Altered serum metabolome as an indicator of paraneoplasia or concomitant cancer in patients with rheumatic disease.

OBJECTIVES: A timely diagnosis is imperative for curing cancer. However, in patients with rheumatic musculoskeletal diseases (RMDs) or paraneoplastic syndromes, misleading symptoms frequently delay cancer diagnosis. As metabolic remodelling characterises both cancer and RMD, we analysed if a metabolic signature can indicate paraneoplasia (PN) or reveal concomitant cancer in patients with RMD. METHODS: Metabolic alterations in the sera of rheumatoid arthritis (RA) patients with (n=56) or without (n=52) a history of invasive cancer were quantified by nuclear magnetic resonance analysis. Metabolites indicative of cancer were determined by multivariable regression analyses. Two independent RA and spondyloarthritis (SpA) cohorts with or without a history of invasive cancer were used for blinded validation. Samples from patients with active cancer or cancer treatment, pulmonary and lymphoid type cancers, paraneoplastic syndromes, non-invasive (NI) precancerous lesions and non-melanoma skin cancer and systemic lupus erythematosus and samples prior to the development of malignancy were used to test the model performance. RESULTS: Based on the concentrations of acetate, creatine, glycine, formate and the lipid ratio L1/L6, a diagnostic model yielded a high sensitivity and specificity for cancer diagnosis with AUC=0.995 in the model cohort, AUC=0.940 in the blinded RA validation cohort and AUC=0.928 in the mixed RA/SpA cohort. It was equally capable of identifying cancer in patients with PN. The model was insensitive to common demographic or clinical confounders or the presence of NI malignancy like non-melanoma skin cancer. CONCLUSIONS: This new set of metabolic markers reliably predicts the presence of cancer in arthritis or PN patients with high sensitivity and specificity and has the potential to facilitate a rapid and correct diagnosis of malignancy.

Metabolomics analysis of human spermatozoa reveals impaired metabolic pathways in asthenozoospermia.

BACKGROUND: Infertility is a major health issue, affecting 15% of reproductive-age couples with male factors contributing to 50% of cases. Asthenozoospermia (AS), or low sperm motility, is a common cause of male infertility with complex aetiology, involving genetic and metabolic alterations, inflammation and oxidative stress. However, the molecular mechanisms behind low motility are unclear. In this study, we used a metabolomics approach to identify metabolic biomarkers and pathways involved in sperm motility. METHODS: We compared the metabolome and lipidome of spermatozoa of men with normozoospermia (n = 44) and AS (n = 22) using untargeted LC-MS and the metabolome of seminal fluid using 1H-NMR. Additionally, we evaluated the seminal fluid redox status to assess the oxidative stress in the ejaculate. RESULTS: We identified 112 metabolites and 209 lipids in spermatozoa and 27 metabolites in the seminal fluid of normozoospermic and asthenozoospermic men. PCA analysis of the spermatozoa's metabolomics and lipidomics data showed a clear separation between groups. Spermatozoa of asthenozoospermic men presented lower levels of several amino acids, and increased levels of energetic substrates and lysophospholipids. However, the metabolome and redox status of the seminal fluid was not altered inAS. CONCLUSIONS: Our results indicate impaired metabolic pathways associated with redox homeostasis and amino acid, energy and lipid metabolism in AS. Taken together, these findings suggest that the metabolome and lipidome of human spermatozoa are key factors influencing their motility and that oxidative stress exposure during spermatogenesis or sperm maturation may be in the aetiology of decreased motility in AS.

Distinct immune-effector and metabolic profile of CD8+ T cells in patients with autoimmune polyarthritis induced by therapy with immune checkpoint inhibitors.

OBJECTIVES: Rheumatic immune-related adverse events (irAE) such as (poly)arthritis in patients undergoing immune checkpoint inhibitor (ICI) treatment pose a major clinical challenge. ICI therapy improves CD8+ T cell (CD8) function, but CD8 contributes to chronic inflammation in autoimmune arthritis (AA). Thus, we investigated whether immune functional and metabolic changes in CD8 explain the development of musculoskeletal irAE in ICI-treated patients. METHODS: Peripheral CD8 obtained from ICI-treated patients with and without arthritis irAEs and from AA patients with and without a history of malignancy were stimulated in media containing 13C-labelled glucose with and without tofacitinib or infliximab. Changes in metabolism, immune-mediator release, expression of effector cell-surface molecules and inhibition of tumour cell growth were quantified. RESULTS: CD8 from patients with irAE showed significantly lower frequency and expression of cell-surface molecule characteristic for activation, effector-functions, homing, exhaustion and apoptosis and reduced release of cytotoxic and proinflammatory immune mediators compared with CD8 from ICI patients who did not develop irAE. This was accompanied by a higher glycolytic rate and ATP production. Gene-expression analysis of pre-ICI-treated CD8 revealed several differentially expressed transcripts in patients who later developed arthritis irAEs. In vitro tofacitinib or infliximab treatment did not significantly change the immune-metabolic profile nor the capacity to release cytolytic mediators that inhibit the growth of the human lung cancer cell line H838. CONCLUSIONS: Our study shows that CD8 from ICI-treated patients who develop a musculoskeletal irAE has a distinct immune-effector and metabolic profile from those that remain irAE free. This specific irAE profile overlaps with the one observed in CD8 from AA patients and may prove useful for novel therapeutic strategies to manage ICI-induced irAEs.

A simple method for quantifying de novo lipogenesis rate and substrate selection in cell cultures by 13 C NMR isotopomer analysis of the crude lipid fraction.

PURPOSE: De novo lipogenesis (DNL) is critical for cell growth and maintenance, and acetyl-CoA precursors can be derived from different substrates. We developed a 13 C NMR analysis of lipid extracts from cultured microglia cells administered with [U-13 C]glucose that informs overall lipogenic activity as well as the contribution of glucose to lipogenic acetyl-CoA. METHODS: BV-2 microglial cell line cultured with glucose and glutamine was provided with [U-13 C]glucose and unlabeled glutamine for 24 h and studied in either the presence or absence of lipopolysaccharide (LPS). Cells were then extracted for lipids and the crude lipid fraction was analyzed by 13 C NMR. 13 C-isotopomer signals in the fatty acid ω - 1 and ω - 2 signals representing consecutive or non-consecutive enrichment of the fatty acid chain by [1,2-13 C2 ]acetyl-CoA were quantified and applied to a probabilistic model of acetyl-CoA precursor and fatty acid enrichment. RESULTS: Glucose contributed 72 ± 2% of lipogenic acetyl-CoA while DNL from all sources accounted for 16 ± 2% of lipid turnover. With LPS, there was a significant decrease in glucose contribution (59 ± 4%, p < 0.05) while DNL was unchanged (11 ± 3%). CONCLUSIONS: A simple 13 C NMR analysis of the crude lipid fractions of BV-2 cells administered with [U-13 C]glucose informs DNL activity and the contribution of glucose to the acetyl-CoA precursors. While DNL was preserved in the presence of LPS, there was redirection of lipogenic acetyl-CoA sources from glucose to other substrates. Thus, in the present article, we describe a novel and simple 13 C NMR analysis approach to disclose the overall lipogenic activity and substrate contribution to DNL, suitable for evaluating DNL rates in cell cultures.

Chenodeoxycholic Acid Has Non-Thermogenic, Mitodynamic Anti-Obesity Effects in an In Vitro CRISPR/Cas9 Model of Bile Acid Receptor TGR5 Knockdown.

