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Objectives Sodium-glucose cotransporter 2 inhibitors (SGLT2i) modulate lipid metabolism and improve cardiovascular morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). The exact cardioprotective mechanism of SGLT2i is unclear. We evaluated the effects of SGLT2i on postprandial lipids, lipoprotein concentrations, glucose and fatty acids. Design A placebo-controlled randomized, proof-of-concept study. Methods Fourteen male patients with T2DM on intensive insulin regimen were randomly and double-blind allocated to 12 weeks dapagliflozin (10 mg) or placebo. Postprandial effects were assessed with an 8-h standardized oral fat loading test. Results Mean glycated A1c did not change by dapagliflozin, but the mean daily insulin dose was significantly reduced. Although dapagliflozin did not affect fasting or postprandial levels of glucose and insulin, it increased the postprandial levels of glucagon. While fasting levels of free fatty acids and beta-hydroxybutyrate (bHBA) were unchanged, dapagliflozin significantly increased the postprandial bHBA response. This was seen in the context of increased postprandial glucagon levels by dapagliflozin, without influencing postprandial insulin or glucose levels. Dapagliflozin did not affect fasting or postprandial plasma cholesterol and triglycerides nor postprandial inflammatory markers. Fasting apolipoprotein B48 was decreased without affecting the postprandial response. Markers of inflammation and vascular function did not change. Conclusion Treatment with dapagliflozin of patients with T2DM led to a reduction of fasting chylomicron remnants and increased postprandial ketone bodies compared to placebo suggesting enhanced hepatic fatty acid oxidation. The latter may have been caused by decreasing the insulin–glucagon ratio. The beneficial clinical effects seen in the trials using dapagliflozin most likely are not due to effects on postprandial inflammation nor postprandial lipemia.

Epithelial-to-mesenchymal transition (EMT) is key to tumor aggressiveness, therapy resistance, and immune escape in breast cancer. Because metabolic traits might be involved along the EMT continuum, we investigated whether human breast epithelial cells engineered to stably acquire a mesenchymal phenotype in non-tumorigenic and H-RasV12-driven tumorigenic backgrounds possess unique metabolic fingerprints. We profiled mitochondrial–cytosolic bioenergetic and one-carbon (1C) metabolites by metabolomic analysis, and then questioned the utilization of different mitochondrial substrates by EMT mitochondria and their sensitivity to mitochondria-centered inhibitors. “Upper” and “lower” glycolysis were the preferred glucose fluxes activated by EMT in non-tumorigenic and tumorigenic backgrounds, respectively. EMT in non-tumorigenic and tumorigenic backgrounds could be distinguished by the differential contribution of the homocysteine-methionine 1C cycle to the transsulfuration pathway. Both non-tumorigenic and tumorigenic EMT-activated cells showed elevated mitochondrial utilization of glycolysis end-products such as lactic acid, β-oxidation substrates including palmitoyl–carnitine, and tricarboxylic acid pathway substrates such as succinic acid. Notably, mitochondria in tumorigenic EMT cells distinctively exhibited a significant alteration in the electron flow intensity from succinate to mitochondrial complex III as they were highly refractory to the inhibitory effects of antimycin A and myxothiazol. Our results show that the bioenergetic/1C metabolic signature, the utilization rates of preferred mitochondrial substrates, and sensitivity to mitochondrial drugs significantly differs upon execution of EMT in non-tumorigenic and tumorigenic backgrounds, which could help to resolve the relationship between EMT, malignancy, and therapeutic resistance in breast cancer.

Advances in metabolomics analysis and data treatment increase the knowledge of complex biological systems. One of the most used methodologies is gas chromatography-mass spectrometry (GC-MS) due to its robustness, high separation efficiency, and reliable peak identification through curated databases. However, methodologies are not standardized, and the derivatization steps in GC-MS can introduce experimental errors and take considerable time, exposing the samples to degradation. Here, we propose the injection-port derivatization (IPD) methodology to increase the throughput in plasma metabolomics analysis by GC-MS. The IPD method was evaluated and optimized for different families of metabolites (organic acids, amino acids, fatty acids, sugars, sugar phosphates, etc.) in terms of residence time, injection-port temperature, and sample/derivatization reagent ratio. Finally, the method's usefulness was validated in a study consisting of a cohort of obese patients with or without nonalcoholic steatohepatitis. Our results show a fast, reproducible, precise, and reliable method for the analysis of biological samples by GC-MS. Raw data are publicly available at MetaboLights with Study Identifier MTBLS5151.

