Betaine homocysteine S-methyltransferase emerges as a new player of the nuclear methionine cycle

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with D-galactosamine (Gal) or buthionine sulfoximine (BSO) treatments induces BHMT nuclear translocation, an effect that is prevented by administration of N-acetylcysteine (NAC) and glutathione ethyl ester (EGSH), respectively. Unexpectedly, the hepatic nuclear accumulation induced by Gal associates with reduced nuclear BHMT activity and a trend towards increased protein homocysteinylation. Overall, our results support the involvement of BHMT in nuclear homocysteine remethylation, although moonlighting roles unrelated to its enzymatic activity in this compartment cannot be excluded.This work was supported by grants of the Ministerio de Economía y Competitividad (BFU2008-00666 and BFU2009-08977 to MAP; SAF2012-36519 and SAF2015-68590R to DPS) and Instituto de Salud Carlos III (RETIC RIRAAF RD12/0013/0008 and ARADYAL RD16/0006/0021 to DPS).Peer reviewe

Circulating microRNAs fluctuations in exercise-induced cardiac remodeling: A systematic review

MicroRNAs (miRNAs) are small non-coding RNAs that participate in gene expression regulation. It has been observed that circulating levels of miRNAs may fluctuate during exercise, showing numerous cardiac biological and physiological effects such as structural and functional adaptations. We aimed to provide an overview of the currently available information concerning the role of circulating miRNAs in cardiovascular adaptations in response to acute and/or chronic exercise training. Relevant studies published were searched in three databases: PubMed, Web of Science and Scopus. A combination of the following keywords was used: (“microRNA” OR “miRNA” OR “miR” AND “exercise” OR “training” OR “physical activity”) AND “(heart hypertrophy” OR “cardiac remodeling” OR “cardiac muscle mass” OR “cardiac hypertrophy”). Only experimental studies, written in English and conducted in healthy individuals were included. Five articles met the inclusion criteria and were finally included in this systematic review after reviewing both title, abstract and full-text. A total of thirty-six circulating cardiac-related miRNAs were analyzed, but only five of them (miR-1, miR-133a, miR-146a, miR-206 and miR-221) were directly associated with cardiac adaptations parameters, while two of them (miR-1 and miR-133a) were related to cardiac hypertrophy. Most of them were upregulated immediately after a marathon and returned to basal levels at longer times. Therefore, we conclude that, although evidence is still limited, and long-term studies are needed to obtain more robust evidence, exercise is more likely to affect circulating cardiac-related miRNAs level

Long-term omega-3 fatty acid supplementation prevents expression changes in cochlear homocysteine metabolism and ameliorates progressive hearing loss in C57BL/6J mice

Abstract Omega-3 polyunsaturated fatty acids (PUFAs) are essential nutrients well known for their beneficial effects, among others on cognitive development and maintenance, inflammation and oxidative stress. Previous studies have shown an inverse association between high plasma levels of PUFAs and age-related hearing loss, and the relationship between low serum folate and elevated plasma homocysteine levels and hearing loss. Therefore, we used C57BL/6J mice and long-term omega-3 supplementation to evaluate the impact on hearing by analyzing their auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) thresholds. The omega-3 group showed significantly lower ABR hearing thresholds (~25 dB sound pressure level) and higher DPOAE amplitudes in mid-high frequencies when compared to the control group. These changes did not correlate with alterations between groups in plasma homocysteine or serum folate levels as measured by high-performance liquid chromatography and a microbiological method, respectively. Aging in the control group was associated with imbalanced cytokine expression toward increased proinflammatory cytokines as determined by quantitative reverse transcriptase polymerase chain reaction; these changes were prevented by omega-3 supplementation. Genes involved in homocysteine metabolism showed decreased expression during aging of control animals, and only alterations in Bhmt and Cbs were significantly prevented by omega-3 feeding. Western blotting showed that omega-3 supplementation precluded the CBS protein increase detected in 10-month-old controls but also produced an increase in BHMT protein levels. Altogether, the results obtained suggest a long-term protective role of omega-3 supplementation on cochlear metabolism and progression of hearing loss

Nuevas estrategias para la prevención de la pérdida auditiva neurosensorial en el ratón: ácidos grasos omega-3 y dendrogenina B

