Publications by authors named "Rui A Carvalho"

84 Publications

Blueberry Consumption Challenges Hepatic Mitochondrial Bioenergetics and Elicits Transcriptomics Reprogramming in Healthy Wistar Rats.

Pharmaceutics 2020 Nov 14;12(11). Epub 2020 Nov 14.

Institute of Pharmacology & Experimental Therapeutics & Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal.

An emergent trend of blueberries' (BB) "prophylactic" consumption, due to their phytochemicals' richness and well-known health-promoting claims, is widely scaled-up. However, the benefits arising from BB indiscriminate intake remains puzzling based on incongruent preclinical and human data. To provide a more in-depth elucidation and support towards a healthier and safer consumption, we conducted a translation-minded experimental study in healthy Wistar rats that consumed BB in a juice form (25 g/kg body weight (BW)/day; 14 weeks' protocol). Particular attention was paid to the physiological adaptations succeeding in the gut and liver tissues regarding the acknowledged BB-induced metabolic benefits. Systemically, BB boosted serum antioxidant activity and repressed the circulating levels of 3-hydroxybutyrate (3-HB) ketone bodies and 3-HB/acetoacetate ratio. Moreover, BB elicited increased fecal succinic acid levels without major changes on gut microbiota (GM) composition and gut ultra-structural organization. Remarkably, an accentuated hepatic mitochondrial bioenergetic challenge, ensuing metabolic transcriptomic reprogramming along with a concerted anti-inflammatory pre-conditioning, was clearly detected upon long-term consumption of BB phytochemicals. Altogether, the results disclosed herein portray a quiescent mitochondrial-related metabolomics and hint for a unified adaptive response to this nutritional challenge. The beneficial or noxious consequences arising from this dietary trend should be carefully interpreted and necessarily claims future research.
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http://dx.doi.org/10.3390/pharmaceutics12111094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697217PMC
November 2020

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

Arch Toxicol 2020 12 7;94(12):4067-4084. Epub 2020 Sep 7.

UCIBIO/REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.

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.
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http://dx.doi.org/10.1007/s00204-020-02874-4DOI Listing
December 2020

Effect of Increased Lactate Dehydrogenase A Activity and Aerobic Glycolysis on the Proinflammatory Profile of Autoimmune CD8+ T Cells in Rheumatoid Arthritis.

Arthritis Rheumatol 2020 12 8;72(12):2050-2064. Epub 2020 Nov 8.

University Hospital Heidelberg, Heidelberg, Germany, and Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal.

Objective: CD8+ T cells contribute to rheumatoid arthritis (RA) by releasing proinflammatory and cytolytic mediators, even in a challenging hypoxic and nutrient-poor microenvironment such as the synovial membrane. This study was undertaken to explore the mechanisms through which CD8+ T cells meet their metabolic demands in the blood and synovial membrane of patients with RA.

Methods: Purified blood CD8+ T cells from patients with RA, patients with psoriatic arthritis (PsA), and patients with spondyloarthritis (SpA), as well as healthy control subjects, and CD8+ T cells from RA synovial membrane were stimulated in medium containing C-labeled metabolic substrates in the presence or absence of metabolic inhibitors, under conditions of normoxia or hypoxia. The production of metabolic intermediates was quantified by H-nuclear magnetic resonance. The expression of metabolic enzymes, transcription factors, and immune effector molecules was assessed at both the messenger RNA (mRNA) and protein levels. CD8+ T cell functional studies were performed.

Results: RA blood CD8+ T cells met their metabolic demands through aerobic glycolysis, production of uniformly C-enriched lactate in the RA blood (2.6 to 3.7-fold higher than in patients with SpA, patients with PsA, and healthy controls; P < 0.01), and induction of glutaminolysis. Overexpression of Warburg effect-linked enzymes in all RA CD8+ T cell subsets maintained this metabolic profile, conferring to the cells the capacity to proliferate under hypoxia and low-glucose conditions. In all RA CD8+ T cell subsets, lactate dehydrogenase A (LDHA) was overexpressed at the mRNA level (P < 0.03 versus controls; n = 6 per group) and protein level (P < 0.05 versus controls; n = 17 RA patients, n = 9 controls). In RA blood, inhibition of LDHA with FX11 led to reductions in lipogenesis, migration and proliferation of CD8+ T cells, and CD8+ T cell effector functions, while production of reactive oxygen species was increased by 1.5-fold (P < 0.03 versus controls). Following inhibition of LDHA with FX11, RA CD8+ T cells lost their capacity to induce healthy B cells to develop a proinflammatory phenotype. Similar metabolic alterations were observed in RA CD8+ T cells from the synovial membrane.