Bile acids (BA) have shown promising effects in animal models of obesity. However, the said effects are thought to rely on a thermogenic effect, which is questionably present in humans. A previous work has shown that the BA chenodeoxycholic acid (CDCA) can revert obesity and accelerate metabolism in animal and cell culture models. Thus, the aim of this study was to understand if this obesity reduction is indeed thermogenically-dependent. A CRISPR/Cas9 model of TGR5 (BA receptor) knockdown in 3T3-L1 adipocytes was developed to diminish thermogenic effects. Various parameters were assessed, including mitochondrial bioenergetics by Seahorse flux analysis, oxidative stress and membrane potential by fluorometry, intermediary metabolism by NMR, protein content assessment by Western Blot, gene expression by qPCR, and confocal microscopy evaluation of mitophagy. CDCA was still capable, for the most part, of reversing the harmful effects of cellular obesity, elevating mitophagy and leading to the reduction of harmed mitochondria within the cells, boosting mitochondrial activity, and thus energy consumption. In summary, CDCA has a non-thermogenic, obesity reducing capacity that hinges on a healthy mitochondrial population, explaining at least some of these effects and opening avenues of human treatment for metabolic diseases.

Caloric restriction alters the hormonal profile and testicular metabolome, resulting in alterations of sperm head morphology.

Energy homeostasis is crucial for all physiological processes. Thus, when there is low energy intake, negative health effects may arise, including in reproductive function. We propose to study whether caloric restriction (CR) changes testicular metabolic profile and ultimately sperm quality. Male Wistar rats (n = 12) were randomized into a CR group fed with 30% fewer calories than weight-matched, ad libitum-fed animals (control group). Circulating hormonal profile, testicular glucagon-like peptide-1 (GLP-1), ghrelin and leptin receptors expression, and sperm parameters were analyzed. Testicular metabolite abundance and glycolysis-related enzymes were studied by NMR and Western blot, respectively. Oxidative stress markers were analyzed in testicular tissue and spermatozoa. Expressions of mitochondrial complexes and mitochondrial biogenesis in testes were determined. CR induced changes in body weight along with altered GLP-1, ghrelin, and leptin circulating levels. In testes, CR led to changes in receptor expression that followed those of the hormone levels; modified testicular metabolome, particularly amino acid content; and decreased oxidative stress-induced damage in testis and spermatozoa, although sperm head defects increased. In sum, CR induced changes in body weight, altering circulating hormonal profile and testicular metabolome and increasing sperm head defects. Ultimately, our data highlight that conditions of CR may compromise male fertility.

Differences in the serum metabolome and lipidome identify potential biomarkers for seronegative rheumatoid arthritis versus psoriatic arthritis.

OBJECTIVES: The differential diagnosis of seronegative rheumatoid arthritis (negRA) and psoriasis arthritis (PsA) is often difficult due to the similarity of symptoms and the unavailability of reliable clinical markers. Since chronic inflammation induces major changes in the serum metabolome and lipidome, we tested whether differences in serum metabolites and lipids could aid in improving the differential diagnosis of these diseases. METHODS: Sera from negRA and PsA patients with established diagnosis were collected to build a biomarker-discovery cohort and a blinded validation cohort. Samples were analysed by proton nuclear magnetic resonance. Metabolite concentrations were calculated from the spectra and used to select the variables to build a multivariate diagnostic model. RESULTS: Univariate analysis demonstrated differences in serological concentrations of amino acids: alanine, threonine, leucine, phenylalanine and valine; organic compounds: acetate, creatine, lactate and choline; and lipid ratios L3/L1, L5/L1 and L6/L1, but yielded area under the curve (AUC) values lower than 70%, indicating poor specificity and sensitivity. A multivariate diagnostic model that included age, gender, the concentrations of alanine, succinate and creatine phosphate and the lipid ratios L2/L1, L5/L1 and L6/L1 improved the sensitivity and specificity of the diagnosis with an AUC of 84.5%. Using this biomarker model, 71% of patients from a blinded validation cohort were correctly classified. CONCLUSIONS: PsA and negRA have distinct serum metabolomic and lipidomic signatures that can be used as biomarkers to discriminate between them. After validation in larger multiethnic cohorts this diagnostic model may become a valuable tool for a definite diagnosis of negRA or PsA patients.

Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations.

Mitoxantrone (MTX) is used to treat several types of cancers and to improve neurological disability in multiple sclerosis. Unfortunately, cardiotoxicity is a severe and common adverse effect in MTX-treated patients. Herein, we aimed to study early and late mechanisms of MTX-induced cardiotoxicity using murine HL-1 cardiomyocytes. Cells were exposed to MTX (0.1, 1 or 10 µM) during short (2, 4, 6, or 12 h) or longer incubation periods (24 or 48 h). At earlier time points, (6 and 12 h) cytotoxicity was already observed for 1 and 10 µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12 h to MTX (pH gradients 4-7 and 6-11). Of note, the expression of the regulatory protein 14-3-3 protein epsilon was increased by a factor of two and three, after exposure to 1 and 10 µM MTX, respectively. At earlier time-points, 10 µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48 h), MTX-induced cytotoxicity was concentration and time-dependent, according to the MTT reduction and lactate dehydrogenase leakage assays, while caspase-9, -8 and -3 activities increased at 24 h. Regarding cellular redox status, total glutathione increased in 1 µM MTX (24 h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10 µM MTX, oxidized glutathione was significantly higher than control at 48 h. Moreover, MTX was able to significantly decrease proteasomal chymotrypsin-like activity in a concentration and time-independent manner. In summary, MTX significantly altered proteomic, energetic and oxidative stress homeostasis in cardiomyocytes at clinically relevant concentrations and our data clearly demonstrate that MTX causes early cardiotoxicity that needs further study.

Metabolic dynamics of human Sertoli cells are differentially modulated by physiological and pharmacological concentrations of GLP-1.

Obesity incidence has pandemic proportions and is expected to increase even further. Glucagon-like peptide-1 (GLP-1) based therapies are well-established pharmacological resources for obesity treatment. GLP-1 regulates energy and glucose homeostasis, which are also crucial for spermatogenesis. Herein, we studied the GLP-1 effects in human Sertoli cells (hSCs) metabolism and mitochondrial function. hSCs were cultured in absence or exposed to increasing doses of GLP-1 mimicking physiological post-prandial (0.01 nM) levels or equivalent to pharmacological levels (1 and 100 nM) used for obesity treatment. We identified GLP-1 receptor in hSCs. Consumption/production of extracellular metabolites were assessed, as well as protein levels or activities of glycolysis-related enzymes and transporters. Mitochondrial membrane potential and oxidative damage were evaluated. Glucose consumption decreased, while lactate production increased in hSCs exposed to 0.01 and 1 nM GLP-1. Though lactate dehydrogenase (LDH) protein decreased after exposure to 100 nM GLP-1 its activity increased in hSCs exposed to the same concentration of GLP-1. Mitochondrial membrane potential decreased in hSCs exposed to 100 nM of GLP-1, while formation of carbonyl groups was decreased in those cells. Those effects were followed by an increase in p-mammalian target of rapamycin (mTOR) Ser(2448). Overall, the lowest concentrations of GLP-1 increased the efficiency of glucose conversion to lactate, while GLP-1 concentration of 100 nM induces mTOR phosphorylation, decreases mitochondrial membrane potential and oxidative damage. GLP-1 regulates testicular energy homeostasis and pharmacological use of GLP-1 analogues could be valuable to counteract the negative impact of obesity in male reproductive function.

Response to dietary carbohydrates in European seabass (Dicentrarchus labrax) muscle tissue as revealed by NMR-based metabolomics.

INTRODUCTION: Feed optimization is a key step to the environmental and economic sustainability of aquaculture, especially for carnivorous species. Plant-derived ingredients can contribute to reduce costs and nitrogenous effluents while sparing wild fish stocks. However, the metabolic use of carbohydrates from vegetable sources by carnivorous fish is still not completely understood. OBJECTIVES: We aimed to study the effects of diets with carbohydrates of different digestibilities, gelatinized starch (DS) and raw starch (RS), in the muscle metabolome of European seabass (Dicentrarchus labrax). METHODS: We followed an NMR-metabolomics approach, using two sample preparation procedures, the intact muscle (HRMAS) and the aqueous muscle extracts (1H NMR), to compare the variations in muscle metabolome between the two diets. RESULTS: In muscle, multivariate analysis revealed similar metabolome shifts for DS and RS diets, when compared with the control diet. HRMAS of intact muscle, which included both hydrophobic and hydrophilic metabolites, showed increased lipid in DS-fed fish by univariate analysis. Regardless of the nature of the starch, increased glycine and phenylalanine, and decreased proline were observed when compared to the Ctr diet. Combined univariate analysis of intact muscle and aqueous extracts indicated specific diet related changes in lipid and amino acid metabolism, consistent with increased dietary carbohydrate supplementation. CONCLUSIONS: Due to differential sample processing, outputs differ in detail but provide complementary information. After tracing nutritional alterations by profiling fillet components, DS seems to be the most promising alternative to fishmeal-based diets in aquaculture. This approach should be reproducible for other farmed fish species and provide valuable information on nutritional and organoleptic properties of the final product.

The effects of the obesogen tributyltin on the metabolism of Sertoli cells cultured ex vivo.

Human exposure to environmental contaminants is widespread. Some of these contaminants have the ability to interfere with adipogenesis, being thus considered as obesogens. Recently, obesogens have been singled out as a cause of male infertility. Sertoli cells (SCs) are essential for male fertility and their metabolic performance, especially glucose metabolism, is under a tight endocrine control, being essential for the success of spermatogenesis. Herein, we studied the impact of the model obesogen tributyltin in the metabolic profile of SCs. For that, ex vivo-cultured rat SCs were exposed to increasing doses of tributyltin. SCs proliferation was evaluated by the sulforhodamine B assay and the maturation state of the cells was assessed by the expression of specific markers (inhibin B and the androgen receptor) by quantitative polymerase chain reaction. The metabolic profile of SCs was established by studying metabolites consumption/production by nuclear magnetic resonance spectroscopy and by analyzing the expression of key transporters and enzymes involved in glycolysis by Western blot. The proliferation of SCs was only affected in the cells exposed to the highest dose (1000 nM) of tributyltin. Notably, SCs exposed to 10 nM tributyltin decreased the consumption of glucose and pyruvate, as well as the production of lactate. The decreased lactate production hampers the development of germ cells. Intriguingly, the lowest levels of tributyltin were more prone to modulate the expression of key players of the glycolytic pathway. This is the first study showing that tributyltin reprograms glucose metabolism of SCs under ex vivo conditions, suggesting new targets and mechanisms through which obesogens modulate the metabolism of SCs and thus male (in)fertility.

Metabolic remodeling triggered by salivation and diabetes in major salivary glands.

The metabolic profile of major salivary glands was evaluated by 13 C nuclear magnetic resonance isotopomer analysis (13 C NMR-IA) following the infusion of [U-13 C]glucose in order to define the true metabolic character of submandibular (SM) and parotid (PA) glands at rest and during salivary stimulation, and to determine the metabolic remodeling driven by diabetes. In healthy conditions, the SM gland is characterized at rest by a glycolytic metabolic profile and extensive pyruvate cycling. On the contrary, the PA gland, although also dominated by glycolysis, also possesses significant Krebs' cycle activity and does not sustain extensive pyruvate cycling. Under stimulation, both glands increase their glycolytic and Krebs' cycle fluxes, but the metabolic coupling between the two pathways is further compromised to account for the much increased biosynthetic anaplerotic fluxes. In diabetes, the responsiveness of the PA gland to a salivary stimulus is seriously hindered, mostly as a result of the incapacity to burst glycolytic activity and also an inability to improve the Krebs' cycle flux to compensate. The Krebs' cycle activity in the SM gland is also consistently compromised, but the glycolytic flux in this gland is more resilient. This diabetes-induced metabolic remodeling in SM and PA salivary glands illustrates the metabolic need to sustain adequate saliva production, and identifies glycolytic and oxidative pathways as key players in the metabolic dynamism of salivary glands.

Effects of dietary carbohydrate on hepatic de novo lipogenesis in European seabass (Dicentrarchus labrax L.).

Farmed seabass have higher adiposity than their wild counterparts and this is often attributed to carbohydrate (CHO) feeding. Whether this reflects a reduction in fat oxidation, increased de novo lipogenesis (DNL), or both, is not known. To study the effects of high CHO diets on hepatic TG biosynthesis, hepatic TG deuterium ((2)H) enrichment was determined following 6 days in (2)H-enriched tank water for fish fed with a no-CHO control diet (CTRL), and diets with digestible starch (DS) and raw starch (RS). Hepatic fractional synthetic rates (FSRs, percent per day(-1)) were calculated for hepatic TG-glyceryl and FA moieties through (2)H NMR analysis. Glyceryl FSRs exceeded FA FSRs in all cases, indicating active cycling. DS fish did not show increased lipogenic potential compared to CTRL. RS fish had lower glyceryl FSRs compared with the other diets and negligible levels of FA FSRs despite similar hepatic TG levels to CTRL. DS-fed fish showed higher activity for enzymes that can provide NADPH for lipogenesis, relative to CTRL in the case of glucose-6-phosphate dehydrogenase (G6PDH) and relative to RS for both G6PDH and 6-phosphogluconate dehydrogenase. This approach indicated that elevated hepatic adiposity from DS feeding was not attributable to increased DNL.

Leptin modulates human Sertoli cells acetate production and glycolytic profile: a novel mechanism of obesity-induced male infertility?

Human feeding behavior and lifestyle are gradually being altered, favoring the development of metabolic diseases, particularly type 2 diabetes and obesity. Leptin is produced by the adipose tissue acting as a satiety signal. Its levels have been positively correlated with fat mass and hyperleptinemia has been proposed to negatively affect male reproductive function. Nevertheless, the molecular mechanisms by which this hormone affects male fertility remain unknown. Herein, we hypothesize that leptin acts on human Sertoli cells (hSCs), the "nurse cells" of spermatogenesis, altering their metabolism. To test our hypothesis, hSCs were cultured without or with leptin (5, 25 and 50ng/mL). Leptin receptor was identified by qPCR and Western blot. Protein levels of glucose transporters (GLUT1, GLUT2 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 (MCT4) were determined by Western Blot. LDH activity was assessed and metabolite production/consumption determined by proton nuclear magnetic resonance. Oxidative damage was evaluated by assessing lipid peroxidation, protein carbonilation and nitration. Our data shows that leptin receptor is expressed in hSCs. The concentration of leptin found in lean, healthy patients, upregulated GLUT2 protein levels and concentrations of leptin found in lean and obese patients increased LDH activity. Of note, all leptin concentrations decreased hSCs acetate production illustrating a novel mechanism for this hormone action. Moreover, our data shows that leptin does not induce or protect hSCs from oxidative damage. We report that this hormone modulates the nutritional support of spermatogenesis, illustrating a novel mechanism that may be linked to obesity-induced male infertility.

Glucose uptake and lipid metabolism are impaired in epicardial adipose tissue from heart failure patients with or without diabetes.

Type 2 diabetes mellitus is a complex metabolic disease, and cardiovascular disease is a leading complication of diabetes. Epicardial adipose tissue surrounding the heart displays biochemical, thermogenic, and cardioprotective properties. However, the metabolic cross-talk between epicardial fat and the myocardium is largely unknown. This study sought to understand epicardial adipose tissue metabolism from heart failure patients with or without diabetes. We aimed to unravel possible differences in glucose and lipid metabolism between human epicardial and subcutaneous adipocytes and elucidate the potential underlying mechanisms involved in heart failure. Insulin-stimulated [(14)C]glucose uptake and isoproterenol-stimulated lipolysis were measured in isolated epicardial and subcutaneous adipocytes. The expression of genes involved in glucose and lipid metabolism was analyzed by reverse transcription-polymerase chain reaction in adipocytes. In addition, epicardial and subcutaneous fatty acid composition was analyzed by high-resolution proton nuclear magnetic resonance spectroscopy. The difference between basal and insulin conditions in glucose uptake was significantly decreased (P= 0.006) in epicardial compared with subcutaneous adipocytes. Moreover, a significant (P< 0.001) decrease in the isoproterenol-stimulated lipolysis was also observed when the two fat depots were compared, and it was strongly correlated with lipolysis, lipid storage, and inflammation-related gene expression. Moreover, the fatty acid composition of these tissues was significantly altered by diabetes. These results emphasize potential metabolic differences between both fat depots in the presence of heart failure and highlight epicardial fat as a possible therapeutic target in situ in the cardiac microenvironment.

Adenosine A1 receptors control the metabolic recovery after hypoxia in rat hippocampal slices.

Adenosine is a neuromodulator that protects neurons from hypoxia. This effect is attributed to the ability of adenosine A1 receptors (A1 R) to inhibit excitatory synaptic transmission. However, A1 R activation also protects non-brain tissues from hypoxic insults by controlling metabolism. Thus, we now tested the hypothesis that A1 R-mediated neuroprotection after a hypoxic insult in superfused hippocampal slices also involves the control of neuronal and astrocytic metabolism. A 90-min hypoxia insult increased lactate, alanine, and pyruvate levels and decreased energy charge (EC), phosphocreatine/creatine ratio, and glutamine content. These metabolic modifications were fully recovered after reoxygenation for 3 h. The presence of the A1 R-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine stimulated glycolysis, prevented the hypoxia-induced decrease of EC, and increased the levels of GABA. A1 R blockade further blunted the recovery of metabolism on reoxygenation after hypoxia, as typified by a sustained decreased EC and an increased mitochondrial metabolism, as confirmed by a greater [U-(13) C]glucose oxidation through the tricarboxylic acid cycle. These results demonstrate that A1 R blockade prevents the recovery of hypoxia-induced metabolic alterations during reoxygenation, which indicates that the ability of A1 R to control primary metabolism in the brain tissue may be a hitherto unrecognized mechanism of A1 R-mediated neuroprotection. This study demonstrates that tonic activation of adenosine A1 receptors (A1 R) plays an important role in the reoxygenation recovery of the metabolic alterations caused by transient hypoxia in rat hippocampal slices. This ability of A1 R to inhibit neuronal metabolism may be a key mechanism by which adenosine affords neuroprotection upon acute hypoxia, thus preventing the long-term impairment of neuronal circuits.

Combined dual effect of modulation of human neutrophils' oxidative burst and inhibition of colon cancer cells proliferation by hydroxycinnamic acid derivatives.

Colon cancer is one of the most incident cancers in the Western World. While both genetic and epigenetic factors may contribute to the development of colon cancer, it is known that chronic inflammation associated to excessive production of reactive oxygen and nitrogen species by phagocytes may ultimately initiate the multistep process of colon cancer development. Phenolic compounds, which reveal antioxidant and antiproliferative activities in colon cancer cells, can be a good approach to surpass this problem. In this work, hydroxycinnamic amides and the respective acid precursors were tested in vitro for their capacity to modulate human neutrophils' oxidative burst and simultaneously to inhibit growth of colon cancer cells. A phenolic amide derivative, caffeic acid hexylamide (CAHA) (4) was found to be the most active compound in both assays, inhibiting human neutrophils' oxidative burst, restraining the inflammatory process, inhibiting growth of colon cancer cells and triggering mitochondrial dysfunction that leads cancer cells to apoptosis. Altogether, these achievements can contribute to the understanding of the relationship between antioxidant and anticancer activities and based on the structure-activity relationships (SAR) established can be the starting point to find more effective phenolic compounds as anticancer agents.

Exploring new chemical functionalities to improve aromatase inhibition of steroids.

In this work, new potent steroidal aromatase inhibitors both in microsomes and in breast cancer cells have been found. The synthesis of the 3,4-(ethylenedioxy)androsta-3,5-dien-17-one (12), a new steroid containing a heterocycle dioxene fused in the A-ring, led to the discovery of a new reaction for which a mechanism is proposed. New structure-activity relationships were established. Some 5ß-steroids, such as compound 4ß,5ß-epoxyandrostan-17-one (9), showed aromatase inhibitory activity, because they adopt a similar A-ring conformation as those of androstenedione, the natural substrate of aromatase. Moreover, new chemical features to increase planarity were disclosed, specifically the 3α,4α-cyclopropane ring, as in 3α,4α-methylen-5α-androstan-17-one (5) (IC50=0.11µM), and the Δ(9-11) double bond in the C-ring, as in androsta-4,9(11)-diene-3,17-dione (13) (IC50=0.25µM). In addition, induced-fit docking (IFD) simulations and site of metabolism (SoM) predictions helped to explain the recognition of new potent steroidal aromatase inhibitors within the enzyme. These insights can be valuable tools for the understanding of the molecular recognition process by the aromatase and for the future design of new steroidal inhibitors.

Metabolic fingerprints in testicular biopsies from type 1 diabetic patients.

Diabetes mellitus (DM) is a metabolic disease that has grown to pandemic proportions. Recent reports have highlighted the effect of DM on male reproductive function. Here, we hypothesize that testicular metabolism is altered in type 1 diabetic (T1D) men seeking fertility treatment. We propose to determine some metabolic fingerprints in testicular biopsies of diabetic patients. For that, testicular tissue from five normal and five type 1 diabetic men was analyzed by high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. mRNA and protein expression of glucose transporters and glycolysis-related enzymes were also evaluated. Our results show that testes from diabetic men presented decreased levels of lactate, alanine, citrate and creatine. The mRNA levels of glucose transporter 1 (GLUT1) and phosphofructokinase 1 (PFK1) were decreased in testes from diabetic men but only GLUT3 presented decreased mRNA and protein levels. Lactate dehydrogenase (LDH) and glutamate pyruvate transaminase (GPT) protein levels were also found to be decreased in testes from diabetic men. Overall, our results show that T1D alters glycolysis-related transporters and enzymes, compromising lactate content in the testes. Moreover, testicular creatine content was severely depressed in T1D men. Since lactate and creatine are essential for germ cells development and support, the data discussed here open new insights into the molecular mechanism by which DM promotes subfertility/infertility in human males.

Metabolic evaluations of cancer metabolism by NMR-based stable isotope tracer methodologies.

BACKGROUND: Cancer cells are widely recognized for being able to adapt their metabolism towards converting available nutrients into biomass to increase proliferation rates. MATERIALS AND METHODS: We will review a series of nuclear magnetic resonance (NMR)-based stable isotope tracer methodologies for probing cancer metabolism. RESULTS: The monitoring of such adaptations is of the utmost importance to unravel cancer metabolism and tumour growth. Several major metabolic targets have been recognized as promising foci and have been addressed by multiple studies in recent years. In this work are presented strategies to quantify glycolysis, pentose phosphate pathway, Krebs cycle turnover and de novo lipogenesis by NMR isotopomer analysis. CONCLUSIONS: Being able to adequately define the interplay between metabolic pathways allows the monitoring of their prevalence in tissues and such information is critical for an accurate knowledge of the metabolic distinctive nature of tumours towards devising more efficient antitumorigenic strategies. Discussed methodologies are currently available in the literature, but to date, no single review has compiled all their possible uses, particularly in an interdependent perspective.