Injection of tissues with senescent cells induces changes that mimic aging, and this process is delayed in mice engineered to eliminate senescent cells, which secrete proinflammatory cytokines, including C-C motif chemokine ligand 2 (Ccl2). Circulating levels of Ccl2 correlate with age, but the impact of Ccl2 on tissue homeostasis has not been established. We generated an experimental model by crossbreeding mice overexpressing Ccl2 with progeroid mice bearing a mutation in the lamin A (Lmna) gene. Wild-type animals and progeroid mice that do not overexpress Ccl2 were used as controls. Ccl2 overexpression decreased the lifespan of the progeroid mice and induced the dysregulation of glycolysis, the citric acid cycle and one-carbon metabolism in skeletal muscle, driving dynamic changes in energy metabolism and DNA methylation. This impact on cellular bioenergetics was associated with mitochondrial alterations and affected cellular metabolism, autophagy and protein synthesis through AMPK/mTOR pathways. The data revealed the ability of Ccl2 to promote death in mice with accelerated aging, which supports its putative use as a biomarker of an increased senescent cell burden and for the assessment of the efficacy of interventions aimed at extending healthy aging.

The METTEN study assessed the efficacy, tolerability, and safety of adding metformin to neoadjuvant chemotherapy plus trastuzumab in early HER2-positive breast cancer (BC). Women with primary, non-metastatic HER2-positive BC were randomized (1:1) to receive metformin (850 mg twice-daily) for 24 weeks concurrently with 12 cycles of weekly paclitaxel plus trastuzumab, followed by four cycles of 3-weekly FE75C plus trastuzumab (arm A), or equivalent regimen without metformin (arm B), followed by surgery. Primary endpoint was the rate of pathological complete response (pCR) in the per-protocol efficacy population. pCR rate was numerically higher in the metformin-containing arm A (19 of 29 patients [65.5%, 95% CI: 47.3–80.1]) than in arm B (17 of 29 patients [58.6%, 95% CI: 40.7–74.5]; OR 1.34 [95% CI: 0.46–3.89], P = 0.589). The rate of breast-conserving surgery was 79.3% and 58.6% in arm A and B (P = 0.089), respectively. Blood metformin concentrations (6.2 μmol/L, 95% CI: 3.6–8.8) were within the therapeutic range. Seventy-six percent of patients completed the metformin-containing regimen; 13% of patients in arm A dropped out because of metformin-related gastrointestinal symptoms. The most common adverse events (AEs) of grade ≥3 were neutropenia in both arms and diarrhea in arm A. None of the serious AEs was deemed to be metformin-related. Addition of anti-diabetic doses of metformin to a complex neoadjuvant regimen was well tolerated and safe. Because the study was underpowered relative to its primary endpoint, the efficacy data should be interpreted with caution.

A gain-of-function mutation in the fibroblast growth factor receptor 3 gene (FGFR3) results in achondroplasia (ACH), the most frequent form of dwarfism. The constitutive activation of FGFR3 impaired bone formation and elongation and many signaling transduction pathways. Identification of new and relevant compounds targeting the FGFR3 signaling pathway is of broad importance for the treatment of ACH. Natural plant compounds are the prime sources of drug candidates. Here, we found that the phenol compound (-)-epicatechin isolated fromTheobroma cacaoeffectively inhibits FGFR3’s downstream signaling pathways. Transcriptomic analysis inFgfr3mouse model showed that ciliary mRNA expression was modified and influenced significantly by the Indian hedgehog and PKA pathways. (-)-Epicatechin is able to rescue impairments in the expression of these mRNA that control both the structural organization of the primary cilium and ciliogenesis-related genes. In femurs isolated from a mouse model (Fgfr3Y367C/+) of ACH, we showed that (-)-epicatechin countered the bone growth impairment during 6 days ofex vivocultures. We confirmedin vivothat daily subcutaneous injections of (-)-epicatechin inFgfr3Y367C/+mice increased bone elongation and rescued the primary cilium defect observed in chondrocytes. This modification of the primary cilia promoted the typical columnar arrangement of flat proliferative chondrocytes and thus enhanced bone elongation. The results of the present proof-of-principle study illustrated (-)-epicatechin’s ability may facilitate the development of (-)-epicatechin as a treatment for patients with ACH.