Conclusion: Remodeling glucose and glutamine metabolism in RA CD8+ T cells by targeting LDHA activity can reduce the deleterious inflammatory and cytolytic contributions of these cells to the development of autoimmunity.
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http://dx.doi.org/10.1002/art.41420DOI Listing
December 2020

Different Malabsorptive Obesity Surgery Interventions Result in Distinct Postprandial Amino Acid Metabolomic Signatures.

Obes Surg 2020 Oct 20;30(10):4019-4028. Epub 2020 Jun 20.

Endocrine, Cardiovascular & Metabolic Research, Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Porto, Portugal.

Purpose: Biliopancreatic diversion with duodenal switch (BPD-DS) is an effective weight loss surgical procedure. Yet, BPD-DS is technically difficult to perform and carries a higher risk of nutrient deficiencies as compared with other surgical interventions. Single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) is a modified BPD-DS procedure conceived with the aim of decreasing the technical complexity, while retaining the weight loss efficiency. Whether the two surgical procedures diverge in nutrient absorption rates and malnutrition risk is still matter of debate. Our aim was to determine if postprandial nutrient absorption rates are different in patients subjected to BPD-DS or SADI-S for weight loss.

Materials And Methods: Plasma amino acid metabolomic profiling during mixed-meal tolerance test (MMTT) was performed in subjects (N = 18) submitted to BPD-DS (n = 9) or SADI-S (n = 9) 1.6 ± 0.1 years earlier.

Results: Patients submitted to SADI-S or BPD-DS presented distinct postprandial metabolomic profiles. Postprandial excursions of total and essential amino acids-leucine, isoleucine, and valine-were higher after SADI-S as compared with BPD-DS.

Conclusion: Our study demonstrates that a simplified malabsorptive bariatric surgery procedure SADI-S results in greater essential branched-chain amino acid absorption when compared with the classical BPD-DS intervention. These findings suggest that SADI-S can potentially lower lifetime risk of postoperative protein malnutrition, as well as have a positive impact on systemic metabolism and glucose homeostasis.
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http://dx.doi.org/10.1007/s11695-020-04774-zDOI Listing
October 2020

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

Ann Rheum Dis 2020 04 20;79(4):499-506. Epub 2020 Feb 20.

Medizin 5, Hämatologie, Onkologie und Rheumatologie, UniversitätsKlinikum Heidelberg, Heidelberg, Germany.

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.
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http://dx.doi.org/10.1136/annrheumdis-2019-216374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147174PMC
April 2020

Gastric Bypass with Different Biliopancreatic Limb Lengths Results in Similar Post-absorptive Metabolomics Profiles.

Obes Surg 2020 03;30(3):1068-1078

Endocrine, Cardiovascular & Metabolic Research, Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Porto, Portugal.

Background/aim: Roux-en-Y gastric bypass (RYGB) with a long biliopancreatic limb (BPL) was demonstrated to further improve type 2 diabetes (T2D) outcomes. Whether benefits occur at the cost of a negative impact on nutrient absorption is a matter of debate. Our aim was to evaluate the impact of RYGB BPL length on short-term nutrient absorption.

Methods: Subjects (N = 20) submitted to RYGB with a 2 m BPL (n = 11) or standard BPL (60-100 cm) (n = 9) 4.2 ± 0.4 years earlier underwent a mixed meal tolerance test. Plasma metabolites were analyzed at baseline and after meal by nuclear magnetic resonance (NMR) spectroscopy. Spectra were subject to multivariate analysis (MVA). Partial least square discriminant analysis (PLS-DA) was used to identify metabolites responsible for group discrimination.