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 1-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 01-09-2067La hipoacusia neurosensorial es una de las principales causas de discapacidad a nivel mundial. Su etiología es multifactorial e incluye factores genéticos y ambientales, destacando la exposición a ruido y el envejecimiento. A pesar de sus graves consecuencias a nivel médico, laboral y social, no se dispone de ningún tratamiento eficaz, por lo que la investigación en nuevas terapias es esencial. Las alteraciones del ciclo de la metionina en la cóclea dan lugar a estrés oxidativo e hipoacusia en el hombre y en el ratón, por lo que este ciclo podría constituir una potencial diana terapéutica. Diversos estudios epidemiológicos evidencian que la nutrición es clave en la prevención de la pérdida auditiva y que los ácidos grasos omega-3 tienen efectos beneficiosos sobre la inflamación y el estrés oxidativo. Por otro lado, la intervención con pequeñas moléculas como las dendrogeninas, que han mostrado actividad neuroprotectora in vitro, supone un nuevo enfoque farmacológico en el tratamiento de la hipoacusia. En este trabajo se ha empleado un modelo experimental de ratón para i) caracterizar los cambios que se producen en el ciclo de la metionina en la cóclea con la edad y con la exposición al ruido, ii) evaluar el efecto de los ácidos grasos omega-3 en el tratamiento de la hipoacusia asociada a la edad y, iii) estudiar el efecto de la dendrogenina AF243 en el tratamiento de la hipoacusia inducida por ruido. Los resultados obtenidos sugieren que: i) la expresión coclear de los genes Bhmt, Bhmt2 y Cbs aumenta con la edad, mientras que la de Mtr, Mat2a, Mat2b y Ahcy, disminuye. La exposición al ruido provoca un aumento de los umbrales de ABR asociado a un descenso en la expresión de Bhmt, Bhmt2 y un aumento en la proteína BHMT2, y en la expresión de las citoquinas Il1b e Il6. ii) La suplementación de la dieta con ácidos grasos omega-3 reduce significativamente el incremento de umbrales auditivos con la edad y el aumento de la expresión de citoquinas proinflamatorias en la cóclea. También modula la expresión de los genes del ciclo de la metionina, especialmente Bhmt y Cbs. iii) La administración en oído medio de una dosis única de AF243 protege de forma limitada de la hipoacusia inducida por ruido. Sin embargo, el tratamiento sistémico mantenido mejora ostensiblemente los umbrales auditivos y reduce la expresión de marcadores pro-inflamatorios y de daño celular. Como conclusión, el ciclo de la metionina en la cóclea se modula por la edad y tras el ruido en el ratón, por lo que constituye una posible diana terapéutica. En un modelo de ratón, la suplementación con ácidos grasos omega-3 y la dendrogenina AF243 han demostrado un efecto protector frente al desarrollo de la hipoacusia asociada a la edad e inducida por ruido, respectivamente.Sensorineural hearing loss is one of the main causes of impairment worldwide. It presents a multifactorial etiology that includes genetic and environmental factors. In spite of their critical consequences at medical, occupational and social level, effective treatments are not available; consequently, research in new therapies is essential. Disturbances of the methionine cycle in the cochlea give rise to oxidative stress and hypoacusis, in man and mouse, and therefore, this cycle represents a potential therapeutic target. Several epidemiological studies evidence that nutrition is essential in hearing loss prevention and that ingestion of omega-3 fatty acids have benefits in inflammation and oxidative stress. Otherwise, an intervention with small molecules like dendrogenins, which have shown in vitro neuroprotective activity, provides a novel pharmaceutical approach to treat hearing loss. In this study, a mouse model has been used to i) characterize changes in the cochlear methionine cycle with ageing and after noise exposure, ii) to evaluate the effect of omega-3 fatty acids supplementation in the treatment of age-related hearing loss, iii) to study the effect of dendrogenin AF243 in the treatment of noise induced hearing loss. The results from this study suggest that: i) cochlear expression of Bhmt, Bhmt2 and Cbs increases with age, however Mtr, Mat2a, Mat2b and Ahcy expression decreases. Furthermore, noise exposure provokes an increase in ABR hearing thresholds associated with Bhmt and Bhmt2 decrease expression and an increase in BHMT2 protein and expression of pro-inflammatory cytokines Il1b and Il6. ii) omega-3 supplementation significantly reduces the increase in hearing thresholds associated with ageing together with the cochlear expression of pro-inflammatory cytokines in the cochlea. Moreover, omega-3 supplementation modulates methionine cycle genes expression, particularly Bhmt and Cbs. iii) Noise induced hearing loss is partially protected by a single topical administration of AF243. However, systemic sustained treatment of AF243 before and after noise exposure obviously improves hearing thresholds and expression of pro-inflammatory and cellular damage markers. In conclusion, cochlear methionine cycle is modulated by age and before noise exposure in mice, and therefore suggests a potential therapeutic target. In a mouse model, omega-3 fatty acid supplementation and AF243 dendrogenin have demonstrated a protective role against the development of age-related and noise induced hearing loss, respectively.Esta Tesis Doctoral ha sido realizada gracias a un contrato del proyecto de la Unión Europea FP7-309400 AFHELO

Long-term omega-3 fatty acid supplementation prevents expression changes in cochlear homocysteine metabolism and ameliorates progressive hearing loss in C57BL/6J mice