Hepatic biopsy is the gold standard for staging nonalcoholic fatty liver disease (NAFLD). Unfortunately, accessing the liver is invasive, requires a multidisciplinary team and is too expensive to be conducted on large segments of the population. NAFLD starts quietly and can progress until liver damage is irreversible. Given this complex situation, the search for noninvasive alternatives is clinically important. A hallmark of NAFLD progression is the dysregulation in lipid metabolism. In this context, recent advances in the area of machine learning have increased the interest in evaluating whether multi-omics data analysis performed on peripheral blood can enhance human interpretation. In the present review, we show how the use of machine learning can identify sets of lipids as predictive biomarkers of NAFLD progression. This approach could potentially help clinicians to improve the diagnosis accuracy and predict the future risk of the disease. While NAFLD has no effective treatment yet, the key to slowing the progression of the disease may lie in predictive robust biomarkers. Hence, to detect this disease as soon as possible, the use of computational science can help us to make a more accurate and reliable diagnosis. We aimed to provide a general overview for all readers interested in implementing these methods.

Patients with morbid obesity frequently present non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) associated with pro-atherogenic alterations. Laparoscopic sleeve gastrectomy (LSG) is an effective treatment for weight reduction, and for the remission of hepatic alterations. Using 1H-nuclear magnetic resonance (1H-NMR), we investigated the effects of LSG on lipoprotein and glycoprotein profile in patients with morbid obesity and liver disease. We included 154 patients with morbid obesity (49 non-NASH, 54 uncertain NASH, 51 definite NASH). A blood sample was obtained before surgery and, in patients with definite NASH, one year after surgery. Patients with NASH had increased concentrations of medium and small VLDL particles, VLDL and IDL cholesterol concentrations, IDL, LDL, and HDL triglyceride concentrations, and elevated glycoprotein levels. These changes were more marked in patients with type 2 diabetes mellitus. LSG produced significant decreases in the concentration of VLDL particles, VLDL cholesterol and triglycerides, an increase in the concentration LDL particles and LDL cholesterol concentrations, and a decrease in protein glycation. We conclude that patients with obesity and NASH had significant alterations in circulating levels of lipoproteins and glycoproteins that were associated with the severity of the disease. Most of these changes were reversed post-LSG.

Background & Aims A holistic insight on the relationship between obesity and metabolic dysfunction-associated fatty liver disease is an unmet clinical need. Omics investigations can be used to investigate the multifaceted role of altered mitochondrial pathways to promote nonalcoholic steatohepatitis, a major risk factor for liver disease-associated death. There are no specific treatments but remission via surgery might offer an opportunity to examine the signaling processes that govern the complex spectrum of chronic liver diseases observed in extreme obesity. We aim to assess the emerging relationship between metabolism, methylation and liver disease. Methods We tailed the flow of information, before and after steatohepatitis remission, from biochemical, histological, and multi-omics analyses in liver biopsies from patients with extreme obesity and successful bariatric surgery. Functional studies were performed in HepG2 cells and primary hepatocytes. Results The reversal of hepatic mitochondrial dysfunction and the control of oxidative stress and inflammatory responses revealed the regulatory role of mitogen-activated protein kinases. The reversible metabolic rearrangements leading to steatohepatitis increased the glutaminolysis-induced production of α-ketoglutarate and the hyperactivation of mammalian target of rapamycin complex 1. These changes were crucial for the adenosine monophosphate-activated protein kinase/mammalian target of rapamycin-driven pathways that modulated hepatocyte survival by coordinating apoptosis and autophagy. The signaling activity of α-ketoglutarate and the associated metabolites also affected methylation-related epigenomic remodeling enzymes. Integrative analysis of hepatic transcriptome signatures and differentially methylated genomic regions distinguished patients with and without steatohepatitis. Conclusion We provide evidence supporting the multifaceted potential of the increased glutaminolysis-induced α-ketoglutarate production and the mammalian target of rapamycin complex 1 dysregulation as a conceivable source of the inefficient adaptive responses leading to steatohepatitis. Lay summary Steatohepatitis is a frequent and threatening complication of extreme obesity without specific treatment. Omics technologies can be used to identify therapeutic targets. We highlight increased glutaminolysis-induced α-ketoglutarate production as a potential source of signals promoting and exacerbating steatohepatitis.

Chemokine (C–C motif) ligand 2 (CCL2) has been associated with chronic metabolic diseases. We aimed to investigate whether Ccl2 gene overexpression is involved in the regulation of signaling pathways in metabolic organs. Biochemical and histological analyses were used to explore tissue damage in cisgenic mice that overexpressed the Ccl2 gene. Metabolites from energy and one-carbon metabolism in liver and muscle extracts were measured by targeted metabolomics. Western blot analysis was used to explore the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin pathways. Ccl2 overexpression resulted in steatosis, decreased AMPK activity and altered mitochondrial dynamics in the liver. These changes were associated with decreased oxidative phosphorylation and alterations in the citric acid cycle and transmethylation. In contrast, AMPK activity and its downstream mediators were increased in muscle, where we observed an increase in oxidative phosphorylation and increased concentrations of different metabolites associated with ATP synthesis. In conclusion, Ccl2 overexpression induces distinct metabolic alterations in the liver and muscle that affect mitochondrial dynamics and the regulation of energy sensors involved in cell homeostasis. These data suggest that CCL2 may be a therapeutic target in metabolic diseases.

Background: Obesity is a chronic progressive disease with several metabolic alterations. Nonalcoholic fatty liver disease (NAFLD) is an important comorbidity of obesity that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis or hepatocarcinoma. This study aimed at clarifying the molecular mechanisms underlying the metabolic alterations in hepatic and adipose tissue during high-fat high-sucrose diet-induced NAFLD development in mice. Methods: Twenty-four male mice (C57BL/6J) were randomly allocated into 3 groups (n = 8 mice per group) to receive a chow diet, a high-fat diet (HFD), or a high-fat high-sucrose diet (HF-HSD) for 20 weeks. At sacrifice, liver and adipose tissue were obtained for histopathological, metabolomic, and protein expression analyses. Results: HF-HSD (but not HFD) was associated with NASH and increased oxidative stress. These animals presented an inhibition of hepatic autophagy and alterations in AMP-activated protein kinase/mammalian target of rapamycin activity. We also observed that the ability of metabolic adaptation was adversely affected by the increase of damaged mitochondria. NASH development was associated with changes in adipose tissue dynamics and increased amounts of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids in visceral adipose tissue. Conclusion: HF-HSD led to a metabolic blockage and impaired hepatic mitochondria turnover. In addition, the continuous accumulation of fatty acids produced adipose tissue dysfunction and hepatic fat accumulation that favored the progression to NASH.

Plants, including most food and feed plants, produce a broad range of bioactive chemical compounds. Among these compounds, polyphenols are reported to provide beneficial effects as anti-carcinogenic, anti-atherogenic, anti-inflammatory, immune modulating, anti-microbial, vasodilatory and analgesic. Cocoa (Theobroma cacao), a major, economically important, international crop, has been related to several nutritional benefits, which have been associated with the phenolic fraction. The main subclass of flavonoids found in cocoa is flavanols, particularly (epi)catechins monomers, and their oligomers, also known as procyanidins. In this study, these compounds were isolated by different methodologies as solid phase extraction (SPE), semi-preparative high-performance liquid chromatography (HPLC) and membrane technologies to obtain different polyphenolic profiles by HPLC coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and to test their cytotoxicity. Finally, different polyphenolic profiles were collected, where the combination of both semi-preparative HPLC and SPE technologies provided the most purified fractions. Filtration with membranes and SPE provide extracts with different composition depending on the pore size of membranes and on the solvent, respectively. In addition, the results of toxicity assay indicated low levels in all fractions.

Recently, the relationship between nutrients and health is becoming since natural dietary products as polyphenols are being considered by their potential for the management of several diseases. We aimed to investigate the capacity of Lippia citriodora compounds to modulate AMP-activated protein kinase activity (AMPK) on a hypertrophic adipocyte model. HPLC semi-preparative purification method and reverse phase high performance liquid chromatography coupled to time-of-flight mass detection with electrospray ionization (RP-HPLC-ESI-TOF/MS) were used to obtain de compounds from L. citriodora extract. AMPK activity was measured on the hypertrophic 3T3-L1 adipocyte model by immunofluorescence microscopy. Four compounds of 29 total compounds have been tentatively identified in L. citriodora for the first time by HPLC-ESI-TOF-MS. Phenylpropanoids (verbascoside), iridoids (gardoside) and flavonoids (luteolin-7-diglucoronide) were the best candidates to account for activating AMPK capacity. The combination of specific polyphenols from L. citriodora, which showed strong activating AMPK capacity, could be an alternative in the management of obesity-associated diseases.

International audience; The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.

// Elisabet Cuyas 1,2,* , Sara Verdura 1,2,* , Salvador Fernandez-Arroyo 3,4 , Joaquim Bosch-Barrera 5 , Begona Martin-Castillo 6 , Jorge Joven 3,4 and Javier A. Menendez 1,2 1 Metabolism and Cancer Group, Program Against Cancer Therapeutic Resistance, Catalan Institute of Oncology, Girona, Spain 2 Molecular Oncology Group, Girona Biomedical Research Institute (IDIBGI), Girona, Spain 3 Unitat de Recerca Biomedica, Hospital Universitari de Sant Joan, Institut d’Investigacio Sanitaria Pere Virgili, Universitat Rovira i Virgili, Reus, Catalonia, Spain 4 Campus of International Excellence Southern Catalonia, Tarragona, Catalonia, Spain 5 Medical Oncology, Catalan Institute of Oncology, Girona, Catalonia, Spain 6 Unit of Clinical Research, Catalan Institute of Oncology, Girona, Spain * These authors have contributed equally to this work Correspondence to: Javier A. Menendez, email: // Keywords : cancer stem cells; metabolic maps; tumor heterogeneity Received : July 21, 2017 Accepted : September 29, 2017 Published : October 15, 2017 Abstract Personalized cancer medicine based on the analysis of tumors en masse is limited by tumor heterogeneity, which has become a major obstacle to effective cancer treatment. Cancer stem cells (CSC) are emerging as key drivers of inter- and intratumoral heterogeneity. CSC have unique metabolic dependencies that are required not only for specific bioenergetic/biosynthetic demands but also for sustaining their operational epigenetic traits, i.e. self-renewal, tumor-initiation, and plasticity. Given that the metabolome is the final downstream product of all the –omic layers and, therefore, most representative of the biological phenotype, we here propose that a novel approach to better understand the complexity of tumor heterogeneity is by mapping and cataloging small numbers of CSC metabolomic phenotypes. The narrower metabolomic diversity of CSC states could be employed to reduce multidimensional tumor heterogeneity into dynamic models of fewer actionable sub-phenotypes. The identification of the driver nodes that are used differentially by CSC states to metabolically regulate self-renewal and tumor initation and escape chemotherapy might open new preventive and therapeutic avenues. The mapping of CSC metabolomic states could become a pioneering strategy to reduce the dimensionality of tumor heterogeneity and improve our ability to examine changes in tumor cell populations for cancer detection, prognosis, prediction/monitoring of therapy response, and detection of therapy resistance and recurrent disease. The identification of driver metabolites and metabolic nodes accounting for a large amount of variance within the CSC metabolomic sub-phenotypes might offer new unforeseen opportunities for reducing and exploiting tumor heterogeneity via metabolic targeting of CSC.

The usage of metabolic intermediates as substrates for chromatin-modifying enzymes provides a direct link between the metabolic state of the cell and epigenetics. Because this metabolism-epigenetics axis can regulate not only normal but also diseased states, it is reasonable to suggest that manipulating the epigenome via metabolic interventions may improve the clinical manifestation of age-related diseases including cancer. Using a model of BRCA1 haploinsufficiency-driven accelerated geroncogenesis, we recently tested the hypothesis that: 1.) metabolic rewiring of the mitochondrial biosynthetic nodes that overproduce epigenetic metabolites such as acetyl-CoA should promote cancer-related acetylation of histone H3 marks; 2.) metformin-induced restriction of mitochondrial biosynthetic capacity should manifest in the epigenetic regulation of histone acetylation. We now provide one of the first examples of how metformin-driven metabolic shifts such as reduction of the 2-carbon epigenetic substrate acetyl-CoA is sufficient to correct specific histone H3 acetylation marks in cancer-prone human epithelial cells. The ability of metformin to regulate mitonuclear communication and modulate the epigenetic landscape in genomically unstable pre-cancerous cells might guide the development of new metabolo-epigenetic strategies for cancer prevention and therapy.

Background Obesity can influence hepatic mitochondrial function, and cause non-alcoholic steatohepatitis (NASH). Diagnosis and follow-up rely on invasive liver biopsy so blood-based markers are urgently required. Aim To investigate whether values of circulating metabolites from energy and one-carbon (1-C) metabolism may: (a) reflect hepatic mitochondrial flexibility failure and (b) act as NASH biomarkers. Methods Patients with severe obesity undergoing bariatric surgery (n = 270) were investigated using quantitative targeted plasma metabolomics. Comparisons were with non-obese controls without liver disease (n = 50). Obese patients with NASH (n = 53) and without NASH (n = 130) representing extreme groups of liver disease were assessed to test the diagnostic ability of the measured circulating metabolites. Paired liver biopsy and plasma samples from NASH patients were available 1 year post-surgery and were evaluated to monitor metabolomic changes with liver damage resolution. Results We identified correlations between human liver metabolism and obesity. High-plasma α-ketoglutarate (α-KG) and lactate concentrations in NASH patients indicating citric acid cycle replenishment via glutaminolysis might also be a crucial point in NASH onset. Plasma measurements of α-KG, β-hydroxybutyrate, pyruvate and oxaloacetate reduced the uncertainty in clinical diagnosis of NASH [area under receiver operating characteristic curve (AUC) of 0.826] and predicted NASH resolution without ambiguity (AUC of 0.999). Conclusion Changes in plasma mitochondrial metabolites appear to be associated with NASH. These metabolic responses may be dynamically remodelled following resolution of liver damage through massive weight loss.

Neoadjuvant chemotherapy (NACT) is employed in patients with breast cancer (BC) with the aim of reducing tumor burden and improving surgical outcomes. We evaluated the levels of energy metabolites pre- and post-radiotherapy (RT) in breast cancer (BC) patients who previously received NACT and investigated the alterations of these metabolites in relation to the patient achieving a pathologic complete response to NACT. We included 37 BC patients who were treated with NACT following surgery and analyzed the concentrations of energy balance-related metabolites using targeted metabolomics before and one month after the end of RT. The control group was composed of 44 healthy women. Pre-radiotherapy, patients had significant decreases in the plasma levels of 12 metabolites. RT corrected these alterations and the improvement was superior in patients with a pathologic complete response. Our results highlight the importance of metabolism in the outcomes of patients with BC.

Chemokines, particularly chemokine (C-C- motif) ligand 2 (CCL2), control leukocyte migration into the wall of the artery and regulate the traffic of inflammatory cells. CCL2 is bound to functional receptors (CCR2), but also to atypical chemokine receptors (ACKRs), which do not induce cell migration but can modify chemokine gradients. Whether atherosclerosis alters CCL2 function by influencing the expression of these receptors remains unknown. In a necropsy study, we used immunohistochemistry to explore where and to what extent CCL2 and related receptors are present in diseased arteries that caused the death of men with coronary artery disease compared with unaffected arteries. CCL2 was marginally detected in normal arteries but was more frequently found in the intima. The expression of CCL2 and related receptors was significantly increased in diseased arteries with relative differences among the artery layers. The highest relative increases were those of CCL2 and ACKR1. CCL2 expression was associated with a significant predictive value of atherosclerosis. Findings suggest the need for further insight into receptor specificity or activity and the interplay among chemokines. CCL2-associated conventional and atypical receptors are overexpressed in atherosclerotic arteries, and these may suggest new potential therapeutic targets to locally modify the overall anti-inflammatory response.

Homocisteïna; Càncer de mama; Dieta cetogènica Homocisteína; Cáncer de mama; Dieta cetogénica Homocysteine; Breast cancer; Ketogenic diet Certain dietary interventions might improve the therapeutic index of cancer treatments. An alternative to the “drug plus diet” approach is the pharmacological reproduction of the metabolic traits of such diets. Here we explored the impact of adding metformin to an established therapeutic regimen on the systemic host metabolism of cancer patients. A panel of 11 serum metabolites including markers of mitochondrial function and intermediates/products of folate-dependent one-carbon metabolism were measured in paired baseline and post-treatment sera obtained from HER2-positive breast cancer patients randomized to receive either metformin combined with neoadjuvant chemotherapy and trastuzumab or an equivalent regimen without metformin. Metabolite profiles revealed a significant increase of the ketone body β-hydroxybutyrate and of the TCA intermediate α-ketoglutarate in the metformin-containing arm. A significant relationship was found between the follow-up levels of homocysteine and the ability of treatment arms to achieve a pathological complete response (pCR). In the metformin-containing arm, patients with significant elevations of homocysteine tended to have a higher probability of pCR. The addition of metformin to an established anti-cancer therapeutic regimen causes a fasting-mimicking modification of systemic host metabolism. Circulating homocysteine could be explored as a clinical pharmacodynamic biomarker linking the antifolate-like activity of metformin and biological tumor response. This work was supported by grants from the Ministerio de Sanidad, Servicios Sociales e Igualdad (EC10-125, Ayudas para el Fomento de la Investigación Clínica Independiente to Begoña Martin-Castillo). Work in the Menendez laboratory is supported by the Ministerio de Ciencia e Innovación (Grant SAF2016-80639-P, Plan Nacional de l+D+I, founded by the European Regional Development Fund [EU FEDER], Spain) and by an unrestricted research grant from the Fundació Oncolliga Girona (Lliga catalana d’ajuda al malalt de càncer, Girona).

The risk of non-alcoholic fatty liver disease increases with obesity. Vulnerability to oxidative stress and/or inflammation represents a crucial step in non-alcoholic fatty liver disease progression through abnormal metabolic responses. In this study, we investigated the role of CCL2 gene ablation in mice that were double deficient in low density lipoprotein receptor and in paraoxonase-1. Mass spectrometry methods were used to assess the liver metabolic response in mice fed either regular chow or a high-fat diet. Dietary fat caused liver steatosis, oxidative stress and the accumulation of pro-inflammatory macrophages in the livers of double deficient mice. We observed alterations in energy metabolism-related pathways and in metabolites associated with the methionine cycle and the glutathione reduction pathway. This metabolic response was associated with impaired autophagy. Conversely, when we established CCL2 deficiency, histologic features of fatty liver disease were abrogated, hepatic liver oxidative stress decreased, and anti-inflammatory macrophage marker expression levels increased. These changes were associated with the normalization of metabolic disturbances and increased lysosome-associated membrane protein 2, expression, which suggests enhanced chaperone-mediated autophagy. This study demonstrates that CCL2 is a key molecule for the development of metabolic and histological alterations in the liver of mice sensitive to the development of hyperlipidemia and hepatic steatosis, a finding with potential to identify new therapeutic targets in liver diseases.

We live in a world fascinated by the relationship between disease and nutritional disequilibrium. The subtle and slow effects of chronic nutrient toxicity are a major public health concern. Since food is potentially important for the development of "metabolic memory", there is a need for more information on the type of nutrients causing adverse or toxic effects. We now know that metabolic alterations produced by excessive intake of some nutrients, drugs and chemicals directly impact epigenetic regulation. We envision that understanding how metabolic pathways are coordinated by environmental and genetic factors will provide novel insights for the treatment of metabolic diseases. New methods will enable the assembly and analysis of large sets of complex molecular and clinical data for understanding how inflammation and mitochondria affect bioenergetics, epigenetics and health. Collectively, the observations we highlight indicate that energy utilization and disease are intimately connected by epigenetics. The challenge is to incorporate metabolo-epigenetic data in better interpretations of disease, to expedite therapeutic targeting of key pathways linking nutritional toxicity and metabolism. An additional concern is that changes in the parental phenotype are detectable in the methylome of subsequent offspring. The effect might create a menace to future generations and preconceptional considerations.

// Javier A. Menendez 1,2 , Nuria Folguera-Blasco 3 , Elisabet Cuyas 1,2 , Salvador Fernandez-Arroyo 4 , Jorge Joven 4 and Tomas Alarcon 3,5,6,7 1 ProCURE (Program Against Cancer Therapeutic Resistance), Metabolism and Cancer Group, Catalan Institute of Oncology, Girona, Catalonia, Spain 2 Molecular Oncology Group, Girona Biomedical Research Institute (IDIBGI), Salt, Catalonia, Spain 3 Computational and Mathematical Biology Research Group, Centre de Recerca Matematica (CRM), Barcelona, Spain 4 Unitat de Recerca Biomedica, Hospital Universitari de Sant Joan, IISPV, Universitat Rovira i Virgili, Campus of International Excellence Southern Catalonia, Reus, Spain 5 Institucio Catalana d’Estudis i Recerca Avancats (ICREA), Barcelona, Spain 6 Departament de Matematiques, Universitat Autonoma de Barcelona, Barcelona, Spain 7 Barcelona Graduate School of Mathematics (BGSMath), Barcelona, Spain Correspondence to: Javier A. Menendez, email: // Tomas Alarcon, email: // Keywords : BRCA1, geroncogenesis, metabolism, cancer, metformin, Gerotarget Received : January 22, 2016 Accepted : February 21, 2016 Published : March 02, 2016 Abstract The geroncogenesis hypothesis postulates that the decline in metabolic cellular health that occurs naturally with aging drives a “field effect” predisposing normal tissues for cancer development. We propose that mutations in the cancer susceptibility genes BRCA1 / 2 might trigger “accelerated geroncogenesis” in breast and ovarian epithelia. By speeding up the rate at which the metabolic threshold becomes “permissive” with survival and expansion of genomically unstable pre-tumoral epithelial cells, BRCA haploinsufficiency-driven metabolic reprogramming would operate as a bona fide oncogenic event enabling malignant transformation and tumor formation in BRCA carriers. The metabolic facet of BRCA1 one-hit might involve tissue-specific alterations in acetyl-CoA, α-ketoglutarate, NAD + , FAD, or S-adenosylmethionine, critical factors for de/methylation or de/acetylation dynamics in the nuclear epigenome. This in turn might induce faulty epigenetic reprogramming at the “install phase” that directs cell-specific differentiation of breast/ovarian epithelial cells, which can ultimately determine the penetrance of BRCA defects during developmental windows of susceptibility. This model offers a framework to study whether metabolic drugs that prevent or revert metabolic reprogramming induced by BRCA haploinsufficiency might displace the “geroncogenic risk” of BRCA carriers to the age typical for those without the mutation. The identification of the key nodes that directly communicate changes in cellular metabolism to the chromatin in BRCA haploinsufficient cells may allow the epigenetic targeting of genomic instability using exclusively metabolic means. The validation of accelerated geroncogenesis as an inherited “one-hit” metabolic “field effect” might offer new strategies to therapeutically revisit the apparently irreversible genetic-hereditary fate of women with hereditary breast-ovarian cancer syndrome.

Objectives Methotrexate (MTX) functions as an antiproliferative agent in cancer and an anti-inflammatory drug in rheumatoid arthritis (RA). Although macrophages critically contribute to RA pathology, their response to MTX remains unknown. As a means to identify MTX response markers, we have explored its transcriptional effect on macrophages polarised by GM-CSF (GM-MO) or M-CSF (M-MO), which resemble proinflammatory and anti-inflammatory macrophages found in RA and normal joints, respectively. Methods The transcriptomic profile of both human macrophage subtypes exposed to 50 nM of MTX under long-term and short-term schedules were determined using gene expression microarrays, and validated through quantitative real time PCR and ELISA. The molecular pathway involved in macrophage MTX-responsiveness was determined through pharmacological, siRNA-mediated knockdown approaches, metabolomics for polyglutamylated-MTX detection, western blot, and immunofluorescence on RA and normal joints. Results MTX exclusively modulated gene expression in proinflammatory GM-MO, where it influenced the expression of 757 genes and induced CCL20 and LIF at the mRNA and protein levels. Pharmacological and siRNA-mediated approaches indicated that macrophage subset-specific MTX responsiveness correlates with thymidylate synthase (TS) expression, as proinflammatory TS + GM-MO are susceptible to MTX, whereas anti-inflammatory TS low/− M-MO and monocytes are refractory to MTX. Furthermore, p53 activity was found to mediate the TS-dependent MTX-responsiveness of proinflammatory TS + GM-MO. Importantly, TS and p53 were found to be expressed by CD163 + /TNFα + GM-CSF-polarised macrophages from RA joints but not from normal synovium. Conclusions Macrophage response to MTX is polarisation-dependent and determined by the TS-p53 axis. CCL20 and LIF constitute novel macrophage markers for MTX responsiveness in vitro.

We investigated the alterations in the plasma concentrations of energy-balance-related metabolites in patients with lung (LC) or head & neck (HNC) cancer and the changes on these parameters induced by radiotherapy. The study was conducted in 33 patients with non-small cell LC and 28 patients with HNC. We analyzed the concentrations of 17 metabolites involved in glycolysis, citric acid cycle and amino acid metabolism using targeted gas chromatography coupled to quadrupole time-of-flight mass spectrometry. For comparison, a control group of 50 healthy individuals was included in the present study. Patients with LC or HNC had significant alterations in the plasma levels of several energy-balance-related metabolites. Radiotherapy partially normalized these alterations in patients with LC, but not in those with HNC. The measurement of plasma glutamate concentration was an excellent predictor of the presence of LC or HNC, with sensitivity >90% and specificity >80%. Also, associations with disease prognosis were observed with plasma glutamate, amino acids and β-hydroxybutyrate concentrations. SIGNIFICANCE: This study analyzed the changes produced in the plasma concentrations of energy-balance-related metabolites in patients with lung cancer or head and neck cancer. The results obtained identified glutamate as the parameter with the highest discrimination capacity between patients and the control group. The relationships between various metabolites and clinical outcomes were also analyzed. These results extend the knowledge of metabolic alterations in cancer, thus facilitating the search for biomarkers and therapeutic targets.