Poster session 2

The neonatal mammalian heart is capable of substantial regeneration, however this capacity is lost in the adult heart. Interestingly, this is accompanied by a shift in the metabolic pathways and energetic fuels, from embryonic glycolysis to postnatal mitochondrial oxidative phosphorylation. 

The use of direct reprogramming of resident cardiac fibroblasts by cardiogenic transcription factors, Mef2c, Gata4, and Tbx5 (MGT), can create induced cardiac-like myocytes (iCLMs). Besides holding great promise, direct cardiac conversion still lacks effectiveness. 

We observed that retroviral-induced MGT transduction produced increase expression of cardiac troponin T in mouse embryonic (2%) compared to adult ear (0,5%) fibroblasts. Preliminary results suggests that nutrient composition impacts the efficiency of direct cardiac conversion and that this process is accompanied by profound differences in the histone acetylation and methylation landscape. 

These results suggest that exploring the crosstalk between nutrient signaling, epigenetic and age associated barriers can reveal unprecedented information for improved cardiac regenerative strategies.

Most cancer related deaths are due to metastases. Systemic factors, such as lipid-enriched environments (particularly LDL-cholesterol), favor breast cancer (including triple negative breast cancer, TNBC) metastasis formation. Mitochondria metabolism has been implicated in the invasive behavior of TNBC cells relying on fatty acid oxidation (FAO)-dependent energy production. Thus, reprogramming of mitochondrial metabolism may underlie the LDL effects on TNBC cells. Here we show that LDL augments TNBC cells migration and invasion in vivo and in vitro, and is accompanied by increased mitochondrial mass and altered distribution in migrating cells. Energetic analyses revealed that LDL renders TNBC dependent on fatty acids for mitochondrial respiration, decreasing their capacity to adapt to other fuels, such as glucose and engagement of FAO, but not glycolysis, is required for the LDL-induced migration and mitochondrial remodeling. Moreover, LDL exposed TNBC cells present increased CD36 expression and augmented lipid droplets and reactive oxygen species (ROS) and, importantly, CD36 or ROS blockage abolished the LDL-induced cell migration and mitochondria metabolic adaptations. Our results suggest that LDL reprogramming of TNBC cells mitochondrial metabolism favors migration, a new vulnerability in invasive and metastatic breast cancer. 

Peptide protocols are gaining momentum in aging and disease models. The therapeutic relevance of peptide signaling focused on cellular efficiency, metabolic flexibility, reduction of cellular senescence and optimizing endogenous stem cell quiescence can change the fate of untoward epigenetic influences in aging.   

Improved cellular efficiency has the ability to promote favorable outcomes for metabolic, infectious and immune disease regardless of the etiology. Metabolic flexibility of the cell modulates interaction of cell metabolism and 2 way communication with the innate and adaptive immune system. Loss of metabolic flexibility leads to changes in thermodynamic ratios of nucleotide coenzyme couples NAD/NADH,NADP/NADPH,ADP/ATP and AcetylCoA/CoA  in the mitochondria and cell cytoplasm disrupting cellular homeostasis.  These cellular changes in redox and altered mitochondrial function decreases the cells ability to maintain adequate antioxidant transcription. Increasing Oxidative stress overtakes the cellular capacity for DNA damage for repair, autophagy and mitophagy . P53 tumor suppressor is a master regulator in maintaining cellular Redox, efficiency and immune cell interaction. P53 controls cellular progression to the proinflammatory state of irreversible SASP or to cellular apoptosis. Peptide therapies offer natural, nontoxic directed cell signaling to alter redox, initiate stem cell migration and offer senomodulating and senolytic potentials. Though genotype is not reversible, Peptide therapies may reverse epigenetic changes and alter cellular phenotype for improved cellular function. We will define particular peptides and the molecular pathways to improve cell efficiency, reduce cell senescence and improve stem cell quiescence.

Mitochondrial stress triggers a response in the cell’s mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. We characterized a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. Furthermore, we developed a novel knock-in mouse model and found that mutant CHCHD10 aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, survival of CHCHD10 knock-in mice depended on a protective stress response mediated by OMA1. The OMA1 stress response acted both locally within mitochondria, inhibiting mitochondrial fusion, and signaled globally, activating the integrated stress response. We additionally identified an isoform switch in the terminal complex of the electron transport chain as a novel component of this response. Our results demonstrate that OMA1 is essential for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.

Beat-to-Beat contractions of the heart rely on ATP synthesis in mitochondria and myofilament ATP hydrolysis, which alters intracellular pH (pH_i). pH_i is a crucial regulator of cellular structure and function. However, it remains unknown how cardiac cells handle pH_i on a beat-to-beat basis. We developed a method to measure pH_i and sarcomere contractions simultaneously in cardiomyocytes. We demonstrated the beat-to-beat intracellular acidification in synchrony with cardiomyocyte contractions, termed “pH_i transients”. pH_i transients are inexplicably coupled with cardiomyocyte contractions and tightly regulated. Inhibition of the mitochondrial electron transport chain significantly attenuates pH_i transients. Indeed, beat-to-beat pH_i transients reflect the rhythmic metabolic status. Physiologically, a transient increase in the driving force for proton transport may enhance mitochondrial ATP synthesis. Thus, a cardiac cycle is sculpted not only by action potentials, Ca²⁺ transients, and contractions but also by cyclical changes in pH_i. Our findings reveal new features of pH_i handling in excitable cells. 

Tumors frequently hemorrhage due to abnormal vasculature. Macrophages phagocytose extravasated erythrocytes to metabolize hemoglobin and heme, but the impact of this on macrophage functions in the tumor microenvironment is unknown. Using multiple mouse models, human tumors, and single-cell transcriptomics, we identify a distinct heme-metabolizing iron-rich subset of tumor-associated macrophages (iTAMs) and isolate these cells using their ferromagnetic properties. iTAMs display immunosuppressive and pro-angiogenic gene signatures, and expresses the endothelin receptor B (Ednrb). Deletion of Ednrb in iTAMs reduces tumor growth and vascular density. Mechanistic studies show that heme-mediated degradation of the transcription factor Bach1 drives the global immunosuppressive transcriptional program and Ednrb expression in iTAMs. Correspondingly, intratumoral hemorrhage is associated with reduced inflammatory signature and survival in human sarcomas. These findings show how heme signaling in tumor-associated macrophages regulate tumor growth and reveal macrophage endothelin signaling as a potential target for immunotherapy.   

Despite mounting evidence, the molecular link between metabolism and psychiatric status is not well understood. We show that IMDEA-C1, a metabolite belongind to a family of plant-produced compounds with anti-depressant properties, improves mitochondrial function and metabolic parameters, and extends healthspan. Treatment with IMDEA-C1 induced a transient mitochondrial depolarization, a strong mitophagy response, and the AMPK compensatory pathway both in cultured C2C12 myotubes and in mouse liver, BAT and muscle. Mechanistically, simultaneous modulation of monoamine-oxidase B and GABA-A receptor, targets of IMDEA-C1, reproduced IMDEA-C1-induced mitochondrial improvements. Diet-induced pre-diabetic mice improved their glucose tolerance, liver steatosis and HOMA-IR after treatment with IMDEA-C1. IMDEA-C1 or a combination of MAO-B and GABA_AR modulators extended the lifespan of Caenorhabditis elegans and Drosophila melanosgaster. Finally, 2 year-old mice treated with IMDEA-C1 delayed frailty onset by improving their glycemia, exercise performance and strength. Our results reveal a link between improved psychiatric status and healthspan extension through peripheral mitohormesis.

Stimulation of β₃-adrenergic receptors by agonists such as CL-316,243 augments glucose uptake into brown adipocytes through mechanisms distinct from those utilized by the insulin pathways for systemic glucose disposal. But CL-316,243 also induces free fatty acid level leading to rise in insulin secretion. Here we examine whether the effect of CL-316,243 on blood glucose in vivo is insulin-dependent or not. 

In control lean mouse, CL-316,243 was able to lower plasma glucose levels and to increase markedly 2-deoxyglucose uptake into brown adipose tissue (without detectable rise of 2-deoxyglucose uptake into other tissues). In contrast, CL-316,243 was not able to lower glucose levels in (type 2) diabetic db/db mice, and no augmented 2-deoxyglucose uptake into brown adipose tissue was found in these mice. 

In the model of diet-induced obesity, CL-316,243 had practically lost its glucose-lowering ability. To characterize the significance of UCP1 for blood glucose lowering, we examined wildtype and UCP1 KO mice made insulin-deficient by streptozotocin treatment. CL-316,243 induced energy expenditure in wildtype but not in UCP1 KO mice regardless insulin. In contrast, CL-316,243 reduced blood glucose in normoinsulinemic but not in insulin-deficient mice regardless UCP1. 

Thus we conclude that despite the ability of CL-316,243 to stimulate glucose uptake into brown adipocytes, the systemic glucose-lowering effect of CL-316,243 is fully dependent on the presence of insulin but not of UCP1. Brown adipose tissue even in the absence of its glucose-burning properties (i.e. in UCP1 KO) may be an important mediator of the glucose-lowering effects of b₃-adrenergic stimulation.

Colorectal cancer has a high incidence worldwide, making it the third most common cancer. There is growing evidence to confirm that cancer stem cells (CSCs) are responsible for resistance to treatment, metastasis, tumor heterogeneity, chemotherapy, and radiation therapy. The relationship between proprotein convertases (PC) and cancer has been extensively studied, however, there is little knowledge about the role of PC9 (Proprotein Convertase Subtilisin / Kexin 9, also called NARC1), a member of this family, in this disease. In an attempt to elucidate the relationship between PC9 and CSC, we initially studied it expression both at protein and gene level in different colon cancer cell lines and analyzed the role of PC9 on the cellular function of colon CSC after its inhibition with PF-06446846. We found that CSC express grater amount of PC9 and the inhibition of it significantly decreased CSC viability. On the other hand, the co-expression of CD133, a CSC marker, with PC9, by immunofluorescence in biopsies of primary and metastatic tumors, revealed that PC9 expression was higher in CSCs than in stroma. In turn, the location between the two markers, with greater colocalization in metastases, suggested the existence of a correlation between PC9 and CSC of the colon and rectum. Furthermore, in vivo experiments have promising results. All these indicates that PC9 may play a role in tumor development and spread, potentially leading to future therapy.

Human pluripotent stem cells (hPSCs) can self-renew indefinitely or be induced to differentiate. We previously showed that exogenous glutamine (Gln) withdrawal biased hPSC differentiation toward ectoderm and away from mesoderm. We uncovered that while all three germ lineages are capable of de novo Gln synthesis, only ectoderm generates sufficient Gln to sustain cell viability and differentiation, clarifying lineage fate restrictions under Gln withdrawal. Furthermore, Gln acts as a signaling molecule for ectoderm that supersedes lineage-specifying cytokine induction. In contrast, Gln in mesoderm and endoderm is the preferred precursor of α-ketoglutarate without a direct signaling role. Our work raises a question about whether the nutrient environment functions directly in cell differentiation during development. Interestingly, transcriptome analysis of a gastrulation stage human embryo shows unique Gln enzyme-encoding gene expression patterns may also distinguish germ lineages in vivo. Together, our study suggests that intracellular Gln may help coordinate differentiation of the three germ layers.

Triple-negative breast cancer (TNBC) is associated with a high risk of recurrence and generally a bad prognosis. More than one-third of patients with TNBC will present distant metastases during their disease. There is an urgent need to explore vulnerabilities of TNBC and develop novel therapeutic drugs to improve clinical outcomes for TNBC patients since they lack traditional therapies. We have observed evidence of increased lipid droplets (LD) in tumor microenvironment (TME) in primary tumors in TNBC patients with poor clinical outcome. We found that KO of DNP63, a transcription factor in established TNBC tumors, leads to reduced LD in TME and lipid content using untargeted lipidomics data, particularly triglycerides in tumor infiltrating MDSCs along with reduced tumor progression and metastasis. Interestingly, we found that silencing DNP63 in tumor cells, leads to reduced Diacylglycerol O-acyltransferase 2 (DGAT2) in MDSCs, an enzyme primarily responsible for TG synthesis and LDs. DNp63 KO tumor cells also secrete less inflammatory cytokines such as IL-6, CXCL-1 and CXCL-3, which are also associated with increased lipid uptake. Together, our data indicates that that DNP63 in tumor cells regulates lipid metabolism processes in MDSCs such as lipid uptake and DGAT2 mediated TG synthesis and suggest that targeting DNp63 or DGAT2 may unveil new avenues of targeted treatment for aggressive TNBC subset.

Antibiotic-induced gut dysbiosis is currently a frequent and serious side effect of antibiotic use. Host diet can be a therapeutic target to modulate the structure and function of the microbiome during antibiotic treatment. In this study, we utilize metagenomic and metatranscriptomic sequencing combined with de novo gene assembly to elucidate changes to the microbiome during diet modulation. Further, we characterize the chemical redox environment of the gut ex-vivo to understand how metabolic changes affect microbiome susceptibility to antibiotics. Using a murine model, we found that fiber prebiotics are associated with increased microbial diversity post-antibiotic treatment, as well as metabolic reactions that occur at a lower redox potential. This indicates that fiber prebiotics modulate bacterial metabolism in the gut which can protect these microbes during antibiotic treatment. This transformative research could aid efforts to decrease damage to the microbiome from antibiotics and provide an avenue for fiber therapeutics.

Hepatocellular carcinoma (HCC) is malignant liver cancer leading to continuous death worldwide owing to limited therapies and treatments. In this study, we present a systematic drug repositioning method based on comprehensive integration of molecular signatures in liver tissue and liver cancer cell line. First, we identified robust prognostic genes and two gene co-expression from two independent HCC cohorts. Then, we screened 10 genes as potential target genes for HCC based on the network topology analysis. Further, we developed a drug repositioning method by integrating the shRNA and drug perturbation of liver cancer cell lines and identified potential drugs for HCC. Finally, we evaluated candidate drugs effects through an in-vitro model and observed that two drugs inhibited the protein levels of their corresponding target genes and cell migration. These findings proved the usefulness and efficiency of our approach to improve the drug repositioning researches for cancer treatment and precision medicine.

Renal fibrosis is a final common stage of almost all progressive kidney diseases, and it is independent from aetiology. However, new therapeutic biomarkers in clinical application are demanded. In this study, gene expression profiles of two different mouse models at several time points were involved from public datasets: folic acid (FA) induced nephropathy (GSE65267) and unilateral ureteral obstruction (UUO) induced nephropathy (GSE118339). To detect potential biomarkers of kidney fibrosis, time-series clustering, time point specific differential expressed gene analysis and co-expression network analysis were performed on the two datasets, respectively. As a result, 37 genes were identified as preliminary renal fibrosis biomarkers. This result was further validated by single cell RNA-seq data of UUO mouse model (GSE140023), which showed that 17 of 37 genes were significantly over-expressed in different cell types. Finally, CKAP4 and IFNAR2, which exhibited significant over-expression in non-immune cell types, were recognised as new potential anti-fibrotic targets.

Mitochondrial Fission protein 1 (Fis1) is a highly conserved protein, whose physiological importance in mammals is hitherto unexplored. Despite being required for yeast mitochondrial fission, mammalian Fis1 was suggested to be dispensable for mitochondrial division. Here we report that Fis1 is essential for life and it restrains sterile inflammation. Ad hoc generated Fis1-depleted mice (Fis1hh) display growth retardation, ataxia, kyphosis and hypoactivity, ultimately dying within just 18 days. Ubiquitous Fis1 deletion from adult life was similarly lethal. Mechanistically, Fgf21, a stress mitokine, and several inflammatory cytokines were increased in Fis1hh mice, correlating with the accumulation of danger-activated molecular patterns (DAMPs) and the activation of the cGAS-STING pathway. We traced the activation of this pathway to the accumulation of a population of ultrastructurally deranged and non-respiring mitochondria in asymptomatic Fis1hh pups. Our results nominate Fis1 as an essential component of the pathway restraining DAMPs accumulation and sterile inflammation in vivo.

Reactive oxygen species (ROS) can be pathological, but can also have a signaling role in physiological conditions, such as hypoxia. The carotid body (CB) is the prototypical acute O₂-sensing organ, which mediates cardiorespiratory reflexes that are essential for mammalian survival in hypoxia. We have studied the role of mitochondrial ROS in CB chemoreceptor (glomus) cells during hypoxia using conditional knockout mouse models and redox-sensitive fluorescent probes to monitor real-time rapid changes in ROS production in different subcellular compartments. Changes in mitochondrial ROS during acute hypoxia were highly compartmentalized: increase at the intermembrane space (IMS) and decrease in the matrix. IMS ROS changes were selectively inhibited in mouse mutants with abolition of the responses to hypoxia. ROS inhibit membrane K⁺ channels to induce glomus cell depolarization and transmitter release. Therefore, the IMS ROS signal, generated in complexes I and III, seem to play a key role in responsiveness to hypoxia.

Sarcopenia, the progressive loss of muscle with age, is associated with increased occurrence of fall injury, frailty, functional decline, and mortality. Despite established beneficial effects of exercise on ameliorating sarcopenia and other age-related diseases, the growing obesity epidemic and modern lifestyle make exercise a challenge for most people. One major obstacle to translating the benefits of exercise to humans is the lack of therapeutic interventions that can enhance motivation for exercise. We demonstrate that lowering methylglyoxal pharmacologically reduces body weight and improves glucose tolerance in several mouse models. Interestingly, lowering methylglyoxal also enhanced voluntary physical activity, improved neuromuscular function, and extended lifespan. Furthermore, with age, treated mice showed many physiological changes predicted to decrease the risk of sarcopenia including reduced IGF-1 levels, increased insulin sensitivity, and decreased inflammatory markers. Our findings highlight the therapeutic potential of compounds that protect against AGEs in preventing age-related sarcopenia in aged individuals.

Sepsis is the result of a complex interplay between inflammation, immune activation, metabolic reprogramming, and hypoxia, and is characterized by the presence of glucocorticoid resistance (GCR). Hypoxia inducible factors (HIFs) are involved in the regulation of energy homeostasis and a clear crosstalk is present between HIF and the glucocorticoid receptor (GR). Therefore, we investigated the effect of hypoxia and HIF proteins on the transcriptional activity of GR and if this accounts for (part of) the GCR in sepsis. Hypoxia itself causes a transcriptional reprogramming of the GR which is linked, in part, by alterations in the GR DNA-binding profile. Furthermore, hypoxia activates the hypothalamus-pituitary-adrenal (HPA) axis and causes GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GCR.

Fatty acids (FA) are important signaling-metabolites in health and disease. FA are either derived from nutrition, enzymatic triglyceride catabolism (lipolysis), or de-novo lipogenesis. Unsaturated FA (uFA) stabilize Sterol Regulatory Element-Binding Protein -1c (SREBP-1c), but when uFA are scarce, SREBP-1c is proteolytically activated, and drives expression of lipogenic genes. During fasting, however, lipolysis supplies the organism with FA, and lipogenesis is low. Adipose Triglyceride Lipase (ATGL) is the rate‑limiting enzyme for lipolysis, and it preferentially hydrolyzes uFA. Therefore, we hypothesized that ATGL-derived uFA may protect SREBP-1c from proteolytic activation in the liver. Here we show that (i) uFA liberated by ATGL suppress P‑SREBP‑1c activation, (ii) SREBP‑1c is hyper‑activated in livers of mice lacking ATGL, and (iii) pharmacological inhibition of ATGL selectively activates SREBP‑1c in hepatocytes. Our findings highlight an ATGL/SREBP-1c axis, instrumental to coordinate lipogenesis and lipolysis, whose homeostatic regulation is crucial to avoid severe diseases including diabetes, cardiomyopathy, and even cancer.

Defects in Coenzyme Q (CoQ) metabolism have been associated to primary mitochondrial disorders, neurodegenerative diseases and metabolic conditions. Here, we demostrate in a mouse model with primary CoQ deficiency (Coq9^R239X),  that the defect in CoQ induces a mayor remodeling of the mitochondrial proteome and metabolism in the kidneys, and to lesser extend in the brain. The treatment of Coq9^R239X mice with two analogs of the natural precursor for CoQ biosynthesis, the vanillic acid (VA) and the β-resorcilic acid (β-RA), normalize the mitochondrial proteome and metabolism, specially in the kidneys and liver. In the brain, VA and β-RA treatments lead to the reduction of astrogliosis, neuroinflammation and spongiosis and, consequently, rescuing the phenotype. These results are not only important for the use of analogs of the CoQ biosynthetic precursor in the treatment of CoQ deficiency but also for the therapeutic use of those compounds in more common neurodegenerative and metabolic diseases.

Obesity is a major health problem linked to other diseases like type II diabetes, metabolic syndrome and hepatic steatosis. Some treatments for obesity show low efficiency or side effects. Here, we identify a novel application of ß-resorcylic acid (ß-RA) for the treatment of obesity. Oral supplementation with ß-RA in mice fed under high-fat high-sucrose (HFHS) diet induces weight loss, without loss of muscle mass, in mice with obese phenotype. ß-RA improves glucose homeostasis by reducing insulin/glucagon ratio and GIP levels in plasma; reduces white adipose tissue (WAT) hypertrophy and hyperplasia; and prevents hepatic steatosis. Moreover, HFHS feeding induces alterations in metabolites involved in glycolysis, TCA cycle and urea cycle in WAT, liver and serum; and increases carnitines in WAT and liver. ß-RA results in the metabolic normalization on those tissues. Thus, ß-RA is an efficient and safe therapeutic option to treat and prevent obesity, metabolic syndrome and hepatic steatosis.

A recent literature related lipidomic profile to cystic fibrosis (CF), revealing a complex crosstalk between lipids and inflammatory/immune pathways. CF is a multisystemic disease characterized by a chronic inflammation involving most tissue and organs among which the lung with the impairment of airway secretions. Clinical monitoring of lung disease in CF patients requires the invasive collection of bronchoalveolar lavage fluid. The analysis of salivary biomarkers could represent a non-invasive alternative approach. 

We analyzed cholesterol, non-esterified fatty acids (NEFA) and total fatty acids (TFA) by gas chromatography-mass spectrometry, together with inflammation markers, i.e., interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-α) by ELISA in resting saliva samples from CF patients (n = 69) and healthy subjects (n = 50), relating to lung disease severity and sinonasal complications, i.e., inferior turbinate hypertrophy (NTH) and/or nasal polyposis (NP). 

CF patients showed significantly higher levels of salivary IL-6, IL-8, and TNF-α than healthy subjects. Among CF patients, we found that IL-6 and IL-8 increased in NTH (acute phase of sinonasal disease), while TNF-α decreased in severe pulmonary disease and NP (chronic phase of sinonasal disease). Moreover, CF patients showed higher salivary levels of cholesterol, total NEFA, Unsaturated/Saturated (U/S) NEFA ratio than controls, while the U/S TFA ratio was significantly lower in CF patients, suggesting an increased activity of lingual lipase in CF, according with the literature. Interestingly, U/S NEFA ratio was positively correlated with IL-6 and lung disease severity and may represent a prognostic marker in CF. 

Obesity, a condition resulting from an excess of adipose tissue, is a serious health problem worldwide and an important factor in the development and progression of cardiovascular diseases. It is well established that mitochondrial dysfunction in adipose tissue might contribute to obesity-related diseases. In a cohort of obese patients, we found that adipose tissue of obese subjects showed a reduced expression of PGC1α, an important mitochondrial regulator. High-fat diet feeding in mice also promoted a downregulation in PGC1α expression in adipose tissue that was accompanied by cardiac metabolism alteration. Echocardiographic analysis of mice lacking PGC1α specifically in adipose tissue showed that these mice develop a cardiac dysfunction like the one observed during obesity. However, this cardiomyopathy was not accompanied by diabetes, hypertension, or increased adiposity. Proteomics and metabolomics analysis of plasma from these mice and obese subjects revealed several promising adipokines and metabolites that could be involved in obesity cardiomyopathy. 

The group of dinucleoside polyphosphates includes molecules consisting of two nucleosides (adenosine, cytidine, uridine, guanosine) and a phosphate chain of 2-7 phosphates. Various effects of dinucleoside polyphosphates, like proliferation, promotion of angiogenesis and migration are already known. Especially interesting is the angiogenesis-promoting property. The synthesis of these dinucleoside polyphosphates occurs via a kinase function of vascular endothelial growth factor receptor 2 (VEGF-R2), which is considered one of the most important receptors for tumor growth. 

Through this connection of synthesis by VEGF-R2 and an angiogenesis-promoting property, the dinucleotide polyphosphates could be a relevant factor in the pathogenesis of tumor diseases and thus represent a new relevant field of research.  

Mass spectrometric analyses (LC-QTOF-MS and MALDI-TOF/TOF-MS) of plasma samples from various tumor mouse models, have already provided initial indications of a possible association.

Plasma concentrations of some dinucleoside polyphosphates show a significant difference between healthy and tumor-burdened mice. In mice with tumor burden, the plasma concentration is significantly increased compared to healthy mice. In addition, the previously available data indicate that plasma concentrations of the dinucleoside polyphosphates considered to date are differentially expressed in different tumor types. 

From these data, it can be hypothesized that there are tumor-specific concentration profiles for individual dinucleoside polyphosphates depending on tumor stage and/or tumor type. Furthermore, it has to be clarified whether other so far unknown dinucleoside polyphosphates exist that are involved in tumorigenesis and are modulated via VEGF-R2.

A deregulated angiogenesis is involved in diabetes, psoriasis, retinopathies and cancer, among other prevalent and rare diseases. We have previously reported that dimethyl fumarate, an ester from the Krebs cycle intermediate fumarate, has anti-angiogenic activity. Furthermore, dimethyl fumarate was approved and is used for the treatment of psoriasis and multiple sclerosis. However, it is not known whether dimethyl fumarate is able to modulate endothelial cell metabolism, essential for the angiogenic switch. By means of proteomics, isotope tracing and metabolomics experimental approaches, in this work we studied the effects of dimethyl fumarate in endothelial cell energetic metabolism. Our results show for the first time that dimethyl fumarate promotes glycolysis and diminishes cell respiration. This could be a consequence of a down-regulation of serine and glycine synthesis through inhibition of PHGDH activity in endothelial cells. These results throw new light regarding the mechanism of action of dimethyl fumarate.

Oxytocin-expressing paraventricular hypothalamic neurons (PVN^OT neurons) integrate afferent signals from the gut including cholecystokinin (CCK) to adjust whole-body energy homeostasis. However, the molecular underpinnings by which PVN^OT neurons orchestrate gut-to-brain feeding control remain unclear. Here, we show that mice fed high-fat/high-sugar diet not only fail to suppress their food intake in response to CCK but also to properly activate PVN^OT neurons. While chemogenetic restoration of PVN^OT activity in obese mice sufficiently re-establishes CCK’s anorexigenic effect, selective ablation of PVN^OT neurons entirely blunted such response. Importantly, after PVN^OT neuron ablation, mice develop hyperphagic obesity on standard chow diet, which is readily corrected by repeated administration of exogenous oxytocin. Lastly, by single cell profiling, we identify a specialized subpopulation among PVN^OT neurons with increased κ-opioid signaling under high-fat/high-sugar diet that restrains their CCK-evoked activation. In sum, we suggest a novel mechanism by which hypothalamic oxytocin signaling uncouples from a gut-to-brain satiation pathway under obesogenic conditions.

Neural tissue has high metabolic requirements. Following spinal cord injury (SCI), the damaged tissue suffers from a severe metabolic  impairment, which aggravates axonal degeneration and neuronal loss. Impaired cellular energetic, tricarboxylic acid (TCA) cycle and oxidative phosphorylation metabolism in neuronal cells has been demonstrated to be a major cause of neural tissue death and regeneration failure following SCI. Therefore, rewiring spinal cord cell metabolism may be an innovative therapeutic strategy for the treatment of SCI. In this study, we evaluated the therapeutic effect of the recovery of oxidative metabolism in a mouse model of severe contusive SCI. Oral administration of TCA cycle intermediates, co-factors, essential amino acids, and branched-chain amino acids was started 3 days post-injury and continued until the end of the experimental procedures. Metabolomic, immunohistological, and biochemical analyses were performed on the injured spinal cord sections. Administration of metabolic precursors enhanced spinal cord oxidative metabolism. In line with this metabolic shift, we observed the activation of the mTORC1 anabolic pathway, the increase in mitochondrial mass, and the reduction of oxidative damage which effectively prevented the injury-induced neural cell apoptosis in treated animals. Consistently, we found more choline acetyltransferase expressing motor neurons and increased neurofilament positive corticospinal axons in the spinal cord parenchyma of the treated mice. Interestingly, oral administration of the metabolic precursors increased the number of activated M2-like microglia. Treated SCI animals showed significant, although partial, improvement of the motor functions. Our data demonstrate that rewiring the cellular metabolism may represent an effective strategy to treat SCI.

Tuberculosis (TB) patients suffer from progressive loss of muscle mass, which is attributed to combination of poor nutrition and altered metabolism as part of inflammatory responses. During Mycobacterium tuberculosis (Mtb) infection, host macrophages deploy a dual strategy of nutritional deprivation and intoxication to attenuate pathogen. However, it is not clear how Mtb evades this paradoxical host response. Here, we identify a novel zinc metallophore from Mtb that restores zinc metabolic imbalance, while protecting itself from toxic potential. Mtb transiently induces diisonitrile lipopeptides, named kupyaphores, during the early stages of macrophage infection and in infected mice lungs. Kupyaphore Mtb mutant strain is unable to mobilize host zinc and shows reduced fitness with diminished pathology in mice upon infection. Further, we identify novel Mtb encoded isonitrile hydratase that could mediate intracellular zinc release through covalent modification of kupyaphores. Since systemic zinc redistribution is associated with muscle atrophy, our studies could provide a molecular link between TB-induced altered zinc metallostasis and associated cachexia.

The lung airways are exposed to inhaled toxic substances, resulting in cell damage that is repaired by local expansion of resident club cells. This process demands energy and biological building blocks. Adipose TriGlyceride Lipase (ATGL), liberates energy-rich fatty acids from triglyceride stores, essential metabolites that act as cellular building blocks, and that activate Peroxisome Proliferator-Activated Receptor (PPAR)-α signalling. PPAR-α in turn, drives mitochondrial fatty acid oxidation that provides energy‑equivalents. Here we report that bronchiolar club cells of mice lacking ATGL showed triglyceride accumulation and decreased mitochondrial function. This defect manifested as bronchiolar epithelial thickening and increased airway-resistance. After naphthalene‑induced epithelial injury, a regenerative defect was apparent. Mechanistically, lack of ATGL led to dysfunctional PPAR-α lipid-signalling in regenerating bronchioles, and administration of the specific PPAR-α agonist WY14643 restored club cell mitochondrial function, and regenerative potential. Our data highlight the significance of the cellular fatty acid metabolism for lung health and regeneration.

Folate metabolism is an essential metabolic pathway that provides the precursors for purine, thymidylate and methionine synthesis and has been a major target of anti-cancer drugs. A clinically key yet untargeted enzyme in the pathway is MTHFD1, which catalyzes three distinct reactions in two different domains. Here, we investigate the genetic and chemical dependencies caused by loss of MTHFD1 function by utilizing a large scale chemical screen and genome-wide genetic screens. Surprisingly, we discovered that whereas MTHFD1 knock-out cells were dependent on exogenously supplied adenosine, or other adenine-containing compounds, for survival and growth, genetic ablation of the enzymatic activities in one of the domains of MTHFD1 resulted in adenosine-mediated cell death via induction of DNA-damage and replication stress. We exploited the toxicity phenotype and demonstrated that a MTHFD1 inhibitor lethally synergizes with adenosine in a panel of cell lines, which is therapeutically exciting given the elevated levels of adenosine in the tumor environment. Our data provides new insights on the role of folate metabolism in cellular function as well as novel therapeutic opportunities through inhibition of distinct MTHFD1 activities.

Background/Methods

A transcriptomic analysis revealed high expression of mitochondrial uncoupling protein-2 (UCP2) in lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), compared to healthy controls. We studied tissues/fibroblasts obtained from lung explants of IPF patients and utilized the aging murine model of persistent lung fibrosis following bleomycin injury.  We silenced UCP2 using siRNA in vitro and in vivo.  

Results 

UCP2 expression was increased in lung tissues and fibroblasts of IPF patients. Lung fibroblasts from aged mice expressed higher UCP2 than younger mice, further increased after bleomycin-induced lung injury. Lower steady state ATP levels were detected in isolated fibroblasts and lungs of IPF patients compared to controls. UCP2 silencing in IPF fibroblasts decreased pro-fibrotic markers such as α-SMA and collagen, while decreasing senescence markers. Importantly, UCP2 silencing restored ATP levels and decreased aerobic glycolysis; this was associated with decreased mitochondrial ROS (superoxide and hydrogen peroxide) and increased Nrf2-mediated antioxidant responses. In aged mice after bleomycin injury, therapeutic oropharyngeal administration of UCP2 siRNA resulted in decreased lung collagen and fibrotic remodeling, indicating enhanced resolution capacity.  

Conclusion

Our results suggest that expression of mitochondrial UCP2 in lung fibroblasts increases with age, particularly in IPF. UCP2 drives a senescent fibroblast phenotype resulting in lower ATP levels, higher glycolytic demand, higher ROS, and impaired resolution of bleomycin injury-induced fibrosis in aged mice. Further studies are warranted to study the link between cellular aging, altered metabolism and UCP2, and the therapeutic potential of targeting UCP2 in age-related fibrotic disorders such as IPF.

We investigated the mitochondrial alterations that non-alcoholic fatty liver disease and alcoholic liver disease induce in the livers of mice. Liver mitochondria from ob/ob mice (25wks; obesity and steatosis model) and intragastric alcohol fed mice (4wks) displayed a significant increase (~2 fold) in state III respiration for all respiratory substrates examined in isolated mitochondria, including glutamate/malate, succinate, octanoate, and glycerol-3 phosphate. Increased mitochondrial respiration was likely due to increased expression of respiratory complexes I, IV, V, and other ETC complexes, glycerol phosphate dehydrogenase-2 and medium-chain acyl-CoA dehydrogenase, as observed by western blot analysis. These observations suggest that stress induced by alcohol and obesity cause significant mitochondrial remodeling in the liver. Rather than dysfunction, these alterations were likely adaptations to short-term insults, possibly at the expense of long-term damage. Obtaining better characterization of hepatic mitochondrial remodeling may play an important role in understanding the pathophysiology of liver diseases.

To cover their disproportionately high energy consumption, neurons are equipped with a large amount of mitochondria, the main task of which is oxidative phosphorylation. However, oxidative phosphorylation is accompanied by the incomplete reduction of molecular oxygen, resulting in the formation of reactive oxygen species (ROS). Thus, this highly oxidative metabolism enriches molecules that contribute to the neuron's demise. Consuming a lot of energy rich substrates with simultaneous ROS production on the one hand, and not being dependent on neuronal glycolysis on the other, gives rise to the idea that neurons metabolize glucose preferentially through the pentose-phosphate-pathway (PPP).  The PPP is thought to contribute up to 70% to the pool of cellular reduction equivalents in the form of NADPH/H, which is essential for coping with oxidative stress in the cell.

To examine an alternative glucose usage via the PPP in neurons, we study the effects of neuronal and glial dsRNA-mediated knock-downs of enzymes involved in the oxidative phase of the PPP. Making use of novel, genetically encoded, biosensors for NADPH and H₂O₂, we can demonstrate that neuronal ROS concentration increases as a consequence of less NADPH/H production. Over time, that failure in providing reduction equivalents is accompanied by neurodegenerative phenotypes in the adult Drosophila brain. The combination of tools to monitor different metabolic parameters in living tissues and the anatomical data gives further insights about the differential importance of metabolic pathways between neurons and glia and thus the complexity of metabolic interactions between these two cell types. 

Lectins have big potential for their therapeutic applications such as immunomodulatory, anticancer, antibacterial, and other activities. ~ 40 kDa hemagglutinins with LysM domain from purple coneflower roots haven’t been investigated and tested on animal models in vivo. Aim of experiment: to test the activity of purified hemagglutinins from purple coneflower roots on immune system reactions in vivo using Balb/c mice blood samples. 

Materials and methods: 3 animal groups were selected: animals (n=15) in negative control group got 50 μL peritoneal injections of physiological solution, animals in tincture group (n=15) got 50 μL peritoneal injection of purple coneflower root tincture, animals in hemagglutinin group (n=15) got 5 μg lectin peritoneal injection. Injections were repeated 4 times. On 5th week animals were sedated and euthanized. Lymphocyte number in blood samples were counted manually in hemocytometer and lymphocyte formula were defined manually from blood smear.

Conclusion: lectin fraction stimulated mice immunity as well as Echinacea purpurea root tincture.

The large intestine harbors an extensive collection of microorganisms which play a unique role in host physiology. The beneficial or detrimental outcome of host-microbiome coexistence depends largely on the balance between specialized regulators and responders within the intestinal CD4+ T cell population. We found that ulcerative colitis-like changes in the large intestine after infection with the protist Blastocystis ST7 are associated with reduction of anti-inflammatory Treg cells and simultaneous expansion of pro-inflammatory Th17 responders. This reorganization of the CD4+ T cell compartment depended on the tryptophan metabolite indole-3-acetaldehyde (I3AA) produced by this single cell eukaryote. I3AA negatively impacted the Treg subset in vivo and iTreg development in vitro by modifying sensing of TGFβ stimuli by these cells, and concomitantly affecting recognition of self-flora antigens by conventional CD4+ T cell clones. Over-exuberant TCR signaling, manifested by increased TCR-dependent CD69 expression and down-regulation of the co-inhibitory molecule PD-1, was orchestrated by the parasite-derived I3AA. This constitutes a new mechanism dictating CD4+ fate decisions, shining new light on the ability of protist members of the microbiome, and tryptophan metabolites derived from them or other sources, as modulators of the adaptive immune compartment, particularly in the context of gut inflammatory disorders.

Vitamin A, fat-soluble micronutrient, plays an indispensable role in embryogenesis and development, but was also reported to regulate immune responses. In mammals, Vitamin A-enriched tissues, such as liver, gut or fat, comprise various immune cells that contribute to maintenance of tissue homeostasis and immune tolerance. Here, we investigated the effect of vitamin A metabolite, all-trans retinoic acid (atRA), on murine NK cells that are frequently recruited to tissues during inflammatory responses. We show that atRA induces transcriptional, phenotypic and functional reprogramming of NK cells leading to reduced production of inflammatory cytokines. Vitamin A-exposed NK cells affected dendritic cell maturation and were able to skew T cell differentiation towards regulatory phenotype. Our findings suggest that atRA supports regulatory functions of NK cells, which might contribute to tissue tolerance and reduced inflammatory responses in vitamin A-enriched environments.

RESOLUTE, a public-private partnership funded by the Innovative Medicine Initiative, aims to advance knowledge and research on solute carrier transporters (SLCs) by generating reagents and tools as well as performing diverse functional analyses for the family of 446 proteins. The SLC family remains understudied both on the level of individual members and their functional relationships, with a third of them having unknown substrates. Using a targeted metabolomics approach covering 200 metabolites, we analyzed cell lines with inducible overexpression of a single human SLC. To date we have acquired metabolic profiles for more than 250 SLCs and observe statistically significant changes of metabolites covering a diverse set of metabolic pathways for roughly half of the SLCs. While we observe the accumulation of known substrates for some SLCs, we mainly investigate downstream metabolic consequences. Similarity clustering of metabolic signatures indicates functional relationships between SLCs and contributes to their deorphanization.

The prognosis is poor in human acute myeloid leukemia (AML) due to the high frequency of relapses, mainly caused by the growth of relapse-initiating chemoresistant leukemic cells (RICs). AML cells, similar to numerous cancer cells, can reprogram their metabolism. My laboratory and others demonstrated that RICs have an increased oxidative metabolism and relies on fatty acids and amino acids metabolism to survive to chemotherapy. We hypothesized that autophagy could be responsible of this reprogramming and resistance. Autophagy is a catabolic process allowing degradation and recycling of damaged proteins and organelles to feed metabolism and support cancer cell biology. Earlier studies on autophagy and cancer mainly focused on tumor autophagy. Similar to tumor autophagy, host/microenvironment autophagy has also been recently implicated in growth promotion of several cancer by supplying substrates required for their growth. However, the exact mechanisms by which autophagy controls the metabolic dialog between AML cells and their microenvironment and the consequences on therapeutic resistance still remain unknow. Thus, I aim to investigate whether tumor and/or host autophagy can support AML growth and therapeutic resistance by modulating tumor metabolism. Using innovating in vitro and in vivo models for autophagy deficiency, I wish to 1) evaluate the role of tumor and host autophagy in tumor growth and resistance to therapy and 2) identify the underlying mechanisms. This knowledge will provide a deeper understanding on the molecular mechanisms involved in AML resistance especially in vivo and help to develop new selective treatments eradicating RICs to overcome AML patient relapses.

Liver sinusoidal endothelial cells (LSECs) display unique morphological features, such as the presence of fenestrae and lack of basal membrane. At the same time, LSECs maintain the major functional characteristics of ECs at other locations. Generally, ECs are known to rely for ATP production mainly on glycolysis. Surprisingly, our results demonstrate that mitochondrial ATP production prevails in LSECs. To further explore this peculiarity of LSECs, we studied the dependence of primary murine LSEC energy metabolism on fatty acid oxidation. Investigations were carried out using the Seahorse XF technique for the evaluation of mitochondrial respiration and glycolysis and untargeted mass spectrometry–based proteomics for the analysis of proteins involved in energy metabolism pathways. The most effective in supporting mitochondrial respiration were medium-chain fatty acids. In turn, long-chain fatty acids were preprocessed before entering mitochondria, which is supported by: (1) a high abundance of peroxisomal β-oxidation enzymes, (2) low levels of CPT1, and most importantly, (3) the lack of the effects of etomoxir on mitochondrial respiration in LSECs exposed to palmitate. Surprisingly, exogenously added L-carnitine supported mitochondrial respiration even under inhibition of the mitochondrial pyruvate carrier by UK-5099 and without exogenous fatty acids, suggesting an important, but unexplained so far role of L-carnitine in LSECs energy metabolism. In conclusion, our results suggest that in LSECs, representing a unique type of ECs, peroxisome–derived medium– and short–chain fatty acids contribute to mitochondrial respiration. This phenomenon might be instrumental for the harmless utilization of fatty acids in LSECs in the liver microenvironment.  

Glycolysis is essential for the classical activation of macrophages (M1), but how glycolytic pathway metabolites engage in this process remains to be elucidated. Based on studies that used UK5099 as a MPC inhibitor and showed reduction in key inflammatory cytokines, the mitochondrial route has been considered to be of significance for M1 activation. Herein, using a genetic depletion model, we found that MPC is dispensable for metabolic reprogramming and the activation of M1. Compared to wild type mice, mice with conditional knockout of MPC in myeloid cells have similar degree of inflammatory responses in a LPS induced septic model. While UK5099 reaches maximal MPC inhibitory capacity at approximately 2-5μM, higher concentrations are required to inhibit inflammatory cytokine production in M1 and this is independent of MPC expression. Taken together, MPC-mediated metabolism is dispensable for classical activation of macrophages and UK5099 inhibits inflammatory responses due to effects other than MPC inhibition.

The nonessential amino acid asparagine can only be produced by the enzymatic activity of asparagine synthetase (ASNS) that uses glutamine and aspartate as substrates. While ASNS and asparagine have been implicated in the response to numerous metabolic stressors in cultured cells, the in vivo relevance of this enzyme in stress-related pathways remains unexplored. Here, we demonstrate that ASNS is expressed in a small population of hepatocytes localized around the central veins of the liver and specialized in glutamine production and metabolite detoxification. We showed that ASNS expression is strongly increased following CCl4 injection and is required to protect pericentral hepatocytes from apoptosis induced by this toxin. The upregulation of ASNS involves a novel and non-canonical pathway where the nuclear receptor, liver receptor homolog 1 (LRH-1; NR5A2), is recruited to the promoter of ASNS and drives expression of this enzyme. Importantly, we discovered that supplementation with asparagine, the product of ASNS, is sufficient to dampen pericentral damage during acute liver injury. Taken together, our data implicate endogenous asparagine production in pericentral hepatocytes as an adaptive mechanism to constrain the widespread apoptotic wave that follows toxin administration. These findings highlight potential therapeutic opportunities for amino acids, such as asparagine, in the management of acute liver injury.

c-Met and insulin receptor (IR) belong to the family of receptor tyrosine kinases and amongst share the highest homology in kinase domain. To clarify the role of high glucose and high insulin (HGHI) in hepatocellular carcinoma (HCC) progression, we focused on IR and c-Met crosstalk and show that both expressions and activations of IR and c-Met are increased in induced HCC cells. Co-IP, immunofluorescence and proximity ligation assay studies reveal that c-Met and IR can heterodimerize when cells are treated with HG and/or HI, suggesting c-Met activation is a consequence of transphosphorylation, hence related downstream signaling activation. Glucose metabolism related genes’ expressions are altered with HGHI treatment, and c-Met inhibition. Furthermore, actin network is reorganized upon induction; motility, invasion, spheroid formation capacities of HCC cells are increased, which are reversed by c-Met/IR inhibition. Overall, obesity-related cancer progression where IR has pivotal role, can be suppressed by c-Met inhibition.

Introduction

NAFLD (>5% liver fat), is a multi-faceted and increasingly prevalent disease, present in 25% of the world population, yet no therapeutic treatment options are currently available. These subjects exhibit a dysbiotic gut microbiome, which via metabolites in the portal vein can lead to liver injury. The liver is a sexually dimorphic organ, with marked differences in biochemical profile and gene expression between men and women. This could explain the differences in NAFLD clinical phenotypes and disease progression between the sexes. To identify sex-specific mechanisms that drive the pathogenesis of this disease, we employed a multi-omic analysis in a cohort of 300 subjects. 

Methods

We conducted fecal metagenomics sequencing, RNA-sequencing of liver, jejunum and fat tissues, and untargeted plasma metabolomics in a cohort of 300 subjects who were eligible for bariatric surgery. We analyzed the NAFLD signature in a sex-matched subset of the 300 subjects, and a stratified analysis in males and females. Due to our unique study design, we were able to identify NAFLD specific signatures that are unique to NAFLD, not obesity.

Results

We demonstrate several sex-specific NAFLD signatures across all datasets, which could explain the difference in disease pathophysiology between men and women. We additionally validate several NAFLD specific genes, metabolites and microbes that are shared by both men and women.  

Conclusion

This multi-omic study of the sex-specific NAFLD signature enhances our understanding of this complex disease, and highlights potential treatment targets. We show the importance of stratifying NAFLD studies by sex to aid in therapeutic development.

Late-stage diagnosis exacerbates cancer mortality. While genomics-based liquid biopsies promise non-invasive cancer diagnostics, they detect fewer than 20% early-stage cancers. Biofluidic glycosaminoglycan profiles (GAGomes) are biomarkers of cancer metabolism. We investigated if GAGomes can be used for early detection of 14 cancer types by analyzing a total of 2064 plasma and/or urine cancer vs. healthy samples. We found cancer-specific changes in biofluidic GAGomes and developed machine learning models for early detection of cancer. Our models detected any-type cancer with an area-under-the-curve (AUC) = 0.83-0.93 with up to 62% sensitivity to stage I disease at 95% specificity and predicted the putative cancer location with 89% accuracy. In a validation cohort of 50–69-year-old cancer-free adults, GAGomes predicted any-type cancer within three months with 61% sensitivity (45% in stage I) at 95% specificity. Overall, GAGomes are promising multicancer early-detection biomarkers, potentially tripling the number of detectable stage I cancers.

Glucose tolerance represents a complex phenotype in which many tissues play important roles and interact to regulate metabolic homeostasis. We performed analysis of ¹³C₆-glucose tissue distribution, which maps the metabolome and lipidome across 12 metabolically relevant mouse organs and plasma, with integrated ¹³C₆-glucose-derived carbons tracing during oral glucose tolerance test (OGTT). We measured time profiles of water-soluble metabolites and related lipids and integrated the global metabolite response into metabolic pathways. The polar metabolomics results are available as GTTAtlas interactive web application (https://gttatlas.metabolomics.fgu.cas.cz). Here we focused on the time-resolved analysis of plasma and tissue lipidome which consisted of up to 2,300 lipid species. Hierarchical clustering of the time profiles of lipid species revealed 3-6 clusters per organ. Lipid structure enrichment analysis of these clusters highlighted structural and functional patterns specific to the organs. These patterns can be linked to the activities of metabolic pathways during the transition from fasted state to the state of glucose surplus, and to the state when the glucose bolus is gone after 4 hours. Next, ¹³C-isotopic enrichment was integrated for each lipid to explore the distribution of glucose carbons among the lipid species. Timeline profiling of ¹³C-labeled fatty acids and triacylglycerols across tissues suggests that brown adipose tissue was synthesizing ¹³C-labeled lipids at 30 minutes after the glucose bolus, followed by subcutaneous adipose tissue. In contrast, labeled lipids appeared in the liver and epididymal adipose tissue after 120 minutes. Our work demonstrates the dynamics of mouse lipidome during the transition from fasted to glucose-fed state.

In classical models of tumorigenesis, mutations affecting cancer growth act within the cell’s jurisdiction in a cell-autonomous manner. Most cancers express connexins, proteins that form gap junctions, through which cells can exchange small molecules via linking their cytoplasm. We hypothesized that the diffusive exchange of metabolites across gap junctions can blur phenotypic differences between coupled cells, therefore certain loss-of-function mutations in one cell could be compensated by access to functional proteins in neighbouring cells. To test this, we used colorectal cancer cells and inactivated critical genes in pH regulation (SLC9A1), glycolysis (ALDOA), or mitochondrial metabolism (NDUFS1). We found that the detrimental effects of these mutations were partially rescued by Cx26-dependent coupling onto wild-type cells. Therefore, mutations that lead to defective handling of metabolites are less likely to undergo negative selection. Diffusive coupling may explain why certain genes described as essential are not lethal when mutated in human cancers.

L-asparaginase (ASNase) is one of the crucial components of acute lymphoblastic leukemia therapy. ASNase hydrolyzes Asn and Gln and therapeutic ASNase concentration transforms all Asn and Gln to Asp and Glu, respectively. The precise mechanism how ASNase affects leukemic cells metabolism, is not known.

Many studies highlight tricarboxylic acid (TCA) cycle importance in Asp production and hence in sustaining cell viability and proliferation. Our results show that even though TCA cycle is diminished after ASNase treatment, NALM6 and REH leukemic cell lines are able to maintain intracellular Asp and Glu levels. While high Asp and Glu extracellular concentrations are by-products of ASNase treatment, we discovered that both cell lines are able to import Asp and Glu from the media. Next, we found out that high Glu (but not Asp) doses, help both cell lines to survive in Asn‑/Gln‑depleted conditions. Moreover, inhibitor of Asp/Glu transporters moderately sensitized the NALM6 cell line to ASNase. We also found out that sulfasalazine, an inhibitor of Cystine/Glu antiporter xCT, in combination with ASNase altered the Glu metabolism in both cell lines. We assume that activation of oxidative stress after ASNase causes higher demand for Cys, essential substrate for the synthesis of glutathione. When Cystine/Glu antiporter is inhibited, cells use more extracellular Glu to produce Cys. This is the first study describing the transport of Glu into leukemic cells and its survival advantage after ASNase treatment, which is now under investigation.

This work was supported by 20-27132S and NU20J-03-00032.

Metabolic reprogramming is a well-known cancer hallmark, with high levels of lactate being a common feature of cancers. The function of this metabolite remains however unclear. Here, we use a Drosophila brain tumor model to investigate the role of lactate on tumor development. Analysis of these tumors revealed that lactate levels are also altered in this context. Interestingly, we found that tumor volume is significantly reduced if tumor cells have impaired lactate transport, indicating that lactate exchanges with the extracellular media are crucial for tumorigenesis. Furthermore, we found that Lactate Dehydrogenase (LDH) is upregulated in a sub-set of tumor cells but only in large tumors, possibly as a mechanism to sustain tumor growth. Consistently, downregulation of LDH in tumor cells does not suppress tumor formation but strongly decreases tumor volume. We are currently exploring the mechanisms behind LDH upregulation and addressing the dynamics of lactate between tumors and their surroundings.

Mitochondrial cardiomyopathies are fatal diseases, with no effective treatment. Alterations of heart mitochondrial function activate the mitochondrial integrated stress response (ISRmt), a transcriptional program affecting cell metabolism, mitochondrial biogenesis, and proteostasis. In humans, mutations in CHCHD10, a mitochondrial protein with unknown function, were recently associated with dominant multi-system mitochondrial diseases, whose pathogenic mechanisms remain to be elucidated.  Here, in CHCHD10 knock-in mutant mice, we identify an extensive cardiac metabolic rewiring triggered by proteotoxic ISRmt. The stress response arises early on, before the onset of bioenergetic impairments, triggering a switch from oxidative to glycolytic metabolism, enhancement of transsulfuration and one carbon (1C) metabolism, and widespread metabolic imbalance. In parallel, increased NADPH oxidases elicit antioxidant responses leading to heme depletion. As the disease progresses, the adaptive metabolic stress response fails, resulting in fatal cardiomyopathy. Our findings suggest that early interventions to counteract metabolic imbalance could ameliorate mitochondrial cardiomyopathy associated with proteotoxic ISRmt.

Favorable environmental conditions promote organism’s growth and reproduction, while unfavorable conditions result in resource allocation toward defense against the environmental challenges. The choice between these programs is controlled by the hypothalamic-pituitary axes. While growth hormone (GH) is largely implicated in the control of somatic growth, the plasma level of GH is also elevated in adverse environments, such as food deprivation. Our work addresses this dichotomy. Using in vivo and in vitro models we were able to demonstrate that the outcome of growth hormone signaling is modulated by secondary signals reporting on the metabolic state of the organism. Notably, we showed that the glycolysis and somatostatin signaling pathways modify GH receptor signaling in hepatocytes. Lastly, we demonstrated that GHR signaling leads to distinct transcriptional programs in the fed vs fasted liver which reflect the needs of the organism. Our work suggests that growth hormone differentially promotes pro-growth vs. adaptive programs depending on the organismal state.

Almost all bacteria require iron for growth and survival. To compete successfully for this essential nutrient, bacteria developed very efficient iron uptake systems. Iron supplementation and iron food fortification may impact the composition of the gut microbiota and exacerbate inflammation in inflammatory bowel disease (IBD). We fed mice with diets supplemented with ferrous sulphate at different doses (5, 50 and 500 mg of iron/Kg chow) and with different iron formulations (ferrous sulphate, ferrous bisglycinate, ferric ethylenediaminetetraacetic acid (FEDTA), and heme), and analyzed the effects on the composition of the gut microbiota by 16S ribosomal RNA gene sequencing. Using the dextran sodium sulphate (DSS)-induced colitis mouse model, we investigated the effects of iron supplementation in colitis severity, as well as the use of the probiotic Escherichia coli Nissle 1917 (EcN) in combination with iron supplementation. Iron supplementation with ferrous sulphate at different doses induced shifts in the gut microbial communities and had a protective effect on DSS-induced colitis. However, depending on the iron formulation used in the diets, iron supplementation in colitis was either beneficial (ferrous sulphate and ferrous bisglycinate) or highly detrimental (ferric EDTA and heme). Finally, the beneficial effect of the probiotic EcN in the DSS-induced colitis model was potentiated by oral iron supplementation. 

These results suggest that the iron formulations used to treat iron deficiency may influence the gut microbiota and colitis severity. In addition, the beneficial action of probiotics in IBD may be enhanced by oral iron supplementation.

Current treatment of childhood acute lymphoblastic leukemia (ALL) significantly improved survival of patients but still about 15% of them undergo relapse. Relapses are associated with resistance to the chemotherapeutic agents applied in the therapy. One of the crucial drugs which mechanism of resistance is still under investigation is L-asparaginase (ASNase). We previously showed that ASNase caused metabolic rewiring which participated in the resistant phenotype of leukemic cells. Herein we investigated another aspect of the resistance which is in vivo environment. We used co-culture of leukemic cells and mesenchymal stem cells (MSCs) representing bone marrow and ASNase-pretreated culture media to mimic the in vivo half-life of the drug. We showed that leukemic cells survival was increased in comparison to “classical” in vitro treatment while sustaining the low glycolysis and high fatty acid oxidation. What was altered were the mTOR-biosynthetic pathways. After ASNase treatment the downstream targets of mTOR, pS6 (mediator of protein synthesis) and CAD (mediator of nucleotide synthesis) were inhibited whereas in this model the effect was significantly less profound.  We also showed that asparagine was released from MSCs which is normally depleted after ASNase and is responsible for the cytostatic effect of the drug. In conclusion, asparagine efflux from the MSCs reactivated biosynthetic pathways of leukemic cells after ASNase treatment, enabling increased survival of leukemic cells. Moreover, ASNase-triggered bioenergetic rewiring was independent of in vivo environment and presents a potential therapeutical target for resistant patients. This work was supported by GAČR GA20-27132S and GAUK 1262120.

N-arachidonoyl-phosphadidylethanolamine (NAPE) is the major precursor lipid metabolized via direct and indirect pathways, ultimately producing the endocannabinoid anandamide (AEA). Besides its neurological role AEA is thought to kill tumor cells by apoptotic or necroptotic mechanisms. Various NAPE metabolizing enzymes have been proposed as tumor suppressors, therefore we aimed to identify NAPE metabolites with antitumor role. Using a matrigel embedded 3D spheroid model we show that NAPE strongly counteracts mesenchymal type of tumor cell invasion. Amoeboid type of invasion is little affected, unless ROCK inhibition converts invasion to mesenchymal type. Dissecting the pathways by targeted analytics and lipidomics we revealed that NAPE is mainly metabolized to lyso-NAPE, then glycero-phospho-AEA (GP-AEA), and finally to AEA. Applying diether-NAPE with fatty acids inaccessible for the major metabolic route, largely abolished the antitumor effect, suggesting lyso-NAPE and GP-AEA are directly antitumorigenic. Direct assessment of lyso-NAPE and GP-AEA proved to be effective against tumor invasion distinguishable from AEA effect. Administering lyso-pNAPE, which is to bypass GP-AEA production but normally yielding AEA, was shown to be less effective than lyso-NAPE against mesenchymal tumor invasion. Altogether, these data suggest that lyso-NAPE and GP-AEA are novel antitumor NAPE metabolites of mesenchymal tumor invasion.  

Adaptive immunity requires the expansion of antigen-specific T cells, a process called clonal selection. Despite our understanding of how proximal signaling events scale with T cell receptor (TCR) affinity for an antigen, how affinity controls the downstream cell biology of clonal expansion is incompletely understood. We have identified NAD+ biosynthesis as a central mechanism of clonal selection. We find TCR affinity dose-dependently tunes the expansion of cellular NAD+ levels upon T cell activation without altering the NAD+/NADH ratio, by concurrently promoting the NAD+ salvage pathway and inhibiting NAD+ consumption. Using flow cytometry sorting of T cells based on cellular NAD+ levels, we demonstrate that initial NAD+ pool size predicts proliferative potential, prior to the first division, and can equalize expansion capacity of low and high affinity cells. Mechanistically, we show NAD+ biosynthesis coordinates population dynamics by regulating mitochondrial ATP production to control: 1) the rate of cell death; 2) cell cycle kinetics by modulating cMyc stability. Moreover, we find that while both cell death and cMyc destabilization are induced upon inhibiting the NAD+ salvage pathway, delayed restoration of NAD+ biosynthesis 24 hours later can reverse these phenotypes and recover normal expansion, even after TCR stimulation is removed. Finally, we show that globally enhancing NAD+ biosynthesis in T cells in vivo results in altered clonal selection, permitting the expansion of lower affinity T cells to the same numbers as controls. Altogether, these findings provide the first comprehensive biochemical for how TCR affinity coordinates the cell biology clonal selection.

Mounting evidence suggests a prominent role for gut microbiota in pathophysiological pathways influencing central nervous system and glucose homeostasis. In this regard, microbial metabolite acetate is a short-chain fatty acid, reported to mediate host glucose metabolism, insulin secretion and central appetite regulation. However its metabolic effects have not been fully studied in humans. 

For the APRAISE study, 60 individuals were selected to either have a microbiota with either high or low intestinal AP from a sample of 429 individuals from the HELIUS cohort study. Postprandial glucose and insulin responses were measured during a high-fiber standardized mixed meal test (SMMT). Additionally, central craving and reward responses to virtual food cues and the receipt of palatable food were measured using fMRI.

Low acetate producers showed higher plasma glucose levels 20 minutes after a mixed meal test and consistently higher plasma glucose fluctuations. Using linear mixed models, we showed that intestinal AP significantly influenced postprandial glucose and insulin response (p = 0.0088 and 0.0025 respectively). In addition, high-calorie food stimuli invoked a higher CNS response in de caudate nucleus in the high AP group, whereas anticipation of receiving palatable food invoked a higher CNS response in the putamen in the low AP group.

Congruent with previous studies, these findings indicate that high AP is beneficial in postprandial glucose metabolism and central appetite regulation. Additional analyses should determine whether this correlation exists for obese or diabetic subjects.

Studying sporadic age-associated neurodegenerative diseases is challenging due to the inaccessibility of vital patient-derived human brain cells for analysis and experimental modulation. While disease models based on iPSCs posed big hopes, reprogramming erases age-associated phenotypes, including defects in nuclear shuttling, epigenetic age, and adult-specific splicing. In contrast, direct conversion of patient fibroblasts to induced neurons (iNs) preserves cellular signatures of aging. Because old age is the major risk factor for sporadic Alzheimer’s disease (AD), iNs are a potentially very useful and predictive model system to study disease pathologies in a patient-specific manner. We previously showed that sporadic AD phenotypes can be modeled in age-equivalent iNs, but were absent in rejuvenated iPSC-derived neurons from the same patients. Here, using a cohort of 10 sporadic AD patients and 10 age-matched control donors, we identified a pathological hypo-mature neuronal state in iNs, which was associated with a re-activation of programmed cell death competence and a metabolic drift to aerobic glycolysis. Similar metabolic changes are believed to be partially causative for the loss of cell identity in cancer cell transformation. We further found that pathological isoform switching of the glycolytic enzyme pyruvate kinase (PKM) towards the cancer-associated PKM2 isoform conferred both metabolic alterations in AD iNs, and induced transcriptional hypo-maturity following nuclear translocation. Chemical modulation of PKM2 was able to restore a healthy neuronal metabolic signature, and increase neuronal resilience against cell death. Thus, the metabolic enzyme PKM represents a plausible target for age-dependent sporadic AD.

The metabolic needs for postnatal growth of the human nervous system are vast. Mutations in the mitochondrial enzyme glutamate pyruvate transaminase 2 (GPT2) in humans cause postnatal undergrowth of brain, and cognitive and motor disability. We demonstrate that GPT2 governs critical metabolic mechanisms intrinsic to neurons required for neuronal growth, including neuronal alanine synthesis and replenishment of tricarboxylic acid (TCA) cycle intermediates. Neuron-specific deletion of Gpt2 in mice is sufficient to cause motor abnormalities and death pre-weaning. Alanine biosynthesis is profoundly diminished in Gpt2-null neurons, such that alanine must be exogenously supplied for neuron growth in vitro. Dietary alanine supplementation rescues animal survival and improves the metabolic profile of Gpt2-null brain. In surviving Gpt2-null animals, we observe smaller upper and lower motor neurons, and death of lower motor neurons in vivo. We conclude that impairment of Gpt2-mediated metabolism causes selective vulnerabilities in large and long-projecting neurons such as motor neurons.

The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cysteine depletion not only induces amino acid response, but also triggers ferroptosis, a non-apoptotic cell death. Here we report that, unlike general amino acid starvation, cysteine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine that elicits the adaptative ATF4 response. Lysosomal cystine accumulation sensitizes ferroptosis by attenuating ATF4 induction. Untargeted metabolomics revealed the aryl hydrocarbon receptor (AhR) as the lysosome-nucleus signaling pathway. Intriguingly, AhR senses lysosomal cystine via the kynurenine pathway. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent CysRx to convert cytosolic cysteine to lysosomal cystine. By blocking the adaptive ATF4 response, CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, lysosomal nutrient reprogramming has the potential to induce ferroptosis in cancer without systemic perturbation.

The gallbladder stores bile between meals and empties into the duodenum upon demand, thereby being exposed to the intestinal-microbiome. This raises the need for antimicrobial-factors, among them mucins produced by cholangiocytes. Here, we show that the less frequent tuft cells  corelease acetylcholine and cysteinyl leukotrienes upon selective optogenetic-stimulation, with acetylcholine triggering exocytosis of mucin granules from cholangiocytes through the muscarinic receptor M3, and cysteinyl leukotrienes causing bladder-contraction through their cognate receptor CysLTR1. We identify the short chain fatty acid propionate, a major metabolite of intestinal bacteria activating tuft cells via the short chain free fatty acid receptor 2 and downstream signalling involving the cation channel TRPM5. Our results establish gallbladder tuft cells as sensors of a microbial product, initiating two independent innate defence mechanisms through cotransmission. Acetylcholine, best characterized as a neurotransmitter, serves here as a paracrine-factor triggering epithelial-defence, and cysteinyl leukotrienes, known from immune effector cells, trigger the muscular-contraction.

Beyond its well-established role in bone and mineral homeostasis, vitamin D (VD) has a biological activity also on skeletal muscle, and VD deficiency often occurs in conditions associated with skeletal muscle loss. Whether VD supplementation effectively counteracts muscle wasting is still debated. 

Our in vitro investigations on the direct effects of VD metabolites on skeletal muscle-derived cells have revealed an unexpected scenario: calcidiol (25VD) and its precursor cholecalciferol (VD3) protect from atrophy induced by pro-cachectic cytokines, calcitriol (1,25VD) is atrophic per se. In addition, 24,25-dihydroxy VD could have divergent effects, either atrophic or hypertrophic, depending on its concentration.

We hypothesize that the effects of VD3 supplementation in vivo depend on the relative expression of distinct VD hydroxylases producing the different pro- or anti-atrophic metabolites, thus explaining the contrasting outcomes of VD3 supplementation in preserving skeletal muscle in different physiopathological conditions.

Cancer cells reprogram their metabolism to maximize macromolecule biosynthesis for cellular growth and proliferation. The E2F transcription factor-1 (E2F1) is frequently altered in cancer and is involved in a multitude of functional processes including cell cycle control as well as metabolic processes. Our data indicate that E2F1 binds the promoter of several glutamine metabolic genes. Interestingly, gene expression levels of glutamine metabolic genes are strongly increased in mouse embryonic fibroblasts (MEFs) lacking E2F1 (E2f1^-/-) compared to E2f1^+/+ MEFs. In addition, we confirm that  E2f1^-/- MEFs take up more glutamine from the media, are more efficient in metabolizing glutamine and producing precursors for proliferation. Mechanistically, we observe a co-occupancy of E2F1 and MYC on glutamine metabolic promoters and silencing of MYC decreases the expression of glutamine metabolic genes in E2f1^-/- MEFs. Altogether, our results suggest that E2F1 is a regulator of glutamine metabolism and highlights potentially new targets for cancer interventions.

Cytomegalovirus (CMV) infection in the intestinal mucosa is a well-established disease complication of IBD and is associated with increase hospitalization and proctocolectomy rates. The underlying molecular mechanism leveraging increased susceptibility to CMV infection in IBD are poorly understood. Using genetic models of the IBD risk gene and regulator of the ER-stress response Xbp1, we show that chronic ER-stress debilitates cGAS/STING signalling and CMV virus control in intestinal epithelial cells. Mechanistically, we find that ER-stress induces a profound switch in serin-glycin metabolism aiming to counterbalance reactive oxygen species. Specific interception of de-novo serin synthesis or serin-/glycin deprivation increases ROS and lipid peroxidation, resulting in impaired epithelial cGAS/STING signalling, whereas ROS scavenging using n-acetyl cysteine (NAC) fully restores epithelial cGAS/STING signalling and CMV virus control. Altogether our findings define a novel molecular mechanism on how chronic intestinal inflammation increases susceptibility to cytomegalovirus infection as disease complication in IBD.

The rare population of hematopoietic stem cells (HSCs) continuously supplies all blood cell types in homeostasis and emergency hematopoiesis. While dormancy is a fundamental property that protects HSCs from exhaustion and malignant transformation throughout life, the mechanisms controlling the switch between dormancy and awakening of HSCs remain elusive. We identified that CD38 ecto-enzymatic activity promotes murine and human HSCs’ dormancy. The product of nicotinamide adenine dinucleotide (NAD+) conversion by CD38 – cyclic adenosine diphosphate ribose (cADPR), regulates the expression of the transcription factor c-Fos via an increase of cytoplasmic Ca²⁺ concentration. In turn, c-Fos induces the expression of cell cycle inhibitor p57^Kip2, thereby promoting HSCs’ quiescence. Furthermore, CD38 controls human HSC dormancy, despite its lack of expression on them, providing cADPR in a paracrine manner from neighboring CD38+ cells. Together, we discovered the previously unknown CD38/NAD+/cADPR/Ca²⁺/c-Fos/p57^Kip2 axis, which regulates HSCs’ dormancy, irrespective of whether HSCs themselves express CD38 or not.   

Metabolic competition in cancer occurs on a systemic level, triggering systemic metabolic dysfunction, eventually leading to cachexia. Cachexia is a wasting syndrome characterized by devastating skeletal muscle atrophy that dramatically increases mortality in various diseases, most notably in cancer patients with a penetrance up to 80%. Knowledge regarding the mechanism of cancer-induced cachexia remains very scarce, making cachexia an unmet medical need. In this study, we discovered strong alterations of iron metabolism in the skeletal muscle of both cancer patients and tumor-bearing mice, characterized by decreased iron availability in the mitochondria. We found that modulation of iron levels directly influences myotube size in vitro and muscle mass in otherwise healthy mice. Importantly, the sole modulation of the iron importer TFR1 was sufficient to regulate muscle mass through hypertrophy or atrophy according to its levels. Furthermore, iron supplementation was sufficient to preserve both muscle function, mass, prolong survival both in tumor-bearing mice and non-cancer models, and even rescue strength in human subjects within unexpectedly short time frame. Importantly, we found that iron supplementation was sufficient to refuels mitochondrial oxidative metabolism and energy production, allowing the restoration of muscle mass homeostasis. Overall, our findings provide new mechanistic insights in cancer-induced skeletal muscle wasting, and support targeting iron metabolism as a potential therapeutic option for muscle wasting diseases.

The intermediates of the tricarboxylic acid (TCA) cycle possess several different cellular roles. The anaplerotic reactions are essential to prevent the energetical collapse of the cell by depleting the TCA intermediates. Pyruvate carboxylase (PC) was shown to play a crucial role in sustaining the viability and agressivness of a few types of cancer cells in vitro. We applied immunodetection methods to evaluate the expression of PC among different types of human brain tumors. Furthermore, the effect of the PC inhibition on cellular metabolism and viability of cultured glioblastoma cells was determined. Our results revealed that human astrocytoma, glioblastoma, oligodendroglioma, and meningioma types of tumors ubiquitously express PC. In addition, the inhibition of PC in cultured glioblastoma cells stimulated the metabolic changes and negatively affected their viability. The supplementation of the culture media with several intermediates of the TCA cycle reverted a lethal effect of PC inhibition. Therefore, it could be supposed that PC expression among brain tumor-forming cells is essential for sustaining their metabolism and viability. 

This work was supported by the Slovak Research and Development Agency under Contract No. APVV-18-0088; and VEGA 1/0255/20.

Obesity is a public-health concern worldwide, with increasing prevalence, commorbidities, complications and costs for the society. Effective long-term treatments are challenging and often require multiple approaches, with recent studies highlighting the potential of increased thermogenesis as a promising pharmacological target. We evaluated the effects of either succinate (involved in brown adipose tissue activation) and/or liraglutide (GLP-1 agonist) treatment in phenotypes, as well as hypothalamic and serum metabolites, of C57BL/6J mice treated with high-fat diet. Succinate treatment increased core temperature of mice (measured by infrared camera). Interestingly, only 2 metabolites were significantly modulated in the hypothalamus (PC 40:7 and TG 48:2) by liraglutide treatment, while 19 were changed in the serum, being one in the high-fat fed group and 18 in those that received succinate treatment. RT-PCR of white adipose tissue revealed changes in Ucp1, Hif1a, Scd1, Nrf1 and inflammatory markers, indicating a peripheral effect of these treatments.

MANF regulate body weigh and in pregnancy body weight change.

Loss of 3p21.2 gene eterozigosis has been point out as causative in breast cancer (1) 

MANF gene is located in 3p21.2 and that may be causative in breast cancer  and its comorbility with psychiatric disorders.

(1)J Hum Genet . 2002;47(9):453-9.  doi: 10.1007/s100380200064. 

Chromosome 3p and breast cancer, Qifeng Yang 1, Goro Yoshimura, Ichiro Mori, Takeo Sakurai, Kennichi Kakudo

Tumor-reactive CD8 T cells often acquire a state of exhaustion due to multiple factors imposed by the tumor microenvironment. T cell exhaustion with functional impairment interferes with the role of T cells in tumor rejection. However, it became clear that certain subsets of exhausted CD8 TILs have superior functional capacities. Particularly, precursor exhausted T cells with self-renewal capacity and functional persistence are closely tied to durable responses to checkpoint blockade and adoptive transfer therapy. Yet their differentiation and their developmental requirements are poorly understood. By taking advantage of single-cell profiling technologies, we revealed that precursor exhausted T cells have enhanced mitochondrial activity in tumors resected from cancer patients. Next, we used a new human ex vivo T cell exhaustion model to dissect the specific metabolic pathways in the development of precursor exhausted T cells. Our ex vivo model uncovered that early precursor exhausted T cells preferentially rely on amino acids and fatty acids rather than glycolysis. Our ongoing work utilizing CRISPR/Cas9-based genetic screens will expand our basic knowledge on T cell metabolism and may lead to the discovery of novel metabolism-based therapeutic strategies.

Increased de novo lipogenesis (DNL) is a hallmark of nonalcoholic fatty liver disease (NAFLD) in obesity, but the macronutrient source for >80% carbon backbone for fatty acid synthesis has not been determined. Here we take an integrated approach to dissect nutrient metabolism, both ex vivo and in vivo. We discover a castling effect of glucose and glutamine metabolism through ex vivo isotope tracing studies that limits the entrance of glucose carbon into the glutamine-dominated tricarboxylic acid cycle (TCA) and DNL pathways. In vivo comparative tracing studies with a high carbohydrate drink (glucose/amino acid, 3:1, w/w) reveal glucose contributed ~45% of carbon via indirect pathway toward newly synthesized fatty acids, while amino acids contributed ~30%. These results confirm dietary amino acids are twice more efficient than glucose in labeling the hepatic acetyl-CoA and fatty acid pool, and together they account for over 70% of hepatic DNL substrate. Both glucose and glutamine carbon flux into DNL pathways are increased in obese hepatocytes, and metabolic rerouting of substrate carbon toward glycogen synthesis and energy production through GYS2 and GLUD1 overexpression improves hepatic steatosis. Together, our study demonstrates an under-appreciated role of amino acids in fatty acid synthesis, and reveals the quantitative contribution of glucose and amino acid carbon toward hepatic DNL and the development of hepatic steatosis in obesity.

Ketogenic dietary interventions (KDIs) are highly efficient approaches to ameliorate progression of autosomal dominant polycystic kidney disease (ADPKD) in animal models. To promote translation to the clinical setting, we have joint forces to develop a pipeline for the design of clinical projects. Based on a Retrospective Case Series Study enrolling 131 ADPKD patients that adhered to KDIs we can show that KDIs could indeed be beneficial for the overall well-being of ADPKD patients. Our proof-of-principle trial RESET-ADPKD now underlines feasibility and biochemical efficacy of two KDIs (ketogenic diet and 3-day water fasting). The currently ongoing randomized controlled clinical trial KETO-ADPKD extends this approach to 63 patients and has recently completed recruitment. Safety, efficacy and adherence are tightly monitored by monthly visits including MRI based kidney volumetry, metabolic tests and questionnaires. We can now present the first insights into the role of KDIs in human ADPKD based on these three studies.

Nonalcoholic fatty liver disease (NAFLD) is a complex disease involving alterations in multiple biological processes regulated by the interactions between obesity, genetic background and environmental factors including the microbiome. To decipher hepatic steatosis (HS) pathogenesis by excluding critical confounding factors including genetic variants and diabetes, we characterized 56 heterogeneous NAFLD patients by generating multi-omics data including plasma metabolomics, inflammatory proteomics data as well as oral and gut metagenomics. We revealed the altered global metabolic and inflammatory processes, and their connections with the species' abundances in the oral and gut microbiome. We integrated these multi-omics data using biological networks and identified the key features associated with HS. We finally predicted HS using these key features and validated our findings in a follow-up cohort, where we characterized 22 subjects with varying degrees of HS.

Alzheimer’s (AD) and Parkinson’s diseases (PD) are complex diseases with highly variable patient responses to treatment. Due to the growing evidence for ageing-related clinical and pathological commonalities between AD and PD, these diseases have recently been studied in tandem. In this study, we analysed transcriptomic data from AD and PD patients, and stratified these patients into three subclasses with distinct gene expression and metabolic profiles. Through integrating transcriptomic data with a genome-scale metabolic model and validating our findings by network exploration and co-analysis using a zebrafish ageing model, we identified retinoids as a key ageing-related feature in all subclasses of AD and PD. We also demonstrated that the dysregulation of androgen metabolism by three different independent mechanisms is a source of heterogeneity in AD and PD. Our work highlights the need for stratification of AD/PD patients and development of precision medicine approaches.

Dendritic cells (DCs) are central regulators of both innate and adaptive immune processes. In addition to immune stimuli, DC also integrate metabolic signals to tune their function. We previously demonstrated that fatty acids induce a distinct immunometabolic phenotype in TLR-activated DC, characterized by high IL-23 production and reduced glycolysis. Here we present integrated analysis of metabolic reprogramming in these cells and demonstrate that glutamine and related amino acids affect IL-23 production and its exacerbation by palmitic acid. TLR-mediated activation increases expression of key amino acid-related enzymes such as Asns, while decreasing Gls, Glud1, and Got2. Palmitic acid modulates these changes, notably decreasing expression of glutamine transporter Slc1a5. Depletion experiments show that glutamine or aspartic acid deficiency phenocopies fatty acid treatment of TLR-activated DCs with respect to IL23 production. These results shed new light on the complex role of amino acid metabolism in the tuning of innate immune responses.

Sarcopenia is the age-related progressive loss of skeletal muscle and one of the public health concerns of elderly people. Here, based on gene coexpression network analysis, we identified disease-associated genes (e. g. ANXA2, CLMP, CYC1 and SDHB) across three different sarcopenia cohorts and highlighted their role of increased extracellular matrix organisation and decreased mitochondrial energy metabolism. Furthermore, we identified transcription factors, including SP1, showing regulation of muscle expression of genes in the sarcopenia-associated modules. Further, we identified potential effective drugs (e. g. withaferin-a) by in-silico drug repurposing. In conclusion, our integrative analysis facilitates the understanding of the pathophysiologic mechanisms of sarcopenia and improvement of novel treatment approaches.

In this study, we performed an eQTL analysis from 100 Japanese patients with clear cell renal cell carcinoma (ccRCC). Survival analysis and weighted correlation network analysis (WGCNA) were used to find potential pathogenic eGenes and analyze the function of eGenes. A predictive machine learning model was built based on the genotype data of cis-eQTLs from pathogenic eGenes and was validated in the TCGA ccRCC cohort (n = 289). In summary, a total of 805 significant eGenes were identified from the Japanese ccRCC cohort. Of these eGenes, 117 were identified as pathogenic eGenes with a total of 579 significant cis-eQTLs, while EPHX2, PYGM, TUBA1C, APRT and CHMP1A showed hubness in the WGCNA network. The study provides a comprehensive reference to ccRCC with identified key genes and eQTLs as potential drug targets. Moreover, the proposed machine learning model could be potentially used for the prediction of ccRCC patient survival.

The role of gut microbiota in humans is of great interest, and metagenomics provides key opportunities for extensively analysing bacterial diversity in health and disease. Despite increasing efforts to expand microbial gene catalogues and an increasing number of metagenomes assembled genomes, there has been few investigations in pan-metagenomics and in-depth functional analysis across different geographies and diseases. Here, we explored 5,708 human gut metagenome samples across 19 countries and 23 diseases, performing compositional, functional cluster, and integrative analysis. We identified Fusobacterium nucleatum and Anaerostipes hadrus with the highest frequencies, enriched and depleted respectively, across different disease cohorts. Distinct functional distributions were observed in the gut microbiome of westernized and non-westernized populations. Additionally, a random forest machine learning model explained by SHapley Additive exPlanations (SHAP) identified key species as disease biomarkers. This compositional and functional analysis are presented in an open-access Human Gut Microbiome Atlas (www.microbiomeatlas.org), allowing for exploration of the richness, diseases, and regional signatures of the gut microbiota across different cohorts.

Pyruvate kinase catalyze PEP to pyruvate. It is the last step of glycolysis and destination of pyruvate, Lactate or OXPHOS, choose cells metabolism. Also, pyruvate kinase can have two different structure, dimer or tetramer form. Dimer form has lower enzymatic activity but also can be nuclear localization and work as co-transcription factor that express many genes related to cancer development. Our group showed PKL (pyruvate kinase liver), the isotype expressed in Liver, is one of key gene on the liver cancer and NAFLD (Non Alcoholic Fatty Liver Disease). PKL is involved in de novo fatty acid genesis in liver cells. We developed new in vitro model that synthesize and accumulation lipid droplet via fatty acid synthesis pathway. With this new model, we successfully screened new drug candidate for NAFLD. High performing PKL activators reduced TAG level and inhibit protein expression on fatty acid synthesis pathway. Our study will be a milestone for drug development of NAFLD.

Cancer immunotherapy, including checkpoint blockade and adoptive transfer of tumor-reactive T cells, represents a paradigm shift in the treatment of malignancies in recent years. Nevertheless, a significant portion of patients are refractory to cancer immunotherapies, which may be in part due to the persistent impairment of anti-tumor effector functions in T cells, a phenomenon referred to as T cell exhaustion. Emerging evidence reveal that alterations in epigenetic landscape are keys events to drive development of T cell exhaustion under chronic antigenic stresses. However, it remains elusive how T cells engage epigenetic reprogramming to orchestrate exhausted state. Here, we examined the mitochondrial fitness in CD8⁺ TILs. We found that tumor-infiltrating T cells with accumulation of damaged mitochondria display more severe exhausted phenotypes, including decreased proliferation capacity, reduced cytokine production and up-regulation of co-inhibitory receptors. Importantly, we found that the accumulation of dysfunctional mitochondria is controlled by the affinity of TCR-pMHCI interaction, and also supported by the PD-1 expression. Moreover, the combination of glucose deprivation, hypoxia and TCR signaling in vitro can drastically weaken T cell immunity with the accumulation of dysfunctional mitochondria. Ultimately, supplementation with nicotinamide riboside enhances T cell mitochondrial fitness and improved responsiveness to anti-PD-1 treatment. Taken together, our study suggests that mitochondrial fitness is pivotal for orchestrating T cell-mediated anti-tumor immunity and the accumulation of dysfunctional mitochondria could instruct epigenetic reprogramming for T cell exhaustion. Our findings also provide pillars for better harnessing T cell immune responses with metabolic regulations for cancer treatment.

ABC transporters represent major players of chemoresistance in a broad range of tumors and novel functions in cancer progression are emerging. Particularly, ABCAs are physiologically involved in cholesterol/lipid homeostasis, which is often deregulated in cancer cells.

We previously identified ABCA6 as indicator of good prognosis in Ewing sarcoma (ES) patients. In ES PDX-derived cell lines model, intracellular cholesterol content inversely correlated with ABCA6 expression and the overexpression of ABCA6 impaired migration and anchorage independent-growth and induced an increase in the sensitivity to doxorubicin. Additionally, simvastatin treatment of cells that low expressing ABCA6, impaired the migratory ability and sensitivity to doxorubicin enhanced. Combined treatment experiments reported a synergistic effect between doxorubicin and simvastatin in the same model. 

Our results suggest that ABCA6 may have a role in cancer progression likely modulating homeostasis cholesterol and statins might be used as treatment in combination with classical chemotherapy.

[This work was supported by Italian Ministry of Health (RF-2016-02361373)]

Leucine zipper-like transcriptional regulator 1 (LZTR1) is a protein that belongs to the BTB-Kelch superfamily and interacts with the Cullin3 (CUL3)-based E3 ubiquitin ligase complex. Mutations in LZTR1 have been linked to several diseases such as glioblastoma, schwannomatosis, and Noonan syndrome (NS). NS is part of a broader group of pathologies called RASopathies in which one of the key features is dysfunctional metabolism, characterized by higher resting energy expenditure and a lean phenotype.

To evaluate how Lztr1 loss may contribute to organism-wide metabolic aging, a pilot study has been conducted to test the energy expenditure of global Lztr1 +/- (HET) compared to wild-type (WT) mice with the Oxymax/CLAMS – an indirect calorimetry-based system. Our analysis indicates that loss of Lztr1 triggers a specific remodeling of the metabolic flux at a young age that fails during aging and leads to dramatic changes in body weight and composition with a pronounced depletion of body fat. Comparison of the liver proteome by mass spectrometry (LC-MS) further revealed major differences between old HET and WT mice with a strong focus in the mitochondrial compartment.

From these preliminary results, we conclude that Lztr1 influences overall metabolism and that Lztr1-depletion is associated with specific metabolic shifts both in mitochondrial functionality and energy expenditure profiles.

Pancreatic ductal adenocarcinoma (PDAC) cells reprogram their transcriptional and metabolic state to sustain their energetic needs in the nutrient-poor tumor microenvironment. We employed in vivo CRISPR screening in an orthotopic PDAC model to identify modulators of aggressive PDAC growth in vivo. The screening identified a previously uncharacterized ISL LIM homeobox 2 (ISL2) gene as a candidate tumor suppressor among some anticipated growth modulators. We confirmed that depletion of ISL2 leads to enhanced cell proliferation and tumor growth in vitro and in vivo. Conversely, the exogenous expression of ISL2 or CRISPR-mediated epigenetic upregulation of the endogenous loci led to reduced cell proliferation, supporting the hypothesis that ISL2 is a candidate tumor suppressor. Importantly, we find that ISL2 is a nuclear, chromatin-associated transcription factor that is epigenetically silenced through DNA methylation in a significant fraction of PDAC tumors. Critically, higher DNA methylation of ISL2 or its reduced expression correlates with poor patient survival. Mechanistically, ISL2 binds to thousands of genes, and its depletion increases antioxidant capacity, oxidative phosphorylation (OXPHOS) and fatty acid metabolism. Critically, ISL2 depleted cells have heightened stemness potential, and we find PPARg as a critical modulator of cell proliferation in ISL2 depleted cells. Notably, ISL2 depleted cells are sensitive to small molecule inhibitors of mitochondrial complex I  in vitro and in vivo. Collectively, these findings nominate ISL2 as a putative tumor suppressor candidate whose inactivation leads to an aggressive PDAC growth through increased mitochondrial metabolism and antioxidant capacity, which creates a potentially exploitable therapeutic vulnerability.

Cancer subtypes have developed specific adaptations in terms of energetics and metabolism, which could be used to identify vulnerabilities that can be targeted through pharmacology or dietetics. However, it has been a challenge to correlate between genetic profile and metabolic phenotypes.

Our team developed the Massively Correlated Biclustering (MCbiclust) bioinformatic tool to detect correlated gene biclusters and stratify breast tumors according to their mitochondrial gene expression profiles. On similarly stratified breast cancer cell lines, we evaluated mitochondrial function, metabolic profile by functional imaging, mass spectrometry and other biochemical approaches.

We have found that the breast cancer subtypes identified using MCbiclust have differential glutamine utilization, associated with adaptive changes in mitochondrial function. These metabolic differences were also confirmed in more physiologically relevant models, namely spheroids derived from the identified cancer cell lines. Enzymatic activities and protein expression analysis on glutathione and thioredoxin systems also revealed that these subtypes have different expression patterns of cellular redox machinery.

Overall, we show that MCbiclust sorts breast cancer cell lines and patient samples based on gene expression profile, thus identifying mitochondrial tumor subtypes with specific metabolic phenotypes in a way that does not strictly match traditional breast cancer classifications. Interestingly, our method could be exploited to discover new biomarkers in order to develop personalized cancer treatments.

Amino acid (AA) availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA-D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralog pairs (RagA/B, RagC/D) are referred to as functionally-redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically-modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how AA regulate mTORC1. Our findings reveal key qualitative differences between Rag paralogs in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.

Acetyl-coenzyme A (acetyl-CoA) plays an important role in metabolism, gene expression, signaling, and other cellular processes, via transfer of its acetyl group to the proteins and metabolites. However, reprogramming of acetyl-CoA metabolism and its molecular mechanisms remain poorly understood. Here, we investigated and uncovered the impact of global acetyl-CoA metabolism and protein acetylation in cancer, in particular in a mouse model and patient samples of hepatocellular carcinoma (HCC). We show that acetyl-CoA levels are reduced in HCC due to down-regulation of the main acetyl-CoA synthesis pathways including branched chain amino acid (BCAA) catabolism, fatty acid oxidation (FAO), and pyruvate catabolism, leading to hypo-acetylation mainly of non-histone proteins including many enzymes in metabolic pathways.Mechanistically, high levels of the transcription factors TEAD2 and E2A repress genes encoding enzymes of the acetyl-CoA synthesis pathways.  Importantly, repression of acetyl-CoA metabolism promoted oncogenic dedifferentiation and proliferation.  Our findings suggest that transcriptional reprogramming of acetyl-CoA metabolism and reduced protein acetylation are determinants of cancer.

Infection with SARS-CoV-2 induces both a potent cellular and humoral immune response. Unfortunately, various mutants ofthis virus have emerged that manage to mostly escape antibody recognition. Therefore, it is critically important to understand if SARS-CoV-2 convalescent individuals develop functional memory CD8 T cells that are capable of protection from subsequent tinfections. Here we performed a comprehensive phenotypic and functional analysis of antigen-specific CD8 T cells in SARS-CoV-2–infected individuals 3 and 6 months’ post infection and in vaccinated individuals. Mice with a humanized immunesystem using cells from SARS-CoV-2 convalescent donors were infected with mCMV-strains carrying dominant SARS-CoV-2or Influenza epitopes, to assess the in vivo recall capacity of antigen-specific cells. We demonstrate that both SARS-CoV-2 infection and vaccination elicit potent antigen-specific memory CD8 T cell response. However, the overall magnitude of theanalyzed antigen-specific response was higher after vaccination. Importantly, convalescent individuals developed SARS-CoV-2-specific memory T cells that persisted for at least six months. Expression of CD3, CD57 and NKG2D was lower in the individuals vaccinated against COVID-19 compared to SARS-CoV-2–infected individuals whereas expression of CD27 was higher after COVID-19 vaccination. In addition, we observed distinct phenotypic profiles of SARS-CoV-2 and Influenza-specific memory CD8 T cells. Our findings indicate that both infection and vaccination induce a potent memory CD8 T cell response against SARS-CoV-2, but that there are key functional difference between these methods of memory induction.

Tumor resistance to anticancer drugs is a well-known clinical phenomenon that impinges on patients' quality of life and survival. We recently discovered a new mechanism of adaptation that involves translational regulation of metabolism in response to therapies that target oncogenic kinases (e.g. EGFR/HER2, BCR/ABL, BRAF). Translation of mRNAs encoding metabolic regulators, including those involved in serine (PHGDH, PSAT1), aspartate (PC), and asparagine (ASNS) synthesis, was dependent on the mTORC1/eIF4F/4E-BP axis. Pathway dysregulation caused by 4E-BP1/2 depletion induces metabolic plasticity and partial resistance to combinations of kinase inhibitors and biguanides, suggesting that translational regulation of metabolic genes via the mTORC1/4E-BP pathway plays a major role in energy stress response in cancer and that the efficiency of cancer strategies targeting metabolic vulnerabilities is dependent on the translation machinery. 

Intriguingly, we found that drugs that interfere with the translation machinery (such as molecules targeting the translation initiation complex eIF4F) are very effective against BRAF inhibitor-resistant melanoma cells. Classic translational targets of mTORC1/eIF4F, encoding for cell cycle and pro-survival proteins, are downregulated by these inhibitors. Metabolic characterization of BRAFinh-resistant melanoma cells treated with eIF4Finh shows a strong metabolic rewiring, particularly for TCA cycle intermediates and derivate metabolites. Bioenergetic assessment using Seahorse technology indicates a strong reduction in respiration, drastically amplified by the combination with BRAFinh vemurafenib. Our studies highlight (i) the importance of the crosstalk between mRNA translation and metabolic regulation and (ii) that direct inhibition of translation represents an appealing therapeutic strategy for clinical cases of targeted therapy resistance.  

Background - Succinate excreted from cells during hypoxia and metabolic overload is sensed by SUCNR1/GPR91 as an auto- and paracrine signal of metabolic stress.

Overarching working-hypothesis: On metabolically stressed cells SUCNR1 functions as an autocrine, Gi-signaling, dampening sensor while on neighboring M2 macrophages SUCNR1 orchestrates tissue repair/remodeling through Gq-mediated hyperpolarization of the M2 phenotype. Adipose - Gi mediated antilipolytic effect of SUCNR1 is part of the autocrine dampening component and Gq-mediated SUCNR1 activation of M2 macrophages is the initial signal in the formation of the repairing adipogenic niche. Skeletal muscle - SUCNR1 in a similar manner initiates the remodeling/repair through M2s macrophages whereas a dampening autocrine component is not physiological relevant. In Liver and kidney - SUCNR1 is massively expressed on the main metabolically active cells, hepatocytes and proximal tubular cells, respectively; but in the liver also on activated stellate cells.

In general - Succinate/SUCNR1 signaling balances between protection/repair and inflammation.

Regulatory T cells (Tregs) are central for peripheral tolerance and their deregulation is associated to autoimmunity. Autoimmune Tregs display altered mitochondrial metabolism but factors contributing to this phenotype still remain elusive. High salt (HS) intake has been identified to potentially promote autoimmunity by inducing shifts in the immune cell balance, mainly by promoting proliferation and activity of pro-inflammatory cells, like T helper 17 (Th17) and M1 macrophages, and by impairing the functions of anti-inflammatory cells such as Tregs and M2 macrophages. In spite of this, the precise molecular mechanisms that lead to this phenotype are still unknown. The role of metabolic regulation in shaping immune responses has gained increasing attention in recent years. Cellular metabolism is a vital process, which is essential for growth, survival and proliferation, and can be greatly influenced by environmental factors such as diet. Our results show that HS directly interferes with the mitochondrial respiration of Tregs and promotes shifts in metabolite composition, recapitulating phenotypic features of autoimmune Tregs. Metabolic analysis by liquid-chromatography mass spectrometry (LC-MS) in HS-treated Tregs revealed shifts in several key metabolites, essential for the mitochondrial electron transport chain (ETC) activity and for overall oxidative phosphorylation (OXPHOS) pathway. This metabolic disturbance of Tregs could be mimicked by inhibition of the ETC leading to similarly dysfunction in vitro and in vivo. Our results indicate that salt could contribute to metabolic reprogramming observed in autoimmune Tregs.