Results: Principal component analysis and PLS-DA showed a clear separation between plasma metabolites before and 30 min after meal intake in both groups. The metabolites responsible for differences between time points were glucose and branched-chain amino acids. A complete overlap in metabolite species and concentrations was observed at 0 and 30 min time points for both groups, while acetate levels 120 min after the meal intake were significantly higher in subjects submitted to RYGB with a 2-m-long BPL as compared to the group submitted to the standard RYGB procedure.

Conclusions: Post-prandial plasma metabolomics profiles suggest that a 2-m-long BLP RYGB does not have a negative impact on acute metabolite absorption. RYGB BPL length seems to influence post-prandial acetate levels, which could contribute to the additional positive metabolic outcomes.
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http://dx.doi.org/10.1007/s11695-019-04294-5DOI Listing
March 2020

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

Am J Physiol Endocrinol Metab 2020 01 26;318(1):E33-E43. Epub 2019 Nov 26.

Department of Microscopy, Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal.

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 ( = 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.
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http://dx.doi.org/10.1152/ajpendo.00355.2019DOI Listing
January 2020

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

Metabolomics 2018 07 4;14(7):95. Epub 2018 Jul 4.

CFE - Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, 3000-456, Coimbra, Portugal.

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 (H 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.
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http://dx.doi.org/10.1007/s11306-018-1390-4DOI Listing
July 2018

Impact of Caffeine Consumption on Type 2 Diabetes-Induced Spatial Memory Impairment and Neurochemical Alterations in the Hippocampus.

Front Neurosci 2018 9;12:1015. Epub 2019 Jan 9.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Diabetes affects the morphology and plasticity of the hippocampus, and leads to learning and memory deficits. Caffeine has been proposed to prevent memory impairment upon multiple chronic disorders with neurological involvement. We tested whether long-term caffeine consumption prevents type 2 diabetes (T2D)-induced spatial memory impairment and hippocampal alterations, including synaptic degeneration, astrogliosis, and metabolic modifications. Control Wistar rats and Goto-Kakizaki (GK) rats that develop T2D were treated with caffeine (1 g/L in drinking water) for 4 months. Spatial memory was evaluated in a Y-maze. Hippocampal metabolic profile and glucose homeostasis were investigated by H magnetic resonance spectroscopy. The density of neuronal, synaptic, and glial-specific markers was evaluated by Western blot analysis. GK rats displayed reduced Y-maze spontaneous alternation and a lower amplitude of hippocampal long-term potentiation when compared to controls, suggesting impaired hippocampal-dependent spatial memory. Diabetes did not impact the relation of hippocampal to plasma glucose concentrations, but altered the neurochemical profile of the hippocampus, such as increased in levels of the osmolites taurine ( < 0.001) and -inositol ( < 0.05). The diabetic hippocampus showed decreased density of the presynaptic proteins synaptophysin ( < 0.05) and SNAP25 ( < 0.05), suggesting synaptic degeneration, and increased GFAP ( < 0.001) and vimentin ( < 0.05) immunoreactivities that are indicative of astrogliosis. The effects of caffeine intake on hippocampal metabolism added to those of T2D, namely reducing -inositol levels ( < 0.001) and further increasing taurine levels ( < 0.05). Caffeine prevented T2D-induced alterations of GFAP, vimentin and SNAP25, and improved memory deficits. We conclude that caffeine consumption has beneficial effects counteracting alterations in the hippocampus of GK rats, leading to the improvement of T2D-associated memory impairment.
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http://dx.doi.org/10.3389/fnins.2018.01015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333904PMC
January 2019

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

Toxicol Appl Pharmacol 2019 01 6;362:1-8. Epub 2018 Oct 6.

Department of Microscopy, Laboratory of Cell Biology, Abel Salazar Institute of Biomedical Sciences (ICBAS), University of Porto, Porto, Portugal; Unit for Multidisciplinary Research in Biomedicine, Abel Salazar Institute of Biomedical Sciences (UMIB-ICBAS), University of Porto, Porto, Portugal. Electronic address:

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.
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http://dx.doi.org/10.1016/j.taap.2018.10.009DOI Listing
January 2019

Senescence and declining reproductive potential: Insight into molecular mechanisms through testicular metabolomics.

Biochim Biophys Acta Mol Basis Dis 2018 10 29;1864(10):3388-3396. Epub 2018 Jul 29.

Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Centre for Reproductive Genetics Prof. Alberto Barros, Porto, Portugal. Electronic address:

Aging is associated with structural and functional changes in the organism that result in the declining of its functioning. Postponed parenthood has renewed the interest in age-related decline of testicular function and male fertility. Still, little is known about the molecular mechanisms associated with testicular senescence and related decline of fertility. Here we sought to elucidate the molecular basis of metabolic changes associated with testicular aging and reproductive potential using an NMR-based metabolomics approach. Testicular metabolic profiles of rats from 3 to 24 months-of-age were analysed. An age-associated decrease in most antioxidant metabolites, like betaine, creatine and glutathione was observed. Amino acid content changed as early as 6 months-of-age, with an increase in branched chain and aromatic amino acids, accompanied by decrease of nucleotide synthesis (IMP, CMP, ATP). Testicular content of phospholipid precursors (choline, ethanolamine, myo-inositol, glycerol) increased with advanced age and was accompanied by a decrease in the levels of their phosphorylated products, suggesting compromised spermatogenesis. This is the first metabolomics study of testicular tissue of aged rats and we were able to identify metabolites associated with reproductive maturity from the onset to senescence. Our results provide evidence for an influence of aging on global testicular metabolome, as early as 6 months-of-age, with a profound alteration of several key metabolic pathways associated with the male reproductive potential.
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http://dx.doi.org/10.1016/j.bbadis.2018.07.028DOI Listing
October 2018

Methyl-β-cyclodextrin Inclusion Complex with β-Caryophyllene: Preparation, Characterization, and Improvement of Pharmacological Activities.

ACS Omega 2017 Dec 18;2(12):9080-9094. Epub 2017 Dec 18.

Department of Chemistry, CQC, Department of Biochemistry, NMR Spectroscopy Center, Faculty of Sciences and Technology, and REQUIMTE/LAQV, Group of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal.

β-Caryophyllene (BCP) is a sesquiterpene that shows high potential in pharmacological applications. However, these have been drastically limited by the respective volatility and poor water solubility. The present study investigates the formation of inclusion complexes between BCP and methyl-β-cyclodextrin (MβCD) and shows that these complexes promote a significant improvement of the anti-inflammatory, gastric protection, and antioxidant activities relative to neat BCP. It is shown that the solubility of BCP is significantly increased through complexation in phase solubility studies. Inclusion complexes with MβCD in solid state were prepared by three different methods, kneading, rotary evaporation, and lyophilization, with the latter confirmed by differential scanning calorimetry, Fourier transformed infrared spectroscopy, scanning electron microscopy, H NMR spectroscopy, and molecular dynamics studies. This study provides for the first time a full characterization of inclusion complexes between BCP and MβCD and highlights the impact of complex formation upon pharmacological activity.
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http://dx.doi.org/10.1021/acsomega.7b01438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044968PMC
December 2017

Reactive Oxygen Species Deficiency Due to Ncf1-Mutation Leads to Development of Adenocarcinoma and Metabolomic and Lipidomic Remodeling in a New Mouse Model of Dextran Sulfate Sodium-Induced Colitis.

Front Immunol 2018 14;9:701. Epub 2018 May 14.

Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

Inflammatory bowel disease is characterized by chronic relapsing idiopathic inflammation of the gastrointestinal tract and persistent inflammation. Studies focusing on the immune-regulatory function of reactive oxygen species (ROS) are still largely missing. In this study, we analyzed an ROS-deficient mouse model leading to colon adenocarcinoma. Colitis was induced with dextran sulfate sodium (DSS) supplied the drinking water in wild-type (WT) and Ncf1-mutant (Ncf1) B10.Q mice using two different protocols, one mimicking recovery after acute colitis and another simulating chronic colitis. Disease progression was monitored by evaluation of clinical parameters, histopathological analysis, and the blood serum metabolome using H nuclear magnetic resonance spectroscopy. At each experimental time point, colons and spleens from some mice were removed for histopathological analysis and internal clinical parameters. Clinical scores for weight variation, stool consistency, colorectal bleeding, colon length, and spleen weight were significantly worse for Ncf1 than for WT mice. Ncf1 mice with only a 7-day exposure to DSS followed by a 14-day resting period developed colonic distal high-grade dysplasia in contrast to the low-grade dysplasia found in the colon of WT mice. After a 21-day resting period, there was still β-catenin-rich inflammatory infiltration in the Ncf1 mice together with high-grade dysplasia and invasive well-differentiated adenocarcinoma, while in the WT mice, high-grade dysplasia was prominent without malignant invasion and only low inflammation. Although exposure to DSS generated less severe histopathological changes in the WT group, the blood serum metabolome revealed an increased fatty acid content with moderate-to-strong correlations to inflammation score, weight variation, colon length, and spleen weight. Ncf1 mice also displayed a similar pattern but with lower coefficients and showed consistently lower glucose and/or higher lactate levels which correlated with inflammation score, weight variation, and spleen weight. In our novel, DSS-induced colitis animal model, the lack of an oxidative burst ROS was sufficient to develop adenocarcinoma, and display altered blood plasma metabolic and lipid profiles. Thus, oxidative burst seems to be necessary to prevent evolution toward cancer and may confer a protective role in a ROS-mediated self-control mechanism.
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http://dx.doi.org/10.3389/fimmu.2018.00701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960697PMC
June 2019

Mitochondrial Bioenergetics by C-NMR Isotopomer Analysis.

Methods Mol Biol 2018 ;1782:229-247

Centre for Functional Ecology, University of Coimbra, Coimbra, Portugal.

Metabolic reprogramming has been associated to a plethora of diseases, and there has been increased demand for methodologies able to determine the metabolic alterations that characterize the pathological states and help developing metabolically centered therapies. In this chapter, methodologies for monitoring TCA cycle turnover and its interaction with pyruvate cycling and anaplerotic reactions will be presented. These methodologies are based in the application of stable C isotope "tracers"/substrates and C-NMR isotopomer analysis of metabolic intermediates. These methodologies can be applied at several organizational levels, ranging from isolated organelles and organs to whole organisms/humans. For the sake of simplicity, only very simple and well-defined models will be presented, including isolated heart mitochondria and isolated perfused hearts and livers.
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http://dx.doi.org/10.1007/978-1-4939-7831-1_13DOI Listing
February 2019

Assessing Sertoli Cell Metabolic Activity.

Methods Mol Biol 2018 ;1748:157-171

Unit for Multidisciplinary Research in Biomedicine (UMIB), Laboratory of Cell Biology, Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal.

Nuclear magnetic resonance (NMR)-based metabolomics is widely used in the research of metabolic conditions of complex biological systems under various conditions, and its use has been found in the field of male fertility. Here we describe the implementation of total and targeted NMR-based metabolomics in the research on Sertoli cell metabolism. Main principles and techniques of cell medium, cellular extracts, and intact cells are explained, as well as some classical experiments that can give complementary information on the Sertoli cell metabolism.
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http://dx.doi.org/10.1007/978-1-4939-7698-0_12DOI Listing
February 2019

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

Arch Toxicol 2018 Feb 9;92(2):601-610. Epub 2017 Oct 9.

Health Sciences Research Centre (CICS-UBI), University of Beira Interior, Rua Marquês d'Ávila e Bolama, Av. Infante D. Henrique, 6200-001, Covilhã, Portugal.

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.
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http://dx.doi.org/10.1007/s00204-017-2091-xDOI Listing
February 2018

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

NMR Biomed 2017 Feb 26;30(2). Epub 2016 Dec 26.

Centre for Functional Ecology, University of Coimbra, Portugal.

The metabolic profile of major salivary glands was evaluated by C nuclear magnetic resonance isotopomer analysis ( C NMR-IA) following the infusion of [U- 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.
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http://dx.doi.org/10.1002/nbm.3683DOI Listing
February 2017

Methamphetamine Induces Anhedonic-Like Behavior and Impairs Frontal Cortical Energetics in Mice.

CNS Neurosci Ther 2017 Feb 19;23(2):119-126. Epub 2016 Oct 19.

Laboratory of Pharmacology and Experimental Therapeutics/Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Introduction: We recently showed that a single high dose of methamphetamine (METH) induces a persistent frontal cortical monoamine depletion that is accompanied by helpless-like behavior in mice. However, brain metabolic alterations underlying both neurochemical and mood alterations remain unknown.

Aims: Herein, we aimed at characterizing frontal cortical metabolic alterations associated with early negative mood behavior triggered by METH. Adult C57BL/6 mice were injected with METH (30 mg/kg, i.p.), and their frontal cortical metabolic status was characterized after probing their mood and anxiety-related phenotypes 3 days postinjection.

Results: Methamphetamine induced depressive-like behavior, as indicated by the decreased grooming time in the splash test and by a transient decrease in sucrose preference. At this time, METH did not alter anxiety-like behavior or motor functions. Depolarization-induced glucose uptake was reduced in frontocortical slices from METH-treated mice compared to controls. Consistently, astrocytic glucose transporter (GluT1) density was lower in the METH group. A proton high rotation magic angle spinning (HRMAS) spectroscopic approach revealed that METH induced a significant decrease in N-acetyl aspartate (NAA) and glutamate levels, suggesting that METH decreased neuronal glutamatergic function in frontal cortex.

Conclusions: We report, for the first time, that a single METH injection triggers early self-care and hedonic deficits and impairs frontal cortical energetics in mice.
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http://dx.doi.org/10.1111/cns.12649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492743PMC
February 2017

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

Bioorg Med Chem 2016 08 30;24(16):3556-64. Epub 2016 May 30.

Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal; CNC.IBILI, University of Coimbra, Portugal. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmc.2016.05.065DOI Listing
August 2016

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

J Lipid Res 2016 07 31;57(7):1264-72. Epub 2016 May 31.

Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.

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.
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http://dx.doi.org/10.1194/jlr.M067850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918855PMC
July 2016

Exploring new chemical functionalities to improve aromatase inhibition of steroids.

Bioorg Med Chem 2016 06 26;24(12):2823-31. Epub 2016 Apr 26.

Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal; CNC.IBILI, University of Coimbra, Portugal. Electronic address:

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 5β-steroids, such as compound 4β,5β-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.
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http://dx.doi.org/10.1016/j.bmc.2016.04.056DOI Listing
June 2016

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

Am J Physiol Endocrinol Metab 2016 Apr 26;310(7):E550-64. Epub 2016 Jan 26.

Center of Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; Portuguese Diabetes Association, Lisbon, Portugal; Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas; and Arkansas Children's Hospital Research Institute, Little Rock, Arkansas.

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.
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http://dx.doi.org/10.1152/ajpendo.00384.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824138PMC
April 2016

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

J Neurochem 2016 Mar 13;136(5):947-57. Epub 2016 Jan 13.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

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.
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http://dx.doi.org/10.1111/jnc.13512DOI Listing
March 2016

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

Biochim Biophys Acta 2015 Sep 10;1852(9):1824-32. Epub 2015 Jun 10.

Health Sciences Research Centre (CICS-UBI), University of Beira Interior, Covilhã, Portugal; Department of Life Sciences, Faculty of Sciences and Technology and Center for Neurosciences and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbadis.2015.06.005DOI Listing
September 2015

Metabolic fingerprints in testicular biopsies from type 1 diabetic patients.

Cell Tissue Res 2015 Nov 9;362(2):431-40. Epub 2015 Jun 9.

CICS - UBI - Health Sciences Research Centre, University of Beira Interior, 6201-506, Covilhã, Portugal.

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.
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http://dx.doi.org/10.1007/s00441-015-2217-5DOI Listing
November 2015

Do Cyclodextrins Aggregate in Water? Insights from NMR Experiments.

Langmuir 2015 Jun 5;31(23):6314-20. Epub 2015 Jun 5.

§Division of Physical Chemistry, Department of Chemistry, Lund University, PO Box 124, S-221 00 Lund, Sweden.

One decade ago Bonini et al. [Langmuir 2006, 22, 1478-1484] reported the occurrence of aggregates of β-cyclodextrin in aqueous solutions with sizes in the range from 90 nm to a few micrometers. The experimental technique used was cryo-TEM. This work followed a number of previous studies involving other physical parameters, such as viscosities and activity coefficients, the results of which were interpreted in terms of self-aggregation of cyclodextrins. Since then, the ability of cyclodextrins to self-assemble were often used to explain and rationalize the supramolecular mechanisms involving cyclodextrins. Here, the question of aggregation of native cyclodextrins (α-, β-, and γ-) in aqueous solutions is addressed by using (1)H NMR techniques, including NMR diffusometry, relaxometry, and proton peak intensities. Within the detection limit of the NMR experiments, no aggregates of cyclodextrin were observed. If aggregates are present, the fraction of cyclodextrin in aggregates is quite small-less than 1%. However, we cannot exclude the presence of transient clusters involving several cyclodextrin molecules where the lifetime of the cluster is short.
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http://dx.doi.org/10.1021/acs.langmuir.5b01493DOI Listing
June 2015

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

Eur J Clin Invest 2015 Jan;45 Suppl 1:37-43

Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal; Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

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.
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http://dx.doi.org/10.1111/eci.12358DOI Listing
January 2015

Exemestane metabolites: Synthesis, stereochemical elucidation, biochemical activity and anti-proliferative effects in a hormone-dependent breast cancer cell line.

Eur J Med Chem 2014 Nov 28;87:336-45. Epub 2014 Sep 28.

Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto, Portugal; Institute for Molecular and Cell Biology (IBMC), University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Electronic address:

Exemestane is a third-generation steroidal aromatase inhibitor that has been used in clinic for hormone-dependent breast cancer treatment in post-menopausal women. It is known that exemestane undergoes a complex metabolization, giving rise to some already identified metabolites, the 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one (17-βHE) and the 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione (6-HME). In this study, four metabolites of exemestane have been analyzed, three of them were synthesized (6β-spirooxiranandrosta-1,4-diene-3,17-dione (2), 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (3) and 17-βHE (4)) while one was acquired, the 6-HME (6). The stereochemistry of the epoxide group of 2 and 3 has been unequivocally elucidated for the first time on the basis of NOESY experiments. New structure-activity relationships (SAR) have been established through the observation that the substitution of the double bonds by epoxide groups led to less potent derivatives in microsomes. However, the reduction of the C-17 carbonyl group to a hydroxyl group originating 17-βHE (4) resulted in a significant increase in activity in MCF-7aro cells when compared to exemestane (IC50 0.25 μM vs 0.90 μM, respectively). All the studied metabolites reduced MCF-7aro cells viability in a dose and time-dependent manner, and metabolite 3 was the most potent one. Altogether our results showed that not only exemestane but also its main metabolites are potent aromatase inhibitors and reduce breast cancer cells viability. This suggests that exemestane efficacy may also be due to the active metabolites that result from its metabolic transformation. Our results emphasize the importance of performing further studies to expand our understanding of exemestane actions in breast cancer cells.
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http://dx.doi.org/10.1016/j.ejmech.2014.09.074DOI Listing
November 2014

Metabolic effects of hypoxia in colorectal cancer by 13C NMR isotopomer analysis.

Biomed Res Int 2014 1;2014:759791. Epub 2014 Jul 1.

Biophysics Unit, IBILI, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal ; Centre of Investigation on Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, 3001-301 Coimbra, Portugal.

(13)C NMR isotopomer analysis was used to characterize intermediary metabolism in three colorectal cancer cell lines (WiDr, LS1034, and C2BBe1) and determine the "metabolic remodeling" that occurs under hypoxia. Under normoxia, the three colorectal cancer cell lines present high rates of lactate production and can be seen as "Warburg" like cancer cells independently of substrate availability, since such profile was dominant at both high and low glucose media contents. The LS1034 was the less glycolytic of the three cell lines and was the most affected by the event of hypoxia, raising abruptly glucose consumption and lactate production. The other two colorectal cell lines, WiDr and C2BBe1, adapted better to hypoxia and were able to maintain their oxidative fluxes even at the very low levels of oxygen. These differential metabolic behaviors of the three colorectal cell lines show how important an adequate knowledge of the "metabolic remodeling" that follows a given cancer treatment is towards the correct (re)design of therapeutic strategies against cancer.
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http://dx.doi.org/10.1155/2014/759791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100400PMC
March 2015