Under a Creative Commons license.Omega-3 polyunsaturated fatty acids (PUFAs) are essential nutrients well-known for their beneficial effects, among others on cognitive development and maintenance, inflammation and oxidative stress. Previous studies have shown an inverse association between high plasma levels of PUFAs and age-related hearing loss, and the relationship between low serum folate and elevated plasma homocysteine levels and hearing loss. Therefore, we used C57BL/6J mice and long-term omega-3 supplementation to evaluate the impact on hearing by analyzing their auditory brainstem response (ABR) and distortion product otoacustic emissions (DPOAE) thresholds. The omega-3 group showed significantly lower ABR hearing thresholds (∼25 dB SPL) and higher DPOAE amplitudes in mid-high frequencies, when compared to the control group. These changes did not correlate with alterations between groups in plasma homocysteine or serum folate levels as measured by HPLC and a microbiological method, respectively. Ageing in the control group was associated with imbalanced cytokine expression toward increased pro-inflammatory cytokines as determined by RT-qPCR; these changes were prevented by omega-3 supplementation. Genes involved in homocysteine metabolism showed decreased expression during ageing of control animals, and only alterations in Bhmt and Cbs were significantly prevented by omega-3 feeding. Western blotting showed that omega-3 supplementation precluded the CBS protein increase detected in 10 month-old controls, but also produced an increase in BHMT protein levels. Altogether, the results obtained suggest a long term protective role of omega-3 supplementation on cochlear metabolism and progression of hearing loss.R.M.V. is a fellow of the JAE-CSIC predoctoral program, and N.V. is supported by FP7-AFHELO. The authors wish to thank the Genomics and Non-invasive Neurofunctional Evaluation facilities (IIBM, CSIC-UAM) for their technical support. This work was supported by grants of the Ministerio de Economía y Competitividad (SAF2011-24391 and SAF2014-53979-R to I.V.N.; BFU2009-08977 to M.A.P.), the European Union (FP7-AFHELO and TARGEAR to I.V.N.) and Puleva-LACTALIS (to I.V.N., G.V.M. and M.A.P.).Peer reviewe

Cochlear Homocysteine Metabolism at the Crossroad of Nutrition and Sensorineural Hearing Loss

Hearing loss (HL) is one of the most common causes of disability, affecting 360 million people according to the World Health Organization (WHO). HL is most frequently of sensorineural origin, being caused by the irreversible loss of hair cells and/or spiral ganglion neurons. The etiology of sensorineural HL (SNHL) is multifactorial, with genetic and environmental factors such as noise, ototoxic substances and aging playing a role. The nutritional status is central in aging disability, but the interplay between nutrition and SNHL has only recently gained attention. Dietary supplementation could therefore constitute the first step for the prevention and potential repair of hearing damage before it reaches irreversibility. In this context, different epidemiological studies have shown correlations among the nutritional condition, increased total plasma homocysteine (tHcy) and SNHL. Several human genetic rare diseases are also associated with homocysteine (Hcy) metabolism and SNHL confirming this potential link. Accordingly, rodent experimental models have provided the molecular basis to understand the observed effects. Thus, increased tHcy levels and vitamin deficiencies, such as folic acid (FA), have been linked with SNHL, whereas long-term dietary supplementation with omega-3 fatty acids improved Hcy metabolism, cell survival and hearing acuity. Furthermore, pharmacological supplementations with the anti-oxidant fumaric acid that targets Hcy metabolism also improved SNHL. Overall these results strongly suggest that cochlear Hcy metabolism is a key player in the onset and progression of SNHL, opening the way for the design of prospective nutritional therapies

Papel del extremo N-terminal de la betaina homocisteína metiltransferasa en su actividad y oligomerización

Póster presentado al XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Madrid del 3 al 6 de septiembre de 2013.La betaína homocisteína metiltransferasa (BHMT) es una enzima homo-tetramérica del ciclo de la metionina, que cataliza una de las reacciones de remetilación de homocisteína (Hcy), utilizando betaína como donante de grupos metilo y generando dimetilglicina y metionina. El osmolito betaína se adquiere en la dieta o se produce en la oxidación mitocondrial de colina. Así, se recupera uno de los tres grupos metilo utilizados en la síntesis de colina por la ruta de transmetilación. Recientemente, se ha descrito la existencia de una BHMT-2, que utiliza metilmetionina (vitamina U) como donante de grupos metilo, generando dos moléculas de metionina por cada Hcy. Existe una gran conservación se secuencia entre BHMTs (95% idénticas rata vs. humano), sin embargo BHMT y BHMT-2 presentan importantes diferencias. Así, BHMT tiene una extensión de ~30 residuos en su C-terminal, una inserción de 9 aminoácidos (87-96), y su N-terminal constituye una de las zonas más divergentes. Con el fin de averiguar el papel de las diferencias en el N-terminal, hemos llevado a cabo la mutación de residuos de esa zona y su delección en BHMT. Las proteínas mutantes se han producido como proteínas de fusión a inteina en E. coli BL21(DE3), escindiéndose con 2-mercaptoetanol durante la purificación. Se han analizado los efectos en actