Welcome drinks reception & Poster session 1

Maintenance of cellular metabolism and suppressive function is crucial for Foxp3+ Treg cells (Tregs) in maintaining immune tolerance. Yet how Tregs coordinate transcriptional networks and metabolic reprogramming in the process remains elusive. Here we report that Treg BATF couples the TH2 suppressive function and triglyceride (TG) metabolism to restrain allergic inflammation. Mice with Treg-specific ablation of BATF developed an inflammatory disorder characterized by TH2-biased responses, and were predisposed to house dust mite (HDM)-induced airway inflammation. Loss of BATF enabled Tregs to acquire TH2-cell-like characteristics. Moreover, BATF-deficient Tregs elevated levels of cellular TGs. Repressing or elevating cellular TGs respectively restored or exacerbated their defects. Mechanistically, TCR/CD28 co-stimulation enhanced expression of BATF, which sustained IRF4 activity to preserve Treg functionality. Thus, BATF links Treg functional specification and TG metabolism to control allergic responses, suggesting that therapeutic targeting of TG metabolism could be used for the treatment of allergic disease.

Background   

Intracranial aneurysms (IA) is often associated with metabolic comorbidities. However, the role of plasma metabolite as potential biomarkers in patients with stable and ruptured intracranial aneurysms (RIAs) is still remain unidentified.

Aims  

Our study aimed at providing a plasma metabolic profile in different statuses of IA patients, developing an early diagnosis model and identifying the metabolic risk factors associated with rupture.

Methods  

A case-control study of 71 participants was performed, including 44 patients with IA of different statuses and 27 control subjects. Integration analyses of all samples through the plasma pseudotargeted metabolomics method were conducted by UHPLC-TQMS. Significant different metabolites were figured out through bioinformatics analysis. The classifiers based on the machine learning algorithms were used to establish the IA diagnosis model and the rupture prediction model.

Results 

We determined three machine learning- based metabolite panels to identity UIAs and RIAs yielded the area under the curve (AUC) of 0.925 (95% CI 0.852 to 0.998) and 0.972 (95% CI 0.935 to 1.00, RIA vs Control) respectively. Moreover, a risk prediction model of IA rupture yielded AUC of 0.967 (95% CI 0.912 to 1.00, RIA vs UIA). Notably, glutathione is a hallmark biomarker for UIA and the content of uridine, indole acetaldehyde and 5,6-dimethylbenzimidazole decreased significantly with the severity of IA. KEGG pathway analysis revealed glutathione metabolism, tryptophan metabolism, etc. were enriched.

Conclusion

Our study comprehensively characterizes plasma metabolites in different statuses of IA, and demonstrates the potential of metabolic markers as non-invasive diagnostic and risk stratification tools for IA.

Through an untargeted multi-omics approach, we provide a comprehensive first report of the role of the Krebs cycle enzyme fumarate hydratase in regulating macrophage immune responses. Using a pharmacological inhibitor of FH, we have characterised fumarate-dependent and -independent effects of FH inhibition within LPS-stimulated macrophages.

Of note, impaired FH function decreases expression of pro-IL-1β and IL-10, while increasing expression of the cytokines IL-6 and TNFα. Interestingly, FH inhibition also causes mitochondrial hyperpolarisation, leading to ROS production and mitochondrial dsRNA (mtdsRNA) release, which drives the RIG-I/MDA5-dependent induction of type I IFN. This is the first report linking mitochondrial membrane potential to mtdsRNA release. Pharmacological inhibition of FH in an in vivo model of LPS-induced inflammation also increases serum IFN-β levels.

Here we present evidence supporting the role of fumarate hydratase as an enzyme which orchestrates macrophage function and raises the potential of therapeutically targeting FH in the treatment of inflammatory diseases. 

The Tricarboxylic Acid (TCA) cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Of note, TCAi impaired proline and aspartate synthesis but promoted cytosolic GSH synthesis. Heme regulated inhibitor (HRI) and general control nonderepressible 2 (GCN2) are implicated as the ISR kinases responsible for sensing TCA cycle inhibition, reducing total cytosolic translation and promoting ATF4 translation. Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology and amino acid homeostasis.

Hypothalamic interleukin-6 (IL6) exerts a broad metabolic control. Here we demonstrated that IL6 activates the ERK1/2 pathway in the ventromedial hypothalamus (VMH), stimulating AMPK/ACC signaling and fatty acid oxidation in mice skeletal muscle. Bioinformatics analysis revealed that the hypothalamic IL6/ERK1-2 axis is closely associated with fatty acid oxidation- and mitochondrial-related genes in skeletal muscle of isogenic BXD mice strains and humans. We showed that the hypothalamic IL6/ERK1/2 pathway requires the α2-adrenergic pathway to modify the fatty acid skeletal muscle metabolism. To address the physiological relevance of these findings, we demonstrated that this neuromuscular circuitry is required to underpin AMPK/ACC signaling activation and fatty acid oxidation post-exercise. Once the selective downregulation of IL6 receptor in VMH abolished the effects of exercise to sustain AMPK and ACC phosphorylation and fatty acid oxidation in the muscle post-exercise. Altogether, these data demonstrated that IL6/ERK axis in VMH controls fatty acid metabolism in skeletal muscle.

Ceramides are metabolically active lipids implicated in cardiometabolic disease pathogenesis. Using targeted LC-MS/MS we measured serum ceramides and dihydroceramides in the robust Utah Obesity Study (UOS) to evaluate Roux-en-Y gastric bypass (RYGB) mediated cardiometabolic disease risk reduction and ceramides. The UOS consists of three groups with 12-years of follow-up: 418 patients who underwent RYGB, 417 patients who sought but did not undergo RYGB due to insurance coverage, 321 severely obese population controls. Distinct ceramide and dihydroceramide species (e.g., 16,18,22 carbon acyl chains) decreased 2 and 12-years post-surgery. Additionally, Cer(d18:1/16:0) and Cer(d18:1/24:1) demarcate patients that fail to undergo diabetes remission and predict diabetes remission durability over a 10-year period. These associations were independent of body mass. Collectively, these data indicate that ceramide lowering may be a critical mechanism of bariatric surgery’s metabolic benefits and serum ceramide levels could be a risk stratification tool in post-RYGB diabetes management.

Free radicals (FR) are atoms and molecules that contain at least one unpaired electron. They play an important role both in health and disease, but are difficult to detect with high sensitivity and resolution. Nanodiamonds have exceptional biocompatibility and extremely stable fluorescence. Moreover, this fluorescence can be modulated by external magnetic fields. It allows us to use nanodiamonds to sense the magnetic fields generated by FR in live cells with unprecedented resolution. As a proof of concept, we have used this technique, nanodiamond-based magnetometry, in several biological models: detecting nitric oxide formed by murine macrophages; tracking the changes in FR load in re-differentiating colon adenocarcinoma cells; assessing the oxidative stress, caused by cigarette smoke extract in primary lung epithelial cells from healthy donors and chronic obstructive pulmonary disease patients. Our method is highly versatile and can become a useful addition to other single-cell techniques for detecting these elusive metabolites.

Dietary fibers are essential for preserving a healthy and functional gut microbiota and influence the biochemical transformation of host metabolites. Here we show that the soluble dietary fiber oligofructose sustains the production of 6a-hydroxylated bile acids from primary bile acids by gut bacteria when fed western-style diet. Mechanistically, we demonstrated that the effects of oligofructose on 6a-hydroxylated bile acids were microbiota dependent and specifically required functional TGR5 signaling to reduce body weight gain and improve glucose metabolism. Furthermore, we show that the 6a-hydroxylated bile acid hyodeoxycholic acid stimulates TGR5 signaling, in vitro and in vivo, and increases GLP-1R activity to improve host glucose metabolism. Modulation of the gut microbiota with dietary fibers that enrich 6a-hydroxylated bile acid levels might be a promising therapeutic strategy to alleviate obesity and type 2 diabetes.

The shift on energetic demands of proliferating cells during tumorigenesis requires an intense crosstalk between cell cycle and metabolism. Beyond their role in cell proliferation, cell cycle regulators also modulate intracellular metabolism. Cyclin-dependent kinase 4 (CDK4) is upregulated or stabilized in triple-negative breast cancer (TNBC), characterized by its aggressiveness and poor prognosis due to lack of selective therapies. We aimed to determine the metabolic role of CDK4 in TNBC cells. CDK4 deletion deeply affects mitochondrial morphology, leading to hyperfused mitochondria and reduced activity of fission proteins. Surprisingly, pro-apoptotic stimuli fail to induce proper apoptosis in CDK4-depleted or long-term CDK4/6 inhibitors-treated TNBC cells. Mechanistically, CDK4 depletion impairs mitochondrial-ER contacts thus reducing calcium fluxes upon pro-apoptotic stimuli. Taken together, these results suggest that CDK4 inhibition leads to cell death resistance limiting mitochondrial apoptotic function through dampened ER-mitochondrial calcium signalling in breast cancer cells.

Adrenal glands regulate cardiovascular physiology and pathophysiology via the synthesis and secretion of well-known compounds like mineralocorticoids, glucocorticoids and amine peptides. In the current study, we investigated a previously unknown systemic function of adrenal glands, that is the regulation of calcification and mineralization processes via peptidic metabolites.

Bovine adrenal gland homogenates were separated using chromatographic fractionation and the resulting fractions were first assessed for effects on vascular calcification processes in cells, aortic rings and vitamin D3 plus nicotine renal failure rat model, as the pathophysiological part of the calcification paradox. Potential mediators were distinguished by mass spectrometry and comparison with pertinent databases. We identified a 19 aa peptide, named “calcification blocking factor” (CBF), which reduces vascular calcification. CBF is released from the parent protein Chromogranin A, which is released from adrenal glands. CBF reduced the calcium content of cells and aortic rings in calcifying cultures. Pulse pressure as a marker of arterial stiffness of VDN animals treated with CBF significantly decreased. An 8 aa peptide was found to have a better effect on reducing vascular calcification when compared to the original peptide. Moreover, the 8 aa peptide promotes mineralization of bones which is analyzed by bone mineral density quantification, demonstrating the key role of this peptide in the calcification paradox. 

In conclusion, we show that CBF reduces vascular calcification via PIT-1/NF-κB/BMP2/p-SMAD pathway. Further, we identified the active site of this peptide (8aa long), which promotes bone mineralization. These findings suggest a novel function of adrenal glands in the calcification paradox.

Introduction: The kynurenine pathway is a critical immuno-metabolic regulator of inflammation. Enhanced Toll-like receptor signalling in liver tissue is associated with elevated 3HK in experimental AP. Here, we investigated whether 3HK directly potentiates TLR signalling in immune cells and whether 3HK injures primary human cells representative of the key organs damaged during AP.

Methods: Reporter cells engineered to interrogate the role of TLR1-9, NOD1/2, STING-pathway and NLRP3-inflammasome activation were exposed to dose-ranges of 3HK. 3HK-induced apoptosis of primary human small airway epithelial cells (SAEC), renal proximal tubule epithelial cells (HRPTEpC), and hepatocytes (PHH) was investigated. 

Results: 3HK potentiated the response of THP-1 cells to TLR2 (p<0.0001) and TLR4 (p=0.0002) specific ligands without directly activating TLRs1-9, NOD1/2, the STING-pathway or NLRP3-inflammasome. 3HK induced apoptosis and reduced cell viability in SAEC (p=0.0029), HRPTEpC (p=0.0128), and PHH (P<0.0001).

 Conclusions: 3HK plays an important role in propagating organ damage in acute pancreatitis. 

Mitochondria as critical regulators of intracellular signaling and metabolism determine T cell differentiation and function. We previously identified the mitochondrial protein TCAIM (T cell activation inhibitor, mitochondrial) being highly expressed in naïve and quiescent T cells but downregulated upon activation and effector differentiation. CD8⁺ T cells from Tcaim knock‑in and knock‑out mice showed reduced respective enhanced CD69 and Hif1α  up‑regulation, proliferation and effector cell differentiation upon activation. Metabolomics and transcriptional data revealed an impaired mevalonate pathway and cholesterol biosynthesis induction in TCAIM KI CD8⁺ T cells. Furthermore, we found TCAIM to interact with proteins involved in mitochondria‑ER contact formation including RMD3 and VDAC2. Treatment with Erastin, a small molecule that keeps VDAC2 in an open conformation for constant ATP/ADP exchange, completely rescued CD69 and HIF1α expression, proliferation and effector differentiation of TCAIM KI CD8⁺ T cells. Thus, TCAIM seems to repress VDAC2‑mediated ATP/ADP exchange controlling cholesterol synthesis and T cell fate.

Acute O₂ sensing is essential for mammalian homeostasis. The carotid body (CB), the main peripheral chemoreceptor, contains O₂-sensitive glomus cells. Hypoxia generates mitochondrial complex I (MCI) signaling molecules, NADH and reactive oxygen species, which inhibit membrane K⁺ channels to induce glomus cell depolarization and transmitter release. Mice lacking Ndufs2, a component of the ubiquinone/rotenone binding site, have abolished systemic hyperventilation (HVR) and glomus cell responses to hypoxia. We have tested whether the transgenic expression of a yeast NADH dehydrogenase (NDI1) can restore MCI function in Ndufs2-null mice. Ndufs2-null Ndi1 mice show almost complete restoration of the HVR. Glomus cells from Ndufs2-null Ndi1 mice also show strong rotenone-insensitive secretory responses and reversible increases in NADH during exposure to hypoxia. These data indicate that rescue of NADH dehydrogenase activity by NDI1 is sufficient to restore responsiveness to hypoxia and provide important insights into the essential role of MCI in acute O₂ sensing.

Kynurenine monooxygenase (KMO) blockade protects against multiple organ failure caused by acute pancreatitis (AP), but linking metabolism through KMO and systemic inflammation has eluded discovery. Here, we show that the KMO product 3-hydroxykynurenine primes innate immune signalling pathways to exacerbate systemic inflammation and critical illness during experimental AP. Mice that lack Kmo solely in hepatocytes have elevated plasma 3-hydroxykynurenine levels, reduced 3-hydroxykynurenine clearance, and significantly altered hepatic inflammatory signalling pathway gene transcription. 3-hydroxykynurenine enhances interleukin-1ß-induced apoptosis. Critically, elevated 3-hydroxykynurenine potentiates the fatal consequences of experimental AP, and this phenotype can be rescued therapeutically by systemic administration of the highly-selective KMO inhibitor GSK898. Together, our findings establish the KMO product 3-hydroxykynurenine as a regulator of inflammation and the innate immune response to sterile inflammatory injury. During critical illness, excess morbidity and death from multiple organ failure can be rescued by systemic KMO blockade.

The mTOR Complex 1 (mTORC1) kinase controls growth in response to many nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and tissue distribution of Sestrin-mediated leucine sensing are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon leucine deprivation or adapt to a leucine-free diet, but respond normally to diets lacking other amino acids. When deprived of leucine, these mice suffer a rapid loss of white adipose tissue and muscle. The adipose loss is driven by increased FGF21 levels as a result of mTORC1 dysregulation in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation by leucine of mTORC1 activity and FGF21 induction. These results establish the mammalian Sestrins as leucine sensors in vivo and reveal an unexpected intra-tissue spatial organization to nutrient sensing by the mTORC1 pathway.

Metabolic studies suggest that L-lactate lowers food intake and stimulates thermogenesis in rodents and humans. Yet, like many other signaling metabolites, L-lactate is commercially available as a sodium salt and typically administered in vivo as hypertonic aqueous solutions of Na-L-lactate. Surprisingly, most studies have not appropriately controlled for injection osmolarity and sodium content. Here we show that the hypophagia caused by treating mice with hypertonic solutions of Na-L-lactate is reproduced by iso-osmolar injections of Na-D-lactate and NaCl. Moreover, the elevated energy expenditure observed in response to the same treatment disappeared when administering Na-L-lactate as a less concentrated solution. Lastly, the induction of UCP1 in brown adipocytes observed after incubation with Na-L-lactate was mimicked, at least partially, by iso-osmolar NaCl. These findings call for a re-evaluation of L-lactate’s role in metabolic regulation and highlight counterions and osmotic load as underappreciated confounding factors that should be considered in metabolite signaling research.

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease worldwide. We performed network analysis to investigate the dysregulated biological processes in the disease progression and revealed the molecular mechanism underlying NAFLD. Based on network analysis, we identified a highly conserved disease-associated gene module across three different NAFLD cohorts and highlighted the predominant role of key transcriptional regulators associated with lipid and cholesterol metabolism. In addition, we revealed the detailed metabolic differences between heterogenous NAFLD patients through integrative systems analysis of transcriptomic data and liver-specific genome-scale metabolic model. Furthermore, we identified transcription factors (TFs), including SREBF2, HNF4A, SREBF1, YY1 and KLF13, showing regulation of hepatic expression of genes in the NAFLD-associated modules and validated the TFs using data generated from a mouse NAFLD model. In conclusion, our integrative analysis facilitates the understanding of the regulatory mechanism of these perturbed TFs and their associated biological processes.

Sarcopenia is an age-related condition characterized by progressive loss of muscle mass and force. Some studies suggested an accumulation of bioactive lipids such as ceramides in aged skeletal muscle. Considering that ceramides have been shown to inhibit anabolic signalling, we hypothesized that they could contribute to anabolic resistance and muscle mass loss during aging. We aimed to determine if a pharmacological inhibition of de novo ceramide synthesis exerted a geroprotective effect on muscle mass loss. Adult (7 months) and aged (23 months) mice were treated for 6 weeks with myriocin using subcutaneous osmotic mini-pumps. Surprisingly, myriocin treatment significantly increased the cross-sectional area of muscle fibers in young mice. A similar hypertrophic effect was observed in cultured C2C12 myotubes. However, no geroprotective effect was observed in aged mice. The underlying molecular mechanisms are currently being investigated. Our data demonstrate a positive effect of de novo ceramide synthesis inhibition on muscle mass.

Colorectal cancer (CRC) is a multi-stage process, initiated by formation of a benign adenoma, progressing to invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with this progression. To understand metabolic reprogramming across CRC development, we used a unique model system generated via progressive in vitro transformation of a cell line derived from a human colorectal adenoma. This model consists of four cell lines representing the different stages of tumour progression. We show that adenoma cells transition to increased glycolytic metabolism early in their progression to adenocarcinoma, whilst maintaining oxidative metabolism. Stable isotope tracer analysis (SITA) revealed increased use of glutamine to fuel the TCA-cycle as tumour cells progress, whereas glycolysis and TCA-cycle activity remain tightly coupled in early adenoma cells. Through nutrient depletion experiments, we show that late-stage tumour cells are more flexible with respect to fuel source, supporting their ability to proliferate in nutrient-poor environments. Combining SITA with proteomic analysis identified elevated asparagine synthesis in late-stage tumours. We show that loss of ASNS inhibits proliferation in late-stage cells, whereas early adenoma cells are largely resistant to ASNS knockdown. This phenotype can be reversed through asparagine addition. Late-stage cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and TCA activity. Furthermore, resistance in early adenoma cells is due to their ability to engage autophagy; inhibiting autophagy sensitises these cells to ASNS loss. These data suggest ASNS may be a targetable metabolic vulnerability in CRC.

Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumours. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR/Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1^-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy which lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.

The phospholipid cardiolipin (CL) plays a key role in nearly every facet of mitochondrial structure and function, and its loss is linked to a myriad of diseases across numerous tissues. Consequently, prevailing dogma asserts that CL is an irreplaceable feature of cellular physiology. Yet, here, using targeted ablation of CL synthesis in skeletal muscle, we uncover a fiber type-specific, bioenergetic and metabolic adaptation to the loss of this critical phospholipid. Glycolytic muscle unexpectedly retains full respiratory capacity in the absence of CL through doubling mitochondrial content, increasing fatty acid utilization, and globally adopting an oxidative fiber type identity. Moreover, despite this paradoxical shift towards lipid oxidation, CL-deficient muscles consume substantially more glucose than controls. Surprisingly, this effect is independent of changes in insulin signaling or the insulin-sensitizing actions of mitochondrial stress-induced secreted proteins, fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15). The increased glucose uptake in CL-depleted muscles is instead largely driven intrinsically by elevated reactive oxygen species (ROS) and contributes to widespread metabolic re-routing that reinforces antioxidant defense. Thus, these findings reshape our fundamental understanding of mitochondrial biology and raise the potential of leveraging these cell-specific and organismal protective adaptations to mitigate mitochondrial diseases.

Until menopause, women have lower propensity to develop metabolic diseases than men, suggestive of a protective role for sex hormones. Although a functional synergy between central actions of estrogens and leptin has been demonstrated to protect against metabolic disturbances, the underlying cellular and molecular mechanisms mediating this crosstalk have remained elusive. By using a series of embryonic, adult-onset, and tissue/cell-specific loss-of-function mouse models, we document an unprecedented role of hypothalamic Cbp/P300 Interacting Transactivator with Glu/Asp Rich Carboxy-Terminal Domain 1 (Cited1) in mediating estradiol (E2)-dependent leptin actions that control feeding specifically in pro-opiomelanocortin (Pomc) neurons. We reveal that within arcuate Pomc neurons, Cited1 drives leptin’s anorectic effects by acting as a co-factor converging E2 and leptin signaling via direct Cited1-ERα-Stat3 interactions. Together, these results provide new insights on how melanocortin neurons integrate endocrine inputs from gonadal and adipose axes via Cited1, thereby contributing to the sexual dimorphism in diet-induced obesity.

Endometrial cancer (EC) is the most common gynaecological cancer in high-income countries. Elevated body mass index (BMI) is an established risk factor, although the mechanisms underpinning this association remain unclear. We performed Mendelian randomization (MR) using a genome-wide association study of 12,906 cases and 108,979 controls to evaluate the mediating role of 14 molecular risk factors, including nine metabolic traits, in the relationship between BMI and EC. We show that total testosterone (odds ratio (OR) per unit increase: 1.64, 95% CI: 1.43 to 1.88, P = 1.71 x 10-12), bioavailable testosterone (OR per unit increase: 1.46, 95% CI: 1.29 to 1.65, P = 3.48 x 10-9), fasting insulin (OR per unit increase: 3.93, 95% CI: 2.29 to 6.74, P = 7.18 x 10-7), sex hormone-binding globulin (SHBG, OR per unit increase: 0.71, 95% CI: 0.59 to 0.85, P = 2.07 x 10-4) and total serum cholesterol (OR per unit increase: 0.90, 95% CI: 0.81 to 1.00, P = 4.01 x 10-2) have a causal effect on EC risk. Additionally, fasting insulin (19% total effect mediated, 95% CI: 5 to 34%, P = 9.17 x 10-3), bioavailable testosterone (15% mediated, 95% CI: 10 to 20%, P = 1.43 x 10-8), and SHBG (7% mediated, 95% CI: 1 to 12%, P = 1.81 x 10-2) have a mediating role in the relationship between BMI and EC risk. Our analysis provides insight into causal mechanisms linking BMI with EC risk and suggests targeting of metabolic and hormonal traits as a potential prevention strategy.

Cancer cells that metastasize to distant organs must adapt their metabolism to the new nutrient environment. However, a comprehensive understanding of the organ specific nutrient dependencies of metastases is lacking. We performed a pooled loss-of-function CRISPR screen of 407 SLC transporters in an experimental mouse model of breast cancer metastasis to define differences in nutrient utilization between metastasizing cancer cells in the lung and liver. In general, we find that intracellular SLC transporters were more often identified as important for metastasis formation than plasma membrane transporters. One of the highest ranking SLC transporters we identify as a requirement for liver but not lung metastases growth is mitoferrin-1 (Slc25a37), which transports iron into the mitochondria. Mitoferrin-1 loss almost completely ameliorates liver metastasis in experimental models of mouse and human breast cancer metastasis. Consequently, high tumoral mitoferrin-1 expression is associated with liver metastasis in breast cancer patients. Functionally, we discover that mitoferrin-1 enables cancer cells to grow in less perfused tissue areas, which is especially important in the highly zonated liver. Accordingly, we observe that hypoxia and HIF1α stabilization induce mitoferrin-1 expression to promote heme synthesis. While the requirement of heme cofactors for hypoxia-induced proteins has been described, we unexpectedly find that cancer cells utilize heme in hypoxia to support production of the potent lipophilic antioxidant, bilirubin. Treating mice with the lipid peroxidation inhibitor, Liproxstatin-1, rescues mitoferrin-1 deficient liver metastasis growth. In summary, we identify mitoferrin-1 and heme metabolism dependent ROS scavenging as a major liability of liver metastasis formation.

Ageing is characterized by increased glucose production and hepatic steatosis, conditions that are exacerbated in obesity. Glycerol is a substrate for gluconeogenesis (GNG) and can form the backbone of triglycerides (TAG). Glycerol is converted by glycerol kinase (GYK) to glycerol-3-phosphate mainly in the liver. In this study, we first investigated the intersection of glycerol with lipid and carbohydrate metabolism in the context of ageing and obesity. We then developed a liver-specific GYK knock-out (LGYKKO) to perturb glycerol use in downstream metabolic pathways.

C57BL/6J mice were fed normal chow (NC) or Western diet (WD) for 12, 25 or 50 weeks. LGYKKO mice were fed NC or WD for 12 or 25 weeks. To trace glycerol metabolism in these contexts, mice were infused with 13C-glycerol and 13C-glucose followed by metabolomics. Age and obesity augmented glycerol use for glucose production and TAG esterification. Preliminary data show that glucose production and lipid deposition were decreased in LGYKKO. GYK may be a target to ameliorate the pathological features of the metabolic syndrome. 

Epstein-Barr Virus (EBV) is a ubiquitous herpesvirus that typically causes asymptomatic infection but can promote B lymphoid tumors in the immune-suppressed. In vitro, EBV infection of primary B cells stimulates glycolysis during immortalization into lymphoblastoid cell lines (LCLs). Lactate export during glycolysis is crucial for cell proliferation in many cancer cells, and is mediated by monocarboxylate transporters 1-4 (MCT1-4). However, the role of MCT-mediated lactate export remains under-explored in viral lymphomas. We have found that EBV-infected B-cell immortalization is associated with a temporally regulated increase in lactate, as well as MCT1 and MCT4 expression. Notably, dual MCT1/4 inhibition in LCLs led to growth arrest, decreased glycolysis, OXPHOS and NAD+/NADH ratios, and increased glucose-driven glutathione synthesis to counteract elevated reactive oxygen species. Furthermore, MCT1/4 inhibition sensitized LCLs as well as EBV+ and KSHV+ lymphoma cell lines to killing by metformin, suggesting a promising therapeutic approach to targeting herpesvirus-associated viral lymphomas.

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.

The TGFβ superfamily (consisting of TGFβ, BMPs, GDFs, and activins) controls multiple aspects of organismal physiology.

We have previously found that the pseudoreceptor BAMBI (BMP and Activin Membrane-Bound Inhibitor) acts as a rheostat for TGFβ signal, controlling the balance between pro-inflammatory versus anti-inflammatory CD4-T cells. As a consequence, body-whole BAMBI deficient mice are protected from autoimmune/autoinflammatory diseases, as observed in rheumatoid arthritis and mannan-induced psoriatic arthritis (published results in 2016 and 2020, respectively).

We are now investigating the role of BAMBI in adipose tissue biology during aging and obesity. We have found that mice lacking BAMBI show restricted body weight gain and white fat expansion. It is related to reduced adipocyte hypertrophy, lower lipid, and glucose blood levels. Moreover, these animals display differences in the immune cell content of the visceral perigonadal adipose tissue. The protection of obesity is partially mediated by TGFβ ligands and linked to gut microbiota.

Background: Blood pressure (BP) is regulated by plasma metabolites from different neurohumoral and cardiometabolic systems.

Methods: We included 369 subjects from the multiethnic HELIUS cohort. Associations between metabolite profiles (untargeted LC-MS) and BP were assessed with machine learning prediction models. Next, the effects of the best predicting metabolite were investigated in endothelial cells (EC) and vascular smooth muscle cells (VSMC) .

Results: Formylmethionine was the highest ranked predictor for systolic BP and correlated with higher levels of NO pathway metabolites, including dimethylarginine and citrulline. In line with this finding, formylmethionine suppressed the expression of endothelial NOS (eNOS), arginase-2, adherens and tight junction markers in a dose-dependent manner in EC. In VSMC, formylmethionine exposure induced signaling pathways (e.g. ERK, myosin light chain) that regulate contractile activity.

Conclusions: Formylmethionine was associated with higher systolic BP, and may regulate BP by inhibition of eNOS in EC and induction of contractile pathways in VSMC.

Membrane contact sites (MCSs) link organelles to coordinate cellular functions across space and time. Although viruses remodel organelles for their replication cycles, MCSs remain largely unexplored during infections. Here, we design a targeted proteomics platform for measuring MCS proteins at all organelles simultaneously and define functional virus-driven MCS alterations by ancient and rapidly-evolving human viruses: human cytomegalovirus (HCMV), herpes simplex virus (HSV-1), influenza A, and beta-coronavirus HCoV-OC43. Integration with super-resolution and live microscopy reveals time-sensitive contact regulation that facilitates modulation of the metabolism-immunity axis during infection. We uncover a mitochondria-ER membrane encapsulation structure (MENC). As HCMV infection progresses, stabilized MENCs recruit the mitochondrial integrity complex VAP-B and PTPIP51, supporting virus production. In contrast, premature ER-mitochondria tethering activates STING immunity. At peroxisomes, ACBD5-mediated ER contacts balance peroxisome proliferation versus membrane expansion, facilitating pro-viral plasmalogen lipid synthesis. These virus-directed MCS modulations offer molecular fingerprints of organelle remodeling linked to infection and pathogenesis.

Heavy consumption of alcohol has been found to be a risk factor for development of Alzheimer’s Disease (AD). The link between alcohol consumption and AD is not fully understood. We hypothesized that alcohol may promote AD by causing liver injury and modulating the liver-brain axis. To test this hypothesize, we examined the effect of intragastric alcohol feeding on genes in the liver connected with AD, including beta-secretase, presenilin, tau, RAGE, and low-density lipoprotein receptor-1 (LRP1). Only LRP1 expression was found to be significantly decreased in the liver (~40%). LRP1 has been demonstrated to be the major receptor in the liver for removing peripheral beta-amyloid from circulation. Our data suggests chronic alcohol feeding may decrease liver functioning to clear beta-amyloid to promote AD. Our future research objective is to determine the significance of LRP1 expression in the liver on beta-amyloid levels in the body.

Methylglyoxal, an unavoidable degradation product of triose phosphates, is a highly reactive metabolite known to covalently modify a variety of nucleophilic residues and biomolecules. Accumulation of such modifications, collectively known as advanced glycation end-products, is associated with metabolic and aging-related diseases such as diabetes, cancer, and neurodegeneration. Despite these observations, knowledge of where methylglyoxal modifications occur in cells and how the adducts contribute to cellular and disease phenotypes is incomplete. We have recently identified several novel post-translational modifications and metabolites formed by reaction of methylglyoxal with endogenous thiols. We have developed metabolomics and proteomics approaches to study the reactivity landscape of methylglyoxal in vitro and in cells and map where such modifications form. These approaches have allowed us to identify previously unknown mechanisms by which methylglyoxal regulates critical cellular functions.  

Although the link between the chronic inflammation caused by Helicobacter pylori and gastric cancer development is well established, the details of the process are not fully elucidated and the majority of infected individuals remain asymptomatic. In this study we performed RNA-Seq analysis of gastric corpus biopsies from patients at different stages of the gastric precancerous process (N = 28). Using gene set analysis focused on metabolic pathways and reporter metabolites, we found the expression of kynurenine pathway genes to be significantly increased with disease progression. Several kynurenine pathway enzymes were up-regulated, including indoleamine 2,3-dioxygenase 1 (IDO1), tryptophan 2,3-dioxygenase (TDO2), kynurenine 3-monooxygenase (KMO), and kynureninase (KYNU). We subsequently validated this signature in two validation cohorts (N = 261).

The kynurenine pathway is one of the catabolic pathways of tryptophan and the depletion of tryptophan as well as the downstream metabolites and IDO1 itself has been shown to have an inhibitory effect on the proliferation of T lymphocytes, and to be up-regulated in several cancer settings. Using immune cell deconvolution analysis we pin-pointed several immune cell types that strongly correlate with increased kynurenine metabolism, including a decrease in regulatory T-cells and increased macrophage abundances. Finally, using targeted serum metabolomics, we identify an increased kynurenine tryptophan ratio correlating with disease progression. The up-regulation of this metabolic pathway and it’s downstream active metabolites might play a role in the modulation of the immune response to chronic H. pylori infection and will be further validated to elucidate the mechanisms and cell-types involved.

Inflammasomes are cytoplasmic signalling hubs that control the activity of certain proinflammatory cytokines and are thus important in host defense. The NLRP3 inflammasome also drives pathogenic inflammation in numerous diseases, including organismal metabolic disorders. 

A plethora of biologically diverse danger signals as well as cellular mitochondrial dysregulation have been recognized to engage NLRP3 inflammasome activation, but it remains elusive how NLRP3 mechanistically integrates these disparate signals. It has, however, been suggested that NLRP3 detects common metabolic changes or endometabolites induced by these signals. 

Our group recently discovered that the small molecule drug imiquimod induces nucleation of the NLRP3 inflammasome by inhibiting mitochondrial respiration, therefore reducing cellular ATP concentrations and triggering mitochondrial ROS (doi: 10.1016/j.immuni.2016.08.010). Hence, we decided to identify other small molecule inflammasome activators that might mimic cellular metabolic changes or endometabolites sensed by NLRP3 to gain insights into the activation mechanism. 

To this end, we developed a high throughput screening strategy, screened a library of 50,000 compounds, and successfully identified a novel group of small molecule NLRP3 activators that engage a yet unknown mechanism for NLRP3 inflammasome activation. Our unpublished data suggests that these compounds act more proximal to NLRP3 than any other NLRP3 activator described so far and that the mechanism is related to metabolic effects. Identifying the molecular targets and chemical requirements for activity, will shed light on NLRP3 inflammasome activation in the context of cellular metabolic dysregulation. Ultimately, we hope to elucidate the physiological function of NLRP3 in maintaining homeostasis that justifys its evolutionary conservation. 

The NADPH oxidase 4 (Nox4) produces H2O2 and is highly expressed in the kidney. Its expression is reduced in diabetic renal disease, suggesting its role in normal renal function.  To uncover the physiological role of Nox4 in the kidney, we performed global untargeted LC/MS for metabolites in urine, plasma and renal tissue of WT (wild type) and Nox4-/- (tamoxifen-inducible, global Nox4 knockout mice) which were put on a diet with low nutrients. Sulphur metabolites (cystine, cysteine and homocysteine) were reduced in Nox4-/- mice as compared to WT. Metabolic reconstruction using Fastacore showed a significant downregulation on extracellular transport function; metabolism of nucleotides, and folate in Nox4-/- mice. Human single nucleotide polymorphisms in Nox4 gene reduced cystine and homocysteine. The results indicate that the physiological function of Nox4 in the kidney is to control reabsorption and metabolism of sulphur amino acids and folate which are important for redox homeostasis.

Cardiolipin is a mitochondrial phospholipid that has a unique structure which allows it to take part in vital mitochondrial functions. The hallmark of ischemia/reperfusion is increased generation of reactive oxygen species (ROS) which cause cardiolipin peroxidation, however it is known that even in ischemic conditions ROS can be elevated. We have determined that 30-60-minute rat kidney ischemia in vivo causes peroxidation of the most abundant kidney cardiolipin species – tetralinoleoyl cardiolipin (TLCL). During ischemia TLCL was oxidized with up to eight additional oxygen atoms and we observed TLCL with up to four oxygens on a single linoleic acid. 40-minute ischemia caused an average of 7-fold increase of all oxidized TLCL forms along with a decrease in mitochondrial respiration. It is known that peroxidized cardiolipin may be cleaved into fragments that act as signaling and cytotoxic molecules. Therefore, such variety of oxidized cardiolipin products could cause severe damage already under ischemic conditions.

Organic elements make up 99% of an organism but without the remaining inorganic bioessential elements, termed the metallome, no life could be possible. Here, we report the evolution with age of the metallome and copper and zinc isotope compositions in five mouse organs. The aging metallome shows a conserved and reproducible fingerprint. By analyzing the metallome in tandem with the phenome, metabolome and proteome, we show organ-specific, age-dependent, isotopically-typified interactions with a wealth of clinical and molecular traits. The copper isotope composition in liver is age-dependent, extending the existence of aging isotopic clocks. Iron concentration and copper isotope composition relate to predictors of metabolic health, such as body fat percentage and maximum running capacity at the physiological level, and adipogenesis and OXPHOS at the biochemical level. Our results highlight the metallome as an overlooked omic layer and open perspectives for potentially modulating cellular processes using careful and selective metallome manipulation.

Compartmentalization plays a key role in metabolic regulation allowing spatial separation of metabolism in distinct organelles. The functionality of compartmentalization, however, relies on the expression of specific transporters that finely tune the channeling of metabolites across subcellular compartments. The Slc25 superfamily of carrier encodes for mitochondrial transporters that play a key role in metabolism by bridging cytoplasmic and mitochondrial branches of metabolic pathways. Surprisingly, these gatekeepers of metabolism have been largely understudied. Comprehensive tissue distribution analyses of all Slc25 family members across metabolic organs revealed that the expression of one member, SLC25A47, is strictly confined to the liver suggesting an important role in hepatic metabolism. We used a murine loss-of-function (LOF) model to unravel the role of this transporter in mitochondrial and hepatic homeostasis. Slc25a47^hep-/- mice displayed a wide variety of metabolic abnormalities as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration in this organ. This mitochondrial phenotype was associated with robust activation of the mitochondrial stress response (MSR) in the liver, which in turn, induced the secretion of several mitokines, amongst which FGF21 was key in broadcasting the effects of hepatic MSR on systemic physiology. Collectively, our data place SLC25A47 at the center of mitochondrial homeostasis and link this mitochondrial carrier to the mitochondrial stress response.

Ischemia/reperfusion (I/R) causes disturbances in mitochondrial respiratory chain, provokes reactive oxygen species (ROS) production. One promising class of mitochondria-targeted antioxidants are polyphenols. CAPE is polyphenolic compound, which has strong antioxidant activity. We injected CAPE into rat tail 90 minutes before inducing 30-minute kidney ischemia followed by 30-minute reperfusion and analyzed the generation of H₂O₂ as well as the accumulation of CAPE in mitochondria. Our results showed that, CAPE accumulates in kidney mitochondria - 0,4±0,1 µg/mL of CAPE  was detected. Meanwhile, CAPE metabolite caffeic acid (CA) was detected in higher amounts (1,1±0,6 µg/mL). At physiological (0.4 mM) succinate concentration, I/R induced an increase in H₂O₂ generation by 52%. Pretreatment of rats with CAPE reduced the ability of Complex II (SDH) to generate H₂O₂. At saturating (5 mM) succinate concentration H₂O₂ generation after I/R was 34% lower. In conclusion, CAPE shows promising mitochondria-targeted antioxidant activity.

The lifetime risk of kidney disease in patients with Type I diabetes is 10%-30%, implicating genetic predisposition in the cause of diabetic kidney disease (DKD). Here we examine mouse inbred strains that are susceptible (DBA/2J) and resistant (C57BL/6J) to DKD, as well as a panel of recombinant inbred BXD mice, to map quantitative trait loci (QTLs) associated with diabetes-induced podocyte loss in DKD. An expression QTL was identified in the cis-acting regulatory region of the Xanthine oxidoreductase (XOR), a binding site for C/EBPβ. We also uncovered promoter XOR orthologue variants in humans associated with high-risk for DKD. We next introduced the risk variant into the 5’-regulatory region of XOR in DKD resistant mice resulting in increased XOR activity associated with podocyte depletion, albuminuria, oxidative stress and damage restricted to the glomerular endothelium with diabetes, and with aging. Therefore, differential regulation of XOR contributes to phenotypic consequences with diabetes and aging.

The bone marrow provides a specific microenvironment that sustains hematopoiesis as well as different blood cancers. Several studies have shown that non-transformed cells in the tumor microenvironment adapt and support cancer cell survival and proliferation. One mechanism by which niche cells can maintain cancer cells is by providing essential nutrients. Our prior research demonstrated aspartate release from bone marrow stromal cells and aspartate accumulation in the bone marrow. In the current study, we found that both normal hematopoietic progenitors and acute myeloid leukemia (AML) cells express aspartate transporters. AML cells take up aspartate from their microenvironment, and blocking aspartate transport reduces leukemic burden in a mouse model of AML. Current efforts aim at further exploring the therapeutic potential of targeting aspartate metabolism in AML.

Short-term fasting is a nutritional intervention that induces a metabolic rewiring across the body. In this project, we aimed to characterise the physiological response produced by fasting in a B16F10 melanoma syngeneic tumor model. Two cycles of 48h of fasting in combination with 10 mg/kg doxorubicin delayed tumor progression in males, but not in females. This sexual dimorphism is evidenced by an analysis of the altered immune populations six days after the second cycle. We have observed that fasting in males enhanced several anti-tumoral defences (Natural Killer, Natural Killer T cells, Gzmb, Tnfα, Il4rα in tumor) and reduced immune suppressive populations (MDSCs in tumor and peripheral blood). These changes were not observed in females. In addition, fasting increased pro-tumoral exhausted CD8⁺ cells in the tumor. These data indicate that the beneficial effects of fasting in combination with doxorubicin are affected by sex, at least in the B16F10 melanoma model.

Aspirin has a well-established anti-cancer effect, particularly in colorectal cancer, although the mechanisms are not fully understood. We investigated the effects of long-term (52-week) aspirin exposure on colorectal cancer cell lines and used proteomics to identify novel mechanisms of action with the aim to identify tractable metabolic vulnerability. These data and subsequent validation show that aspirin regulates several key enzymes and transporters involved in central carbon metabolism, including GLS1, LAT1, PDK1 and PC. Long-term aspirin exposure impacts glutaminolysis causing a concomitant increase in glucose metabolism, demonstrating reprogramming of nutrient utilisation in the presence of aspirin. This reprogramming leaves the cells vulnerable to further metabolic perturbations. Aspirin treatment increases glutaminase (GLS1) expression and renders cells sensitive to the GLS1 inhibitor - CB-839 in vitroand in vivo. Further, the compensatory increase in glucose incorporation into the TCA cycle leaves the cells sensitive to inhibition of pyruvate import into the mitochondria with UK-5099. Alone, CB-839 and UK-5099 have no effect on colorectal cancer cell phenotype, indeed, targeting glutaminolysis with CB-839 as a monotherapy has had very limited clinical success. These results suggest that, through its impact on metabolism, aspirin use may be a safe and inexpensive way to enhance the efficacy of existing metabolic cancer therapies.

The receptor tyrosine kinase (RTK) KIT is a dominant oncogene in diverse human cancers. However, the specific metabolic underpinnings of KIT-driven oncogenic progression are largely undefined. This lack of understanding has hindered the development of strategies to leverage KIT-driven metabolic dependencies when RTK inhibitors treatment fails. Using systemic mastocytosis (SM) as a model of progression driven by KITD816V, the most frequent KIT mutant in myeloid neoplasms and metabolomics, we found that L-Kynurenine (L-Kyn), a product of tryptophan catabolism, is a plasmatic marker and a metabolic component enabling KIT-driven neoplasms aggressiveness. We found that the inhibition of KIT WT signaling reduced the intracellular concentration of L-Kyn by decreasing L-Kyn transporters expression on mast cells (MCs), whereas a KIT D816V selective inhibitor had no effect. Functionally, L-Kyn enhanced the release of proinflammatory cytokines by MCs in response to acute stimuli and had no effect on proliferation. To gain insight into L-Kyn function in MCs, as L-Kyn is the ligand of the transcription factor AHR, we identified an AHR transcriptomic signature in sorted MCs from patient bone marrow associated to SM aggressiveness and identified genes associated to lipid metabolism as the most up-regulated AHR targets in aggressive SM. Using a Seahorse analysis, we confirmed that L-Kyn increased lipid catabolism in MCs and shifted their metabolism from glucose to fatty acid oxidation. Altogether, this study not only identifies the kynurenine pathway as being regulated by KIT signaling, but also provide a basis for stratifying patients with aberrant KIT and kynurenine signaling for metabolism-targeted therapy.

Metabolism is a key modulator of cellular and organismal ageing. We report that a key determinant of the lifespan of eukaryotic microbes is not only nutrient supply and corresponding intracellular metabolism, but also, the degree of metabolite exchange interactions between cells. Studying chronological ageing in yeast, we observed that metabolites exported by young, proliferating cells, are imported during the stationary phase when cells age chronologically. Using metabolically cooperating yeast communities, as a model to examine the long-term impact of metabolite exchange interactions on cellular and community physiologies, we observe that an increase in cell-cell metabolic interactions significantly extends lifespan. Our results attribute a special role in sharing of methionine as part of the organic sulfur cycle, which reconfigures intracellular metabolism, alters the export of protective metabolites and creates a pro-survival environment. This so far overlooked role to metabolite exchange interaction in cellular ageing, shows that modulation of intracellular metabolism results in a self-generated altered extracellular metabolic environment, which ultimately alters individual and collective cellular survival fates.

Regular exercise enhances brain plasticity and reduces age-related cognitive decline. Each exercise bout triggers a complex adaptive response, coordinated by an intricate interplay of molecules. Metabolites, recognized as both energy substrates and signaling molecules, play an important role in exercise-induced adaptations. The aim of this study was to characterize changes in metabolome  before/after 90-minute run in cerebrospinal fluid (CSF) and plasma, and to explore correlation networks between CSF & plasma metabolome and cognitive functions. Study population included 19 young active adults (M/F 13/6; median age 25(IQR 22-31)yrs, BMI 23.2(IQR 21.7-24.5)kg/m2 &VO2max 47(IQR 38.1-1.2)mL/kg/min). Sampling was performed before (CSF/plasma), immediately after (plasma) and 1-hour after (CSF/plasma) 90-minute run. Targeted, broad-spectrum metabolomics was performed by HPLC-TMS. Cognitive functions were assessed by computerized tests MemTrax, Cogstate. From the top 30 most-changed metabolites in CSF, ~25% decreased and 75% increased after run, with purines & pyrimidines representing 32% and neurotransmiter changes 14% of the metabolic impact. In plasma, metabolites of fatty acids accounted for 46%-60% of the metabolic impact immediately & 1-hour after run, respectively. The correlation analysis revealed limited communication between brain and body and relationships between specific CSF metabolites and cognitive functions. These data support the role of specific metabolic signature in the adaptive response linked to the neuroprotective benefits of endurance exercise.

Grant support: SAS-MOST-JRC2018/10, VEGA-2/0076/22,  APVV-20-0466, COST-CA19101, UCSD Christini Fund, the Lennox Foundation, the JMS Fund, the Malone FF, the Westreich Foundation, the Daniel and Kelly White Family, the UCSD MRF

Chronic vascular inflammation is one of the key drivers of atherosclerosis and cardiovascular diseases and is associated with endothelial dysfunction and arterial stiffness, phenotypes that predict morbidity and mortality. Yet, it is still unclear how inflammation in the vascular wall affects the cell metabolism in situ, as in most cases metabolism of the vascular wall is studied in isolated endothelial or vascular smooth muscle cells cultured alone. This study aimed to examine the functional metabolic responses to inflammatory stimuli in the isolated murine aorta to overcome the limitations of artificial set-ups of cultured vascular cells. We established and optimized functional analysis of vascular bioenergetics in aortic rings ex vivo using Seahorse XFe96 Extracellular Flux Analyzer. It provided new, physiologically relevant information on vascular bioenergetic response to proinflammatory stimuli, under several experimental conditions including e.g., ex vivo stimulation with IL-1β,  IL-6, TNFα or IL-17 in the presence of various bioenergetic substrates in media. We also tested the effects of selective and non-selective LDH-A inhibitors on LDH-A activity in the vascular wall using an LC/MS-based approach and examined the effects of LDH-A inhibition on vascular wall inflammatory response. Altogether, our approach for the analysis of vascular metabolism in the isolated aorta using Seahorse XFe96 Analyzer is reliable, reproducible and uncovers robust functional responses in mitochondrial respiration and glycolysis, thus providing a novel platform for the pharmacology of vascular wall metabolism to better understand pharmacotherapeutic mechanisms and metabolite signalling. This research was funded partly by The National Science Centre grant PRELUDIUM 2021/41/N/NZ5/03396.

Cholangiocarcinoma is a rare cancer of the bile ducts, but incidence and mortality are increasing globally. Tumorigenesis is actively supported by surrounding stromal cells, but interactions with adjacent normal cholangiocytes remain unknown. 

We used a co-culture model to determine the ecological interaction between normal cholangiocyte, MMNK1, and intrahepatic cholangiocarcinoma, CC-SW1. MMNK1 cells exhibit a growth advantage over CC-SW1 cells, inhibiting tumour cell viability and migration. Immunoblotting revealed downregulation of p53 and apoptotic pathways in MMNK1, with concomitant inhibition of vimentin and slug expression in CC-SW1. Metabolomics showed increased aspartate-asparagine conversion, and sustained choline in MMNK1 in co-culture. Furthermore, pathway analysis via RNA-sequencing identified downregulation in gene signatures of intrinsic apoptosis and amino acid biosynthesis in MMNK1, and downregulation of DNA synthesis and cell cycle in CC-SW1.

In conclusion, co-culture with cholangiocarcinoma confers cholangiocytes growth advantages in vitro, linking to p53 and apoptosis inhibition, along with enhanced choline and asparagine metabolism. 

T cell activation and memory generation requires rewiring of the cellular metabolism towards anabolic processes e.g. glycolysis. However, to which degree the cholesterol biosynthesis pathway and its regulators are involved is less clear.  

We previously showed that the mitochondrial protein TCAIM prevents effector T cell differentiation by inhibiting induction of genes controlling cholesterol biosynthesis. Here, we investigated how this impacts memory T cell formation in a sublethal influenza A infection (IAV) mouse model.

TCAIM overexpressing mice show delayed and TCAIM deficient enhanced viral clearance and recovery from acute infection.

In general, we observed a stronger local expansion and contraction of PB1-specific compared to NP-specific effector T cells. TCAIM overexpression nearly completely prevented generation of IAV-specific effector and thus also memory T cells. In contrast, TCAIM deficiency enhanced formation of persisting memory T cells in the lung, which might be caused by Hif-1 alpha / cholesterol-mediated higher proliferative capacity.         

Mitochondrial malfunction is a hallmark of many diseases. We identified, using mouse smoke exposure model, that local alveolar progenitor cells, upregulate the mtDNA-encoded small non-coding RNA, mito-ncR-805.  mito-ncRNA-805 transcript functions as a retrograde signaling molecule between mitochondria and nucleus, with enhanced function during the adaptive stress response.  mito-ncR-805 is a mouse-specific transcript. We identified a region of mito-ncR-805 conserved in mammalian mitochondrial genomes, and generated shorter versions of mouse and human transcripts, which differ in a few nucleotides. We called these small transcripts “functional bit”.  Over-expression of mouse and human functional bits in either mouse or human lung epithelial cells, led to increased activities of Krebs cycle, OXPHOS, faster cell division, stabilized mitochondrial potential, and lowered levels of pro-apoptotic pseudokinase TRIB3. Both ortholog oligoes confer cross-species beneficial effects, indicating a high degree of evolutionary conservation of retrograde signaling via a functional bit of the D-loop transcript, mito-ncR-805, in mammals.

Recognizing the early signs of cancer development is vital for informing early detection, prevention, and improving survival. We constructed a genetic risk score comprised of 72 single nucleotide polymorphisms associated with colorectal cancer to estimate the effects of increased genetic liability to colorectal cancer on circulating metabolites measured by NMR. Linear regression models were applied to examine the relationship between the colorectal cancer genetic risk score and circulating metabolites measured at age 8y, 16y, 18y and 25y in ALSPAC (N = 4,760). The colorectal cancer genetic risk score was associated with up to 35% of the circulating metabolic traits, in particular fatty acids, VLDL, LDL, and IDL subclass lipids. Two-sample Mendelian randomization estimates in an independent sample of adults (UK Biobank, N = 118,466) indicated broadly persistent patterns of disease liability across metabolic traits. This analysis reveals alterations in systemic metabolism which precede the onset of clinically detectable cancer.

Mitochondrial uncoupling proteins (UCPs) are major regulators of cellular metabolism and general redox state. UCPs dysfunction is linked with metabolic disorders and increased oxidative stress, a common cause of male infertility. However, the expression and role of UCPs in human testis remain unknown. Herein, we aimed to characterize the expression and function of UCPs in primary cultures of human Sertoli cells (hSCs). All UCP homologues (UCP1-6) mRNA expression were identified by RT-PCR and UCP1-3 were detected by immunofluorescence. UCPs were inhibited by genipin (0.5, 5, 50, and 100 µM) and cellular viability, proliferation, and ROS production were accessed after 24 h treatment. Mitochondria function was accessed by Seahorse XF Cell Mito Stress assay. Culture media were collected and analysed by 1H-NMR. Our results show that UCPs are regulators of hSCs metabolism and function, suggesting a central role in the crosstalk between metabolic disorders, high oxidative stress and male infertility.

TGF-β1 is the most potent fibrogenic cytokine and promotes excessive extracellular matrix synthesis and deposition in multiple fibrotic conditions. Myofibroblasts reconfigure metabolic networks to support their enhanced synthetic capabilities so that targeting these synthetic vulnerabilities may reveal novel therapeutic strategies. However, tissue culture media composition is highly variable and may influence metabolic networks to a degree which does not replicate the in vivo setting. Here we report that the global fibroblast TGF-β1-regulated transcriptional response is highly influenced by nutrient availability in two common DMEM formulations which differ in glucose, glutamine and pyruvate concentrations. The anti-fibrotic efficacy of targeting glutamine metabolism via inhibition of GPT2 or GLS1, which has seen promising results in the oncology setting, is futher highly dependent on alanine and pyruvate availability, respectively.  Experimental models which replicate nutrient availability and metabolic fidelity within the tissue microenvironment will be critical to identify promising anti-fibrotic therapeutic approaches based on metabolic inhibition.

Asthenozoospermia is a cause of male infertility characterized by a reduction in sperm motility. However, the molecular mechanisms underlying asthenozoospermia remain elusive. Lipids play an important role in sperm morphology and motility. We hypothesized that the lipid profile of asthenozoospermic men is altered as compared to normozoospermic men. Sperm polar lipid content from asthenozoospermic (n=17) and normozoospermic (n=39) men was analyzed by LC-MS. We identified a different lipidomic profile in sperm from asthenozoospermic men and potential sperm lipid biomarkers correlated with sperm motility. Lysophospholipid content was increased in sperm from asthenozoospermic men whereas phosphatidylethanolamines were increased in normozoospermic men. The increased lysophospholipid content in sperm from asthenozoospermic men might be correlated with altered membrane fluidity (and subsequent altered motility), probably resulting from increased inflammation and oxidative stress, which are central in this condition. Overall, sperm lipid composition is indicative of poorer sperm quality and fertilizing potential in asthenozoospermia men.

Carbohydrates, proteins and lipids are essential nutrients to all animals; however, even closely related species and individuals display dramatic variation in diet. Here we explored variation in macronutrient tolerance in Drosophila melanogaster using the Drosophila genetic reference panel. Our study demonstrates that D. melanogaster, often considered a “dietary generalist”, displays marked genetic variation in survival on different diets, notably on high-sugar diet. Our functional validation identify several novel regulators of macronutrient tolerance. We report a role for tailless, a conserved orphan nuclear hormone receptor, in regulating sugar metabolism via insulin-like peptide secretion and CCHamide-2 expression. Moreover, we show that the JNK pathway is required for sugar tolerance, and that loss of a JNK pathway modifier leads to widespread changes in fatty acid and glycolytic metabolites in response to sugar feeding, both in flies and mice. Our study provides support for the use of nutrigenomics in the development of personalized nutrition.

Fluctuation in the intracellular levels of several metabolites has recently been shown to drive switch of cells between distinct functional states. Here, we first observed that downregulation of nicotinamide N-methyltransferase, known to favor glioblastoma (GB) cell aggressiveness, was accompanied by accumulation of its substrate, nicotinamide. We then evaluated the effect of enhanced nicotinamide levels on GB cell functional state. Nicotinamide altered the in vitro and in vivo GB cell properties and induced their transition towards senescence. NAM-induced senescence of GB cells was accompanied by an unexpected downregulation of key enzymes of nicotinamide metabolism. Using GB single-cell transcriptome dataset, we found coherently to our experimental observations that cell with low levels of nicotinamide metabolism enzymes were significantly enriched in gene modules related to senescence. Our findings support that nicotinamide metabolism regulates GB cell senescence and they strengthen the new emerging driver role of metabolism in the regulation of cell functioning states.

Nicotinamide riboside chloride (NR-Cl) is the water-soluble chloride salt of the new form of the pyridine-nucleoside of vitamin B3, which functions as a precursor to nicotinamide adenine dinucleotide (NAD) or NAD⁺, playing an essential role in many biological processes related with energy metabolism, aging and cancer. Little is known about the role of NR supplementation in lung cancer. 

Therefore, we investigate the preventive effect of NR-Cl oral supplementation on the development of lung cancer. We report that NR-Cl supplementation before tumor initiation in a K-RAS-driven lung carcinogenesis model delayed tumor appearance. Consistently, using allograft models of different lung cancer cell lines (LLC1 and TC1), NR-Cl treatment also reduces tumor growth. Furthermore, in vitro NR-Cl treatment also reduced the viability and proliferation of different lung cancer cell lines as well as different markers of aggressiveness and metastasis. Notably, co-treatment with NR-Cl and diverse chemotherapeutic agents, enhancing their antitumor response in different lung cancer cell lines. Our work demonstrates a tumor suppressor role of NR-Cl supplementation, suggesting that NR-Cl may have a therapeutic role against lung cancer.

The complex pathology of Alzheimer’s disease (AD) emphasises the need for comprehensive modelling of the disease on metabolite level, which may lead to the development of efficient treatment strategies. To address this challenge, we analysed transcriptome data of post-mortem human brain samples of healthy elders and individuals with late-onset AD from two cohorts, reflecting to three brain regions. By constructing AD-specific co-expression networks and genome-scale metabolic model, we could identify potential driver genes, metabolites and pathways involved in the progression of AD, reliably. On metabolite level, We observed the depletion of numerous unsaturated fatty acids and neuroprotective metabolites (e.g., vitamins, retinoids) and the increase of neurotoxic metabolites (e.g., β-alanine, bilirubin) in the AD brain. Furthermore, these metabolic changes were reflected as a decline in energy production, myelination and synaptic activity in all brain regions. This study provisions insights about the crucial mechanisms of AD and candidate targets for therapy.

Macrophages in white adipose tissue (WAT) regulate energy, lipid, and glucose metabolism under healthy conditions and contribute to the metabolic dysfunction observed in obesity. Recently, we demonstrated that adipocytes transfer mitochondria to macrophages in vivo and that this process appears to limit the development of obesity. However, the mechanisms that regulate intercellular mitochondria transfer and the function of this process are not fully understood. Here, we show that intercellular mitochondria transfer is sufficient to restore aerobic respiration in metabolically compromised macrophages, suggesting that capturing mitochondria from other cell types may enhance the metabolic fitness of macrophages. By comparing mitochondria transfer in a variety of obesogenic models, we found that dietary long chain fatty acids impair the efficiency of mitochondria transfer to WAT macrophages in vitro and in vivo via a heparan sulfate-dependent mechanism. These data suggest that lipid metabolites may alter macrophage metabolism and function by regulating intercellular mitochondria transfer.

Tumor metabolic phenotypes reflect multiple factors including organ-specific metabolic preferences and local nutrient availability. However, it remains unclear to what extent genetics and the local environment dictate nutrient utilization in human tumors. Here, we utilize a multidisciplinary clinical approach to intraoperatively infuse ¹³C-substrates in over 70 patients with different subtypes of kidney cancer. We demonstrate that glucose utilization patterns vary across kidney cancer subtypes, highlighting the fact that the kidney environment alone cannot account for all aspects of cellular metabolism in kidney cancers. Previous studies determined that glucose oxidation is suppressed in clear cell renal cell carcinoma (ccRCC), the most common and deadly form of kidney cancer. By assessing freshly-cultivated, surgically-resected tissues from tumors and adjacent kidney, we determine that suppressed glucose oxidation in human ccRCC is a tumor-intrinsic property. Infusions of acetate in intact patient tumors and isolated patient mitochondria underscore low TCA cycle turnover in primary ccRCCs. We also show that isolated mitochondria from these tumors display reduced respiration relative to mitochondria from the kidney, indicating that human ccRCC involves metabolic reprogramming at the level of the electron transport chain. Surprisingly, preliminary analysis of some ccRCC metastases show increased glucose oxidation and mitochondrial respiration relative to primary ccRCCs, suggesting a possible divergent metabolic program that affects kidney cancer progression and metastasis. Altogether, our findings indicate metabolic heterogeneity among human kidney cancer subtypes, and reveal an unexpected mitochondrial alteration during ccRCC metastasis in patients.

Birth is the first metabolic challenge that cardiomyocytes overcome as they reshape their fuel preference from glucose to fatty acids (FA) to produce energy postnatally. This adaptation requires a drastic mitochondrial maturation, which is coordinated by unexplored transcriptional circuits. Here, we interrogated the role of myocardial Retinoid X Receptors (RXR), a nutrient-sensing transcription factor, during perinatal metabolic adaptation. We show that newborn mice lacking RXR in embryonic cardiomyocytes (edKO) display an abnormal chromatin landscape that prevented the induction of RXR-dependent signature, which was required to endow mitochondria with FA β-oxidation machinery. Accordingly, edKO hearts show blunted lipid-derived ATP production and enhanced glucose oxidation, which led to lethal cardiac dysfunction after birth. We further demonstrated colostrum γ-linolenic acid (GLA) as the endogenous ligand responsible for driving RXR activity. Altogether, our study identifies the GLA–RXR axis as a key mechanism of maternal control of perinatal mitochondrial maturation and cardiac metabolic adaptation.

Carnosine and related β-alanine–containing peptides are believed to be important antioxidants, neuromodulators, and pH buffers. However, their biosynthetic routes, metabolomics signaling pathways, and therapeutic potential are still being debated. This study describes the first animal model lacking the enzyme glutamic acid decarboxylase–like 1 (GADL1). 

We show that Gadl1−/− mice are deficient in β-alanine, carnosine, and anserine, particularly in the olfactory bulb, cerebral cortex, and skeletal muscle. Gadl1−/− mice also exhibited decreased anxiety, increased levels of oxidative stress markers, alterations in energy and lipid metabolism, and age-related changes. Furthermore, examination of the GADL1 active site indicated that the enzyme may have multiple physiological substrates, including aspartate and cysteine sulfinic acid. Human genetic studies show strong associations of the GADL1 locus with plasma levels of carnosine, subjective well-being, and muscle strength. Together, this shows the multifaceted and organ-specific roles of carnosine peptides and establishes Gadl1 knockout mice as a versatile model to explore carnosine metabolism and its therapeutic potential.

The gliomas represent a heterogeneous group of CNS tumours with histological features of the glial lineage that has its own metabolic characterization. We determinated levels of basal metabolites in blood, their correlation with tumour grade, as well as the feasibility of statistical discrimination in the primary brain tumours. In our study, we identified 60 samples from patients with clinically defined glial tumour and 28 healthy volunteers by NMR analysis. The correlation of the relative concentrations of plasma metabolites with the tumour grade showed that the levels of glucose, phenylalanine, tyrosine, creatine, creatinine and formate were significantly increased. Phenylalanine and tyrosine were both increased exclusively in glioblastoma patients. Based on metabolite levels, an excellent discrimination between plasma from patient's tumours and controls was attainable. Creatine, pyruvate, glucose, formate and citrate were of the highest discriminatory power. This work was supported by the Slovak Research and Development Agency under Contract No. APVV-18-0088.

In cancer cells except for glucose, there are several alternatives substrates, which are supposed to enter their metabolism and sustain their growth. In addittion, to glutamine, leucine is the second most taken up amino acid from media. To confirm leucine-catabolic capacity we cultured cancer cells with 13C-leucine. The cultured cells removed 13C-leucine from their media and released several compounds enriched in 13C atoms, such as 2-oxoisocaproate and citrate. We demonstrated the expression of 3-methylcrotonyl-CoA carboxylase, a unique enzyme from the irreversible part of the leucine catabolic pathway in cultured cancer cell lines and also in human brain tumors. Results from our work demonstrated the ability of cancer cells to expand the use of leucine as a source of acetyl-CoA groups and confirms the role of leucine as a substrate for their metabolism.

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

LAMA2-congenital muscular dystrophy (LAMA2-CMD) is caused by mutations in the LAMA2 gene, encoding the α2 chain of laminins 211/221, components of the extracellular matrix (ECM). Using the dyW mouse model for LAMA2-CMD, we have previously shown that LAMA2-CMD starts in utero between embryonic day 17.5 and 18.5 with a reduction in the pool of muscle stem cells (MuSCs)/myoblasts and impaired muscle growth. To determine the cellular and molecular mechanisms involved in this fetal muscle phenotype we generated a Lama2-deficient C2C12 myoblast cell line to use in parallel with the dyW mouse model. Skeletal muscles from dyW^-/- fetuses and Lama2-deficient C2C12 cells showed altered expression of genes linked to cell cycle regulation and cell survival, with Lama2-deficient C2C12 cells also exhibiting decreased proliferation rate, in comparison to the wildtype counterpart. In addition, we also observed an increase in oxidative stress markers, altered expression of OXPHOS complexes and increased DNA damage. In conclusion, our study implicates defective proliferation, oxidative stress and DNA damage as players in the onset of LAMA2-CMD, therefore establishing a link between ECM components, in particular laminin 211/221, and regulation of cell proliferation and redox metabolism. These results are particularly important since they contribute for a better understanding of the first stages of LAMA2-CMD onset, which is essential to design therapies that specifically target the primary events underlying this incurable, and often lethal, disease.

How intratumor genetic heterogeneity contributes to phenotypic cellular heterogeneity remains largely unexplored. Here, we use human iPSC-derived organoids to dissect the functional consequences of genetic heterogeneity in glioblastoma (GB). Using CRISPR/Cas9, we generated a spectrum of combinations of genetic mutations, which are commonly found in human GB, in iPSCs. The GB organoid (LEGO: Laboratory Engineered Glioblastoma Organoid) derived from these cells recapitulate major features of human GB, and they form GBs in mouse xenograft. We further performed temporal single-cell RNA-Seq, DNA methylation array, metabolomics/lipidomics, and proteomics/phosphor-proteomics analysis of the LEGOs. Our integrative analysis revealed novel fundamental features of GB progression including lineage changes, metabolic reprogramming which is coordinated with DNA methylation, proteomic changes, and novel druggable targets, these features are mostly time-dependent and specific to the combination of mutations. This study provides a research path for realizing genome-based personalized GB therapy using novel advanced models.  

Leucine is an essential, ketogenic amino acid, which belongs to the group of branched-chain amino acids. The leucine catabolism could provide the cells with an amino group and carbon sceleton, which might be converted to acetyl-CoA and ketone bodies. Since ketone bodies are supposed to play several roles in the brain parenchyma, including signaling, we evaluated to which extent are cultured human glial cells to generate ketone bodies. By applying enzymatic and analytical methods, we identified that cultured human astroglial cells removed leucine from their culture media and readily released ketone bodies. In addition, the cells could metabolize lysine only in neglected amounts, stressing the significance of leucine as a ketogenic substrate. Therefore, it could be supposed that leucine catabolism by astroglial cells might be an endogenous source of ketone bodies in brain parenchyma. 

This work was supported by the Slovak Research and Development Agency under the Contract No. APVV-18-0088.

Mitochondria release tricarboxylic acid (TCA) cycle metabolites with an essential role in cellular homeostasis. Succinate dehydrogenase (Sdh), a heterotetrameric complex (SdhA, SdhB, SdhC, SdhD), transports electrons into the mitochondrial respiratory chain and oxidizes succinate to fumarate. We observed a significant decrease of SdhD mRNA expression in chronic liver disease (animals and humans). Here, we targeted SdhD in hepatocytes generating a specific liver SdhD knockout (SdhD^-/-) mice to analyse the metabolic consequences. The LC-MS-based metabolomic analysis in livers from wild-type (WT) and SdhD^-/- mice at different ages showed the following findings: higher levels of succinate, lower ATP/ADP and NADH/NAD ratios, lower levels of aspartate, proline and methionine together with higher levels of lysine, asparagine, glutamine, glutamate and histidine in SdhD^-/- compared to WT animals with clear impact in the phenotype of the mice. Our work highlights the important role of the Sdh in the redox biology and amino-acid homeostasis in hepatocytes.

Metabolic stress evoked by complex I impairment promotes cellular NADH increase in cancer cells. NADH regulates the interaction between Apoptotic Induced Factor 1 (AIFM1) and Mitochondrial intermembrane space import and assembly protein 40 (MIA40). We propose the interaction of MIA40 with AIFM1 to affect signaling during tumorigenesis to overcome apoptosis. We observed that KOs of the respiratory complex I subunit NDUFA13 showed an increased NADH/NAD+ balance, no Complex I activity, 300% increase in AIFM1 expression, resistance to apoptosis, and increased interaction between AIFM1 and MIA40. To investigate whether oxidoreductase activity of MIA40 could be involved in the interaction between MIA40 and AIFM1, we used Flp-ln T-Rex cells containing FLAGMIA40 with C53S and/or C55S mutations. The lack of cysteine 55 drastically reduced the interaction between MIA40 and AIFM1, and thiol-trap analysis indicated possible oxidation of AIFM1 cysteines. These results contribute to our understanding of apoptosis and its mitochondrial basis.

Glioblastoma (GBM) is a highly aggressive brain cancer with a high incidence (3.14 per 100,000). Few patients survive for only five years after diagnosis. Therefore, it is crucial to understand the molecular mechanisms underlying GBM, discover biomarkers for early diagnosis, and identify potential therapeutic targets.

In this study, a computational systems biology approach is used to understand the complexity of GBM. In this context, GBM-specific genome-scale metabolic models (GEMs) were created using RNA-Seq data, and the disease-specific metabolic profile was investigated. In addition, GBM-specific and metabolic network specific Protein-Protein Interaction networks were created. These networks were further evaluated by integrating them into GEMs.

Analysis results will be used to identify new drug targets for the effective treatment of GBM and discover biomarkers for early diagnosis. Moreover, the study could help develop a new methodology to assist clinicians in selecting and prescribing patient-specific available drugs.

We are characterizing the target landscape of metabolites in oxidative phosphorylation, glycolysis and pentose phosphate pathways in human colon cancer cells and associated metastatic cells. To materialize this goal, we exploit our recent tool Proteome Integral Solubility Alteration (PISA) assay as a high-throughput version of Thermal Proteome Profiling or CETSA-MS, for system-wide identification of metabolite targets at physiological concentrations. As a proof of principle, we show ATP to change the solubility/stability of thousand proteins, including most known ATP-linked proteins, in SW480 colon cancer cells. As a next step we identify several known targets of flavin adenine dinucleotide (FAD), along with numerous previously unknown proteins that are engaged by this metabolite and are involved in cellular redox systems. The resulting database will be publicly available online as an expandable resource called MetaTargetMiner. 

Cardiac ischaemia during myocardial infarction or heart transplantation is characterised by extended interruption of oxygen and nutrient supply. Restoring oxygen is essential to prevent cell death, but causes ischaemia reperfusion injury (IRI). To understand the metabolic changes that underlie IRI, I established an anoxic (10 ppm O₂) adult cardiomyocyte model that fully replicates the succinate accumulation and oxidation occurring in vivo during IRI, which hypoxic (0.1% O₂) models do not. Furthermore, this cardiomyocyte model showed that the succinate accumulated during anoxia is released upon reoxygenation via monocarboxylate transporter 1 (MCT1). These findings explain how succinate is released into the circulation during reperfusion from ischaemic tissues as a pathophysiological signal. In addition, the ability for the first time to replicate succinate metabolism during IRI in an in vitro model will enable the exploration of the mechanism of mitochondrial ROS production during IRI and facilitate rapid screening of new therapies against IRI.

The recent efforts for the deciphering of the “molecular language” mediating organ to organ communication has been opening new avenues for the comprehension of organism physiology. In this context, metabolism appears to provide readily interpretable signals to communicate the functional state and reciprocal needs linking different tissues.

We sought to explore how metabolites and metabolic availability influence the metabolic states of distant organs and if they can behave like signaling molecules, by investigating the metabolic networks connecting liver and skeletal muscle.

We created a novel dynamic framework of tissue-specific metabolic networks (liver and muscle) based on the network HGEM, including an extensive manual curation. Using data from GTEx and BRENDA we tuned the networks to represent the tissues. We used equilibrator and chemaxon to compute the ΔG of each reaction.

Preliminary results show that the model queried for specific tasks is able to adjust its metabolism to adapt to the changing conditions.

Although genetic mutations are causal in human cancers, the contribution of metabolite signaling to cancer onset and progression remains less well understood. Multi-omics profiling of human tumors provides insight into how alterations in metabolism regulate malignant protein expression. We applied multi-omics profiling of metabolomics, chromatin accessibility, RNA abundance, protein abundance, and whole-genome sequencing to 36 human thyroid cancer primary tumors, metastases, and patient-matched normal tissue. Through multi-omics integration, we identified that metastases gain metabolite dysregulation associated with increased MAPK signaling. Cancer-specific metabolites showed predicted activation of the MAPK pathway, and metabolite-driven MAPK signaling mediated MAPK transcription factor activity and regulation of cancer gene expression. Taken together, these analyses suggest that metabolites activate metastatic cancer growth signals, and indirectly regulate cancer gene expression. We propose a metabolite signaling model that can be perturbed to identify potential therapeutic targets for metastatic thyroid cancer.

Ageing, obesity and living at thermoneutrality lead to brown adipose tissue (BAT) involution, a process associated with lipid accumulation, loss of mitochondria, a decline in uncoupling protein-1 (UCP1) and other thermogenic proteins, inflammation and insulin resistance. The mechanism that initiates this inflammatory tissue remodeling remains to be elucidated. Here, we show that BAT involution in mice is triggered by ATP secreted by brown adipocytes under conditions of impaired mitochondrial fatty acid oxidation caused by UCP1-deficiency, inhibition of carnitine palmitoyltransferase-I, or thermoneutral housing. Mechanistically, we show that purinergic signaling in myeloid cells via the ATP-activated ion channels P2RX4 and P2RX7 is responsible for BAT involution. Notably, the combined inhibition of these purinergic receptors protects against BAT inflammation, infiltration of immune cells, and thermogenic dysfunction. These results highlight the relevance of extracellular ATP released by brown adipocytes as the paracrine signal for myeloid immune cells to initiate tissue remodeling and BAT involution.

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) identity and lymphangiogenesis remains unknown. Here, we show that autophagy-deficient LEC have impaired responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC reduces expression of main LEC identity markers, like VEGFR3, affects mitochondrial dynamics and causes accumulation of lipid droplets (LDs) in vitro and in vivo. Compromised lipophagy results in defective fatty acid supply to mitochondria, which dwindles fatty acid oxidation, mitochondrial ATP production, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes. Rescuing mitochondria elongation by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC does not alter LD accumulation and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues LEC identity and lymphangiogenesis in LEC-Atg5-/- mice. Our study reveals lipophagy in LEC supports FAO, required to support a mitochondrial-PROX1 gene expression circuit that safeguards LEC identity, responsiveness to lymphangiogenic mediators and lymphangiogenesis.

Vascular aging, a major cause of morbidity and mortality, is marked by decreased nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling. This is partly caused by increased reactive oxygen species (ROS) levels in endothelial cells (EC), believed to be produced by mitochondria. Here, we investigated the effect of chronic treatment with SUL138, an inhibitor of reverse mitochondrial electron transfer which reduces free electron spill-over, in a model of EC-specific aging. EC-specific deletion of DNA repair endonuclease Ercc1 in mice (EC-KO) led to accelerated vascular aging features, marked by reduced endothelium-dependent NO-cGMP vasodilation at 22 weeks of age. Chronic treatment with SUL138 from 14 to 22 weeks of age restored vasodilator function. At 21 weeks of age, EC-KO also displayed reduced renal creatinine filtration and overt proteinuria, which were both attenuated by SUL138 treatment. Therefore, maintaining mitochondrial electron transfer might represent an effective treatment of vascular aging.

In this study, we integrated two large-sized mRNA and protein expression datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM), and validated the GEM by metabolomics data from the MaasHPS cohort. Our study revealed profound alterations in lipid, cholesterol and inositol metabolism, lysosomal lytic activity, and inflammatory activity in unstable, rupture-prone plaques versus stable plaques. Graph-based topological analysis of this network model pinpointed glutamine-glutamate conversion and transfer between cytoplasm and mitochondria as critically dysregulated in unstable plaque, with a sharp drop in the overall plaque glutamate level. Furthermore, the reduced availability of glutamate was associated with macrophage presence and phenotype in plaque, possibly reflecting the inflammation-prone microenvironment in unstable plaque. In conclusion, we established a robust, comprehensive plaque-specific GEM to serve as a referential plaque metabolism atlas.

It is essential to reveal the associations between various omics data for a comprehensive understanding of the altered biological process in human wellness and disease. To date, very few studies have focused on collecting and exhibiting multi-omics associations
in a single database. Here, we present iNetModels, an interactive database and visualization platform of Multi-Omics Biological Networks (MOBNs). This platform describes the associations between the clinical chemistry, anthropometric parameters,
plasma proteomics, plasma metabolomics, as well as metagenomics for oral and gut microbiome obtained from the same individuals. Moreover, iNet-Models includes tissue-and cancer-specific Gene Co-expression Networks (GCNs) for exploring the
connections between the specific genes. This platform allows the user to interactively explore a single feature’s association with other omics data and customize its particular context (e.g. male/female specific). The users can also register their data for sharing and visualization of the MOBNs and GCNs. Moreover, iNetModels allows userswho do not have a bioinformatics background to facilitate human wellness and disease research. iNetModels can be accessed freely at https://inetmodels.com without any limitation.

Treatment with rifaximin-α reduces gut-derived inflammation in cirrhosis and encephalopathy. Here we further explore the effect of the antibiotic treatment by following the profile of Biosynthetic Gene Clusters present in the patients’ microbiome.

Secondary metabolites facilitate the interaction of a microbe with its environment. Commonly, biosynthetic pathways of secondary metabolites are coded in Biosynthetic Gene Clusters and are not expressed under laboratory growth conditions. Although existing evidence has linked antibiotics with secondary metabolism induction in bacteria.

To find out whether there exists a connection between antibiotic administration and the prevalence of genes coding for secondary metabolites, we developed a Bioinformatic Pipeline to detect and compare the presence Biosynthetic Gene Clusters across multiple metagenomic samples. We applied the pipeline to analyze oral and fecal samples from a placebo-controlled trial of 38 patients with cirrhosis. The results provide a description on how the Gene Clusters profile responds to the rifaximin-α treatment.

Diabetes mellitus type 2 (T2D) causes an increased risk of morbidity and mortality in response to viral infection. T2D is characterized by hyperglycemia and is typically associated with insulin resistance and compensatory hyperinsulinemia. CD8 T cells express the insulin receptor and previously we have shown that insulin is able to directly modulate effector CD8 T cell function. We therefore hypothesized memory CD8 T cell responsiveness in context of T2D is negatively impacted by hyperinsulinemia or hyperglycemia. Using a mouse model for T2D we could show that memory CD8 T cell function was significantly reduced in response to re-challenge by viral infection or with melanoma cells. Basal insulin injection of mice increased GLUT-1 expression and glucose uptake in memory CD8 T cell precursors early after infection, which was prevented when these cells were deficient for the insulin receptor. However, neither insulin injection, nor insulin receptor deficiency resulted in a difference in metabolism, memory formation, cytokine production or recall responses of memory CD8 T cells compared to controls. Importantly, in context of obesity, insulin receptor deficiency on CD8 T cells did not affect the functional capacity of memory CD8 T cells. In contrast, we could show in vitro and in vivo that hyperglycemia significantly impairs the antiviral capacity of memory CD8 T cells. Our findings indicate that obesity impairs the memory CD8 T cell response against viral infection and cancer through the detrimental effects of hyperglycemia rather than hyperinsulinemia.

Alterations in the urea cycle, in which arginine is synthesized, are common in cancer. However, little is known about the levels of arginine in these cancers.  Here, we find elevated arginine in hepatocellular carcinoma (HCC) despite suppression of urea cycle enzymes.  Liver tumors accumulate high levels of arginine via elevated uptake and, more importantly, via suppression of arginine-to-polyamine conversion.  Mechanistically, high levels of arginine promote tumorigenesis via metabolic reprogramming.  Furthermore, we identify arginine-binding proteins which regulate metabolic enzyme expression.

mTORC1 is a nodal kinase that integrates a myriad of upstream cues such as nutrient availability, growth factor signals, and sufficiency of oxygen and energy levels, and mediates a corresponding physiological response. One of the key regulators of mTORC1 is the TSC (Tuberous Sclerosis Complex) that acts as a potent negative regulator of mTORC1 signaling by functioning as a GAP (GTPase-activating protein) towards RHEB, a small GTPase that is a direct upstream activator of mTORC1. Under conditions where mTORC1 is active, anabolic processes such as protein synthesis are promoted (via the phosphorylation of S6K1, 4EBP1) while catabolic processes such as autophagy and lysosomal biogenesis are suppressed (via the phosphorylation of ULK1, TFEB, TFE3). Surprisingly, however, recent work demonstrated that in TSC-deficient cells, where mTORC1 is hyperactive towards its canonical substrates like S6K, TFEB is contrarily dephosphorylated and thereby localized in the nucleus to activate lysosomal biogenesis. Importantly, the signaling cascade that mediates this differential mTORC1 effect towards distinct substrates is not known. In this study, we show that the paradoxical activation of TFEB/TFE3 in cells with TSC loss-of-function is driven by RHEB and mTORC1 hyperactivation and is not the result of activation of mTORC1-independent pathways.

Misregulation of the Kynurenine Pathway (KP) has been implicated in many diseases of the central nervous system. Tryptophan 2,3-dioxygenase (tdo-2) is KP enzyme, involved in metabolizing Tryptophan. It has been established that tdo-2 depletion in C. elegans and D. melanogaster rescues motility defects and improved lifespan in ageing wild type and neurodegenerative disease models. However, the precise mechanism of the rescue effect of tdo-2 depletion is not yet fully understood. In this study, we are investigating the precise local and systemic mechanisms induced by tdo-2 knockdown in C. elegans. Using a bespoke tracking pipeline, as well as metabolite measurements, and visualizing KP pathway dynamics using state-of-the-art techniques, we will be able to investigate to what extent the loss of tdo-2 affects the systemic KP in C. elegans.

Working hypothesis: ferritin gene and ace2 gene fusion in patients with very severe Covid 19 as causative of hight ferritin blood levels. 

biblio

ferritin levels and covid-19 Vargas Vargas et al  PAHO 

Proanthocyanidins extracted from Pelargonium sidoides DC. have substantial antioxidant activity and  were proposed as a viable adjunct to periodontal treatment.

The aim: To evaluate the efficacy of proanthocyanidins  in non-surgical periodontal therapy in patients with periodontitis.

Material and Methods: 46 patients with periodontitis and 24 healthy individuals were included in a randomized controlled clinical trial. Patients with periodontitis received treatment -  subgingival  application of collagen hydrogels with proanthocyanidins. Immunological investigation of MMP-3, TIMP-1 concentration in saliva was performed. Frequency of TLR4 rs1927911 gene polymorphism was performed.

Results: Additional use of proanthocyanidins in subgingival instrumentation resulted in additional probing depth reduction. Health individuals had statistically significantly lower MMP-3 concentration in saliva.  Rates of the TLR4 genotypes s in patients with periodontitis were not statistically significant from the control group.

Conclusions:  Adjunctive use of proanthocyanidins in non-surgical periodontal therapy shifted MMP-3 concentration in saliva towards healthy individuals levels more than the subgingival instrumentation alone.

Obesity belongs to one of the independent risk factors of severe COVID-19 outcome. In this study, we aimed to elucidate the role of adipose tissue in SARS-CoV-2 infection. We analyzed autopsy-derived adipose tissue samples from deceased COVID-19 patients. SARS-CoV-2 RNA was detected in 10 out of 19 male individuals in at least one adipose tissue sample analyzed. Viral RNA in adipose tissue was only found in overweight or obese males (BMI ≥ 25). This correlation was not seen in samples of female individuals (n=12). Infection experiments in human stem cell-derived adipocytes revealed replication of SARS-CoV-2 depending on the adipogenic differentiation state and corresponding angiotensin converting enzyme 2 (ACE2) expression. Treatment with the lipase inhibitor tetrahydrolipstatin reduced viral titers by 100-fold in mature adipocytes indicating a dependency on lipid droplet metabolism. Further reduction in viral titers was achieved by combination treatment with atorvastatin, most probably caused by decreased ACE2 expression. Syrian golden hamsters infected with SARS-CoV-2 showed infectious viral titers in adipose tissue accompanied by a pronounced upregulation of isg15. In metabolomic analysis of plasma samples obtained from infected hamsters, we detected an abundance of triglycerides (TG) enriched in polyunsaturated fatty acids (PUFA) and a reduction of TGs enriched in monounsaturated (MUFA) and saturated fatty acids (SFA), typical for de novo lipogenesis (DNL). Plasma samples of COVID-19 patients showed a similar trend towards lower concentration of MUFA, SFA-containing TGs. In conclusion, we show that replication of SARS-CoV-2 in adipose tissue alters the lipid metabolism of hamsters and humans.