PERK is a critical metabolic hub for immunosuppressive function in macrophages.

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity.

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity.

Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila.

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

Omic-Scale High-Throughput Quantitative LC-MS/MS Approach for Circulatory Lipid Phenotyping in Clinical Research.

Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.

Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Urinary metabotype of severe asthma evidences decreased carnitine metabolism independent of oral corticosteroid treatment in the U-BIOPRED study.

INTRODUCTION: Asthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication. METHODS: Baseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12-18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods. RESULTS: A total of 90 metabolites were identified, with 40 significantly altered (p<0.05, false discovery rate <0.05) in severe asthma and 23 by OCS use. Multivariate modelling showed that observed metabotypes in healthy participants and patients with mild-to-moderate asthma differed significantly from those in patients with severe asthma (p=2.6×10-20), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10-4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings. CONCLUSIONS: This is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma.

Metabolomics meets lipidomics: Assessing the small molecule component of metabolism.

Metabolomics, including lipidomics, is emerging as a quantitative biology approach for the assessment of energy flow through metabolism and information flow through metabolic signaling; thus, providing novel insights into metabolism and its regulation, in health, healthy ageing and disease. In this forward-looking review we provide an overview on the origins of metabolomics, on its role in this postgenomic era of biochemistry and its application to investigate metabolite role and (bio)activity, from model systems to human population studies. We present the challenges inherent to this analytical science, and approaches and modes of analysis that are used to resolve, characterize and measure the infinite chemical diversity contained in the metabolome (including lipidome) of complex biological matrices. In the current outbreak of metabolic diseases such as cardiometabolic disorders, cancer and neurodegenerative diseases, metabolomics appears to be ideally situated for the investigation of disease pathophysiology from a metabolite perspective.

Sex-specific alterations in NAD+ metabolism in 3xTg Alzheimer's disease mouse brain assessed by quantitative targeted LC-MS.

Levels of nicotinamide adenine dinucleotide (NAD+) are known to decline with age and have been associated with impaired mitochondrial function leading to neurodegeneration, a key facet of Alzheimer's disease (AD). NAD+synthesis is sustained via tryptophan-kynurenine (Trp-Kyn) pathway as de novo synthesis route, and salvage pathways dependent on the availability of nicotinic acid and nicotinamide. While being currently investigated as a multifactorial disease with a strong metabolic component, AD remains without curative treatment and important sex differences were reported in relation to disease onset and progression. The aim of this study was to reveal the potential deregulation of NAD+metabolism in AD with the direct analysis of NAD+precursors in the mouse brain tissue (wild type (WT) versus triple transgenic (3xTg) AD), using a sex-balanced design. To this end, we developed a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which allowed for the measurement of the full spectrum of NAD+precursors and intermediates in all three pathways. In brain tissue of mice with developed AD symptoms, a decrease in kynurenine (Kyn) versus increase in kynurenic acid (KA) levels were observed in both sexes with a significantly higher increment of KA in males. These alterations in Trp-Kyn pathway might be a consequence of neuroinflammation and a compensatory production of neuroprotective kynurenic acid. In the NAD+ salvage pathway, significantly lower levels of nicotinamide mononucleotide (NMN) were measured in the AD brain of males and females. Depletion of NMN implies the deregulation of salvage pathway critical for maintaining optimal NAD+ levels and mitochondrial and neuronal function.

Merged Targeted Quantification and Untargeted Profiling for Comprehensive Assessment of Acylcarnitine and Amino Acid Metabolism.

Acylcarnitines and amino acids are key players in energy metabolism; however, analytical methods for comprehensive and straightforward quantitative profiling of these metabolites, without derivatization or use of ion-pairing agents, are lacking. We therefore developed a hydrophilic interaction chromatography (HILIC)-based high-resolution mass spectrometry (HRMS) method for the simultaneous quantification of acylcarnitines and amino acids in a single run, while taking advantage of HRMS data acquired in full-scan mode to screen for additional derivatives and other polar metabolites. A single-step metabolite extraction with internal standard mixture (in methanol) warranted high-throughput sample preparation whose applicability was demonstrated on a panel of human biofluids (i.e., blood plasma, CSF, and urine) and brain tissue. Method accuracy was within 90-106% of validated NIST reference plasma concentrations for the panel of measured amino acids. Amino acid and acylcarnitine extraction recoveries were 87-100% on average, depending on the concentration range spiked. The coefficient of variation (CV) was 1-10% and 1-25% for intra- and interday measurements, respectively, with the highest CVs for the metabolites at the limit of quantification, depending on the biofluid. Acylcarnitine and amino acid signatures or chemical composition barcodes of the different biofluids and human brain tissue were acquired and biofluid- and tissue-associated differences were discussed in the context of their respective physiological roles. Significant differences were observed in the amino acid profiles, whereas acylcarnitine composition did not show biofluid-characteristic or brain region-specific pattern. The retrospective exploration of full-scan all-ion-fragmentation data allowed us to extract the information on unsaturated and hydroxylated acylcarnitine species, amines, and purine and pyrimidine metabolites. This merged targeted and untargeted approach provides an innovative strategy for simultaneous and comprehensive assessment of acylcarnitine and amino acid metabolism in clinical research studies using relevant biofluids and tissue extracts.

New in vitro model derived from brain-specific Mut-/- mice confirms cerebral ammonium accumulation in methylmalonic aciduria.

BACKGROUND: Methylmalonic aciduria (MMAuria) is an inborn error of metabolism leading to neurological deterioration. In this study, we used 3D organotypic brain cell cultures derived from embryos of a brain-specific Mut-/- (brain KO) mouse to investigate mechanisms leading to brain damage. We challenged our in vitro model by a catabolic stress (temperature shift). RESULTS: Typical metabolites for MMAuria as well as a massive NH4+ increase were found in the media of brain KO cultures. We investigated different pathways of intracerebral NH4+ production and found increased expression of glutaminase 2 and diminished expression of GDH1 in Mut-/- aggregates. While all brain cell types appeared affected in their morphological development in Mut-/- aggregates, the most pronounced effects were observed on astrocytes showing swollen fibers and cell bodies. Inhibited axonal elongation and delayed myelination of oligodendrocytes were also noted. Most effects were even more pronounced after 48 h at 39 °C. Microglia activation and an increased apoptosis rate suggested degeneration of Mut-/- brain cells. NH4+ accumulation might be the trigger for all observed alterations. We also found a generalized increase of chemokine concentrations in Mut-/- culture media at an early developmental stage followed by a decrease at a later stage. CONCLUSION: We proved for the first time that Mut-/- brain cells are indeed able to produce the characteristic metabolites of MMAuria. We confirmed significant NH4+ accumulation in culture media of Mut-/- aggregates, suggesting that intracellular NH4+ concentrations might even be higher, gave first clues on the mechanisms leading to NH4+ accumulation in Mut-/- brain cells, and showed the involvement of neuroinflammatory processes in the neuropathophysiology of MMAuria.

Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition.

High-resolution mass spectrometry (HRMS)-based metabolomics approaches have made significant advances. However, metabolite identification is still a major challenge with significant bottleneck in translating metabolomics data into biological context. In the current study, a liquid chromatography (LC)-HRMS metabolomics method was developed using an all ion fragmentation (AIF) acquisition approach. To increase the specificity in metabolite annotation, four criteria were considered: (i) accurate mass (AM), (ii) retention time (RT), (iii) MS/MS spectrum, and (iv) product/precursor ion intensity ratios. We constructed an in-house mass spectral library of 408 metabolites containing AMRT and MS/MS spectra information at four collision energies. The percent relative standard deviations between ion ratios of a metabolite in an analytical standard vs sample matrix were used as an additional metric for establishing metabolite identity. A data processing method for targeted metabolite screening was then created, merging m/z, RT, MS/MS, and ion ratio information for each of the 413 metabolites. In the data processing method, the precursor ion and product ion were considered as the quantifier and qualifier ion, respectively. We also included a scheme to distinguish coeluting isobaric compounds by selecting a specific product ion as the quantifier ion instead of the precursor ion. An advantage of the current AIF approach is the concurrent collection of full scan data, enabling identification of metabolites not included in the database. Our data acquisition strategy enables a simultaneous mixture of database-dependent targeted and nontargeted metabolomics in combination with improved accuracy in metabolite identification, increasing the quality of the biological information acquired in a metabolomics experiment.

Metabolomics analysis identifies different metabotypes of asthma severity.

In this study, we sought to determine whether asthma has a metabolic profile and whether this profile is related to disease severity.We characterised the serum from 22 healthy individuals and 54 asthmatics (12 mild, 20 moderate, 22 severe) using liquid chromatography-high-resolution mass spectrometry-based metabolomics. Selected metabolites were confirmed by targeted mass spectrometry assays of eicosanoids, sphingolipids and free fatty acids.We conclusively identified 66 metabolites; 15 were significantly altered with asthma (p≤0.05). Levels of dehydroepiandrosterone sulfate, cortisone, cortisol, prolylhydroxyproline, pipecolate and N-palmitoyltaurine correlated significantly (p<0.05) with inhaled corticosteroid dose, and were further shifted in individuals treated with oral corticosteroids. Oleoylethanolamide increased with asthma severity independently of steroid treatment (p<0.001). Multivariate analysis revealed two patterns: 1) a mean difference between controls and patients with mild asthma (p=0.025), and 2) a mean difference between patients with severe asthma and all other groups (p=1.7×10-4). Metabolic shifts in mild asthma, relative to controls, were associated with exogenous metabolites (e.g. dietary lipids), while those in moderate and severe asthma (e.g. oleoylethanolamide, sphingosine-1-phosphate, N-palmitoyltaurine) were postulated to be involved in activating the transient receptor potential vanilloid type 1 (TRPV1) receptor, driving TRPV1-dependent pathogenesis in asthma.Our findings suggest that asthma is characterised by a modest systemic metabolic shift in a disease severity-dependent manner, and that steroid treatment significantly affects metabolism.

Metabolite Profiling and Stable Isotope Tracing in Sorted Subpopulations of Mammalian Cells.

Biological samples such as tissues, blood, or tumors are often complex and harbor heterogeneous populations of cells. Separating out specific cell types or subpopulations from such complex mixtures to study their metabolic phenotypes is challenging because experimental procedures for separation may disturb the metabolic state of cells. To address this issue, we developed a method for analysis of cell subpopulations using stable isotope tracing and fluorescence-activated cell sorting followed by liquid chromatography-high-resolution mass spectrometry. To ensure a faithful representation of cellular metabolism after cell sorting, we benchmarked sorted extraction against direct extraction. While peak areas differed markedly with lower signal for amino acids but higher signal for nucleotides, mass isotopomer distributions from sorted cells were generally in good agreement with those obtained from direct extractions, indicating that they reflect the true metabolic state of cells prior to sorting. In proof-of-principle studies, our method revealed metabolic phenotypes specific to T cell subtypes, and also metabolic features of cells in the committed phase of the cell division cycle. Our approach enables studies of a wide range of adherent and suspension cell subpopulations, which we anticipate will be of broad importance in cell biology and biomedicine.

E2F transcription factor-1 modulates expression of glutamine metabolic genes in mouse embryonic fibroblasts and uterine sarcoma cells.

Metabolic reprogramming is considered as a hallmark of cancer and is clinically exploited as a novel target for therapy. The E2F transcription factor-1 (E2F1) regulates various cellular processes, including proliferative and metabolic pathways, and acts, depending on the cellular and molecular context, as an oncogene or tumor suppressor. The latter is evident by the observation that E2f1-knockout mice develop spontaneous tumors, including uterine sarcomas. This dual role warrants a detailed investigation of how E2F1 loss impacts metabolic pathways related to cancer progression. Our data indicate that E2F1 binds to the promoter of several glutamine metabolism-related genes. Interestingly, the expression of genes in the glutamine metabolic pathway were increased in mouse embryonic fibroblasts (MEFs) lacking E2F1. In addition, we confirm that E2f1-/- MEFs are more efficient in metabolizing glutamine and producing glutamine-derived precursors for proliferation. Mechanistically, we observe a co-occupancy of E2F1 and MYC on glutamine metabolic promoters, increased MYC binding after E2F1 depletion and that silencing of MYC decreased the expression of glutamine-related genes in E2f1-/- MEFs. Analyses of transcriptomic profiles in 29 different human cancers identified uterine sarcoma that showed a negative correlation between E2F1 and glutamine metabolic genes. CRISPR/Cas9 knockout of E2F1 in the uterine sarcoma cell line SK-UT-1 confirmed elevated glutamine metabolic gene expression, increased proliferation and increased MYC binding to glutamine-related promoters upon E2F1 loss. Together, our data suggest a crucial role of E2F1 in energy metabolism and metabolic adaptation in uterine sarcoma cells.

Inhibition of CERS1 in skeletal muscle exacerbates age-related muscle dysfunction.

Age-related muscle wasting and dysfunction render the elderly population vulnerable and incapacitated, while underlying mechanisms are poorly understood. Here, we implicate the CERS1 enzyme of the de novo sphingolipid synthesis pathway in the pathogenesis of age-related skeletal muscle impairment. In humans, CERS1 abundance declines with aging in skeletal muscle cells and, correlates with biological pathways involved in muscle function and myogenesis. Furthermore, CERS1 is upregulated during myogenic differentiation. Pharmacological or genetic inhibition of CERS1 in aged mice blunts myogenesis and deteriorates aged skeletal muscle mass and function, which is associated with the occurrence of morphological features typical of inflammation and fibrosis. Ablation of the CERS1 orthologue lagr-1 in Caenorhabditis elegans similarly exacerbates the age-associated decline in muscle function and integrity. We discover genetic variants reducing CERS1 expression in human skeletal muscle and Mendelian randomization analysis in the UK biobank cohort shows that these variants reduce muscle grip strength and overall health. In summary, our findings link age-related impairments in muscle function to a reduction in CERS1, thereby underlining the importance of the sphingolipid biosynthesis pathway in age-related muscle homeostasis.

Effect of an eight-week high-intensity interval training programme on circulating sphingolipid levels in middle-aged adults at elevated cardiometabolic risk (SphingoFIT)-Protocol for a randomised controlled exercise trial.

INTRODUCTION: Evidence indicates that sphingolipid accumulation drives complex molecular alterations promoting cardiometabolic diseases. Clinically, it was shown that sphingolipids predict cardiometabolic risk independently of and beyond traditional biomarkers such as low-density lipoprotein cholesterol. To date, little is known about therapeutic modalities to lower sphingolipid levels. Exercise, a powerful means to prevent and treat cardiometabolic diseases, is a promising modality to mitigate sphingolipid levels in a cost-effective, safe, and patient-empowering manner. METHODS: This randomised controlled trial will explore whether and to what extent an 8-week fitness-enhancing training programme can lower serum sphingolipid levels of middle-aged adults at elevated cardiometabolic risk (n = 98, 50% females). The exercise intervention will consist of supervised high-intensity interval training (three sessions weekly), while the control group will receive physical activity counselling based on current guidelines. Blood will be sampled early in the morning in a fasted state before and after the 8-week programme. Participants will be provided with individualised, pre-packaged meals for the two days preceding blood sampling to minimise potential confounding. An 'omic-scale sphingolipid profiling, using high-coverage reversed-phase liquid chromatography coupled to tandem mass spectrometry, will be applied to capture the circulating sphingolipidome. Maximal cardiopulmonary exercise tests will be performed before and after the 8-week programme to assess patient fitness changes. Cholesterol, triglycerides, glycated haemoglobin, the homeostatic model assessment for insulin resistance, static retinal vessel analysis, flow-mediated dilatation, and strain analysis of the heart cavities will also be assessed pre- and post-intervention. This study shall inform whether and to what extent exercise can be used as an evidence-based treatment to lower circulating sphingolipid levels. TRIAL REGISTRATION: The trial was registered on www.clinicaltrials.gov (NCT06024291) on August 28, 2023.

Experimental evolution of metabolism under nutrient restriction: enhanced amino acid catabolism and a key role of branched-chain amino acids.

Periodic food shortage is a common ecological stressor for animals, likely to drive physiological and metabolic adaptations to alleviate its consequences, particularly for juveniles that have no option but to continue to grow and develop despite undernutrition. Here we study changes in metabolism associated with adaptation to nutrient shortage, evolved by replicate Drosophila melanogaster populations maintained on a nutrient-poor larval diet for over 240 generations. In a factorial metabolomics experiment we showed that both phenotypic plasticity and genetically-based adaptation to the poor diet involved wide-ranging changes in metabolite abundance; however, the plastic response did not predict the evolutionary change. Compared to nonadapted larvae exposed to the poor diet for the first time, the adapted larvae showed lower levels of multiple free amino acids in their tissues-and yet they grew faster. By quantifying accumulation of the nitrogen stable isotope 15N we show that adaptation to the poor diet led to an increased use of amino acids for energy generation. This apparent "waste" of scarce amino acids likely results from the trade-off between acquisition of dietary amino acids and carbohydrates observed in these populations. The three branched-chain amino acids (leucine, isoleucine, and valine) showed a unique pattern of depletion in adapted larvae raised on the poor diet. A diet supplementation experiment demonstrated that these amino acids are limiting for growth on the poor diet, suggesting that their low levels resulted from their expeditious use for protein synthesis. These results demonstrate that selection driven by nutrient shortage not only promotes improved acquisition of limiting nutrients, but also has wide-ranging effects on how the nutrients are used. They also show that the abundance of free amino acids in the tissues does not, in general, reflect the nutritional condition and growth potential of an animal.

Molecular insights into sex-specific metabolic alterations in Alzheimer's mouse brain using multi-omics approach.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by altered cellular metabolism in the brain. Several of these alterations have been found to be exacerbated in females, known to be disproportionately affected by AD. We aimed to unravel metabolic alterations in AD at the metabolic pathway level and evaluate whether they are sex-specific through integrative metabolomic, lipidomic, and proteomic analysis of mouse brain tissue. METHODS: We analyzed male and female triple-transgenic mouse whole brain tissue by untargeted mass spectrometry-based methods to obtain a molecular signature consisting of polar metabolite, complex lipid, and protein data. These data were analyzed using multi-omics factor analysis. Pathway-level alterations were identified through joint pathway enrichment analysis or by separately evaluating lipid ontology and known proteins related to lipid metabolism. RESULTS: Our analysis revealed significant AD-associated and in part sex-specific alterations across the molecular signature. Sex-dependent alterations were identified in GABA synthesis, arginine biosynthesis, and in alanine, aspartate, and glutamate metabolism. AD-associated alterations involving lipids were also found in the fatty acid elongation pathway and lysophospholipid metabolism, with a significant sex-specific effect for the latter. CONCLUSIONS: Through multi-omics analysis, we report AD-associated and sex-specific metabolic alterations in the AD brain involving lysophospholipid and amino acid metabolism. These findings contribute to the characterization of the AD phenotype at the molecular level while considering the effect of sex, an overlooked yet determinant metabolic variable.

Evolutionary adaptation to juvenile malnutrition impacts adult metabolism and impairs adult fitness in Drosophila.

Juvenile undernutrition has lasting effects on adult metabolism of the affected individuals, but it is unclear how adult physiology is shaped over evolutionary time by natural selection driven by juvenile undernutrition. We combined RNAseq, targeted metabolomics, and genomics to study the consequences of evolution under juvenile undernutrition for metabolism of reproductively active adult females of Drosophila melanogaster. Compared to Control populations maintained on standard diet, Selected populations maintained for over 230 generations on a nutrient-poor larval diet evolved major changes in adult gene expression and metabolite abundance, in particular affecting amino acid and purine metabolism. The evolved differences in adult gene expression and metabolite abundance between Selected and Control populations were positively correlated with the corresponding differences previously reported for Selected versus Control larvae. This implies that genetic variants affect both stages similarly. Even when well fed, the metabolic profile of Selected flies resembled that of flies subject to starvation. Finally, Selected flies had lower reproductive output than Controls even when both were raised under the conditions under which the Selected populations evolved. These results imply that evolutionary adaptation to juvenile undernutrition has large pleiotropic consequences for adult metabolism, and that they are costly rather than adaptive for adult fitness. Thus, juvenile and adult metabolism do not appear to evolve independently from each other even in a holometabolous species where the two life stages are separated by a complete metamorphosis.

PI5P4Kα supports prostate cancer metabolism and exposes a survival vulnerability during androgen receptor inhibition.

Phosphatidylinositol (PI)regulating enzymes are frequently altered in cancer and have become a focus for drug development. Here, we explore the phosphatidylinositol-5-phosphate 4-kinases (PI5P4K), a family of lipid kinases that regulate pools of intracellular PI, and demonstrate that the PI5P4Kα isoform influences androgen receptor (AR) signaling, which supports prostate cancer (PCa) cell survival. The regulation of PI becomes increasingly important in the setting of metabolic stress adaptation of PCa during androgen deprivation (AD), as we show that AD influences PI abundance and enhances intracellular pools of PI-4,5-P2. We suggest that this PI5P4Kα-AR relationship is mitigated through mTORC1 dysregulation and show that PI5P4Kα colocalizes to the lysosome, the intracellular site of mTORC1 complex activation. Notably, this relationship becomes prominent in mouse prostate tissue following surgical castration. Finally, multiple PCa cell models demonstrate marked survival vulnerability following stable PI5P4Kα inhibition. These results nominate PI5P4Kα as a target to disrupt PCa metabolic adaptation to castrate resistance.