented temporal resolution.

developmental biology↗

bioRxiv2023

A diverse proteome is present and enzymatically active in metabolite extracts

Metabolomics, a foundational tool in metabolism research, relies on the accurate transmittal of biochemical profiles underlying biological phenotypes. Over the years, workflows used in metabolomics have been assumed to remove enzymes to preserve metabolite levels during processing. Here, we uncover a diverse landscape of over 1,000 proteins, strongly enriched for metabolic enzymes, within metabolite extracts generated using common extraction workflows. Moreover, by combining in-extract stable isotope additions and enzyme inhibitors, we demonstrate transaminase activity, which is preventable by protein removal by 3 kDa filtration. We extend these findings to untargeted metabolomics, revealing that both post-extraction formation of glutamate dipeptide and depletion of total glutathione can also be prevented by removing proteins. Finally, we present a simple yet novel workflow that integrates passive filtration for protein removal of crude metabolite extracts as a superior method for broad-coverage metabolomics. Our findings have broad-reaching experimental implications across all fields that use metabolomics and molecular metabolism, especially cancer, immunology, and diabetes research.

biochemistry↗

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

NF1 deficiency drives metabolic reprogramming in ER+ breast cancer

ObjectiveNF1 is a tumor suppressor gene and its protein product, neurofibromin, is the key negative regulator of the RAS pathway. NF1 is one of the top driver mutations in sporadic breast cancer such that 27% of breast cancers exhibit damaging NF1 alterations. NF1 loss-of-function is a frequent event in the genomic evolution of estrogen receptor (ER)+ breast cancer metastasis and endocrine resistance. Individuals with Neurofibromatosis type 1 (NF) - a disorder caused by germline NF1 mutations - have an increased risk of dying from breast cancer [1-4]. NF-related breast cancers are associated with decreased overall survival compared to sporadic breast cancer. Despite numerous studies interrogating the role of RAS mutations in tumor metabolism, no study has comprehensively profiled the NF1-mutant breast cancer metabolome to define patterns of energetic and metabolic reprogramming. The goals of this investigation were (1) to define the role of NF1 deficiency in estrogen receptor-positive (ER+) breast cancer metabolic reprogramming and (2) to identify potential targeted pathway and metabolic inhibitor combination therapies for NF1-deficient ER+ breast cancer. MethodsWe employed two ER+ NF1-deficient breast cancer models: (1) an NF1-mutant MCF7 breast cancer cell line to model sporadic breast cancer, and (2) three distinct, Nf1-deficient rat models to model NF- related breast cancer [1]. IncuCyte proliferation analysis was used to measure the effect of NF1 deficiency on cell proliferation and drug response. Protein quantity was assessed by Western Blot analysis. We then used RNAseq to investigate the transcriptional effect of NF1 deficiency on global and metabolism-related transcription. We measured cellular energetics using Agilent Seahorse XF-96 Glyco Stress Test and Mito Stress Test assays. We performed stable isotope labeling and measured [U-13C]- glucose and [U-13C]-glutamine metabolite incorporation and measured total metabolite pools using mass spectrometry. Lastly, we used a Bliss synergy model to investigate NF1-driven changes in targeted and metabolic inhibitor synergy. ResultsOur results revealed that NF1 deficiency enhanced cell proliferation, altered neurofibromin expression, and increased RAS and PI3K/AKT pathway signaling while constraining oxidative ATP production and restricting energetic flexibility. Neurofibromin deficiency also increased glutamine influx into TCA intermediates and dramatically increased lipid pools, especially triglycerides (TG). Lastly, NF1 deficiency alters the synergy between metabolic inhibitors and traditional targeted inhibitors. This includes increased synergy with inhibitors targeting glycolysis, glutamine metabolism, mitochondrial fatty acid transport, and TG synthesis. ConclusionsNF1 deficiency drives metabolic reprogramming in ER+ breast cancer. This reprogramming is characterized by oxidative ATP constraints, glutamine TCA influx, and lipid pool expansion, and these metabolic changes introduce novel metabolic-to-targeted inhibitor synergies. HIGHLIGHTSO_LINF1 deficiency drives metabolic reprogramming in ER+ breast cancer. C_LIO_LINF1-driven metabolic reprogramming is characterized by oxidative ATP constraints, glutamine TCA influx, and lipid pool expansion. C_LIO_LINF1-deficient ER+ breast cancer cells have increased sensitivity to a combination of RAS and triglyceride synthesis inhibitors. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/568339v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1f4d135org.highwire.dtl.DTLVardef@1d3168forg.highwire.dtl.DTLVardef@16e083forg.highwire.dtl.DTLVardef@df6c1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Mitochondrial citrate metabolism and efflux regulates trophoblast differentiation

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and -ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.

developmental 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

PGC-1β maintains mitochondrial metabolism and restrains inflammatory gene expression

Metabolic programming of the innate immune cells known as dendritic cells (DCs) changes in response to different stimuli, influencing their function. While the mechanisms behind increased glycolytic metabolism in response to inflammatory stimuli are well-studied, less is known about the programming of mitochondrial metabolism in DCs. We used lipopolysaccharide (LPS) and interferon-{beta} (IFN-{beta}), which differentially stimulate the use of glycolysis and oxidative phosphorylation (OXPHOS), respectively, to identify factors important for mitochondrial metabolism. We found that the expression of peroxisome proliferator-activated receptor gamma coactivator 1{beta} (PGC-1{beta}), a transcriptional co-activator and known regulator of mitochondrial metabolism, decreases when DCs are activated with LPS, when OXPHOS is diminished, but not with IFN-{beta}, when OXPHOS is maintained. We examined the role of PGC-1{beta} in bioenergetic metabolism of DCs and found that PGC-1{beta} deficiency in DCs indeed impairs mitochondrial respiration. PGC-1{beta}-deficient DCs are more glycolytic compared to controls, likely to compensate for reduced OXPHOS. PGC-1{beta} deficiency also causes decreased capacity for ATP production at steady state and in response to IFN-{beta} treatment. Loss of PGC-1{beta} in DCs leads to increased expression of genes in inflammatory pathways, and reduced expression of genes encoding proteins important for mitochondrial metabolism and function. Collectively, these results demonstrate that PGC-1{beta} is a key positive regulator of mitochondrial metabolism in DCs.

immunology↗

bioRxiv2021

Small molecule SWELL1-LRRC8 complex induction improves glycemic control and nonalcoholic fatty liver disease in murine Type 2 diabetes

Type 2 diabetes (T2D) is associated with insulin resistance, impaired insulin secretion from the pancreatic {beta}-cell, and nonalcoholic fatty liver disease (NAFLD). SWELL1 (LRRC8a) ablation impairs adipose and skeletal muscle insulin-pAKT2 signaling, {beta}-cell insulin secretion and glycemic control - suggesting that SWELL1-LRRC8 complex dysfunction contributes to T2D pathogenesis. Here, we show that ICl,SWELL and SWELL1 protein are reduced in adipose and {beta}-cells in murine and human T2D. Combining cryo-electron microscopy, molecular docking, medicinal chemistry, and functional studies, we define a structure activity relationship to rationally-designed active derivatives (SN-40X) of a SWELL1 channel inhibitor (DCPIB/SN-401), that bind the SWELL1-LRRC8 hexameric complex, restore SWELL1-LRRC8 protein, plasma membrane trafficking, signaling and islet insulin secretion via SWELL1-dependent mechanisms. In vivo, SN-401 and active SN-40X compounds restore glycemic control and prevents NAFLD by improving insulin-sensitivity and insulin secretion in murine T2D. These findings demonstrate that small molecule SWELL1 modulators restore SWELL1-dependent insulin-sensitivity and insulin secretion in T2D and may represent a first-in-class therapeutic approach for T2D and NAFLD.

physiology↗

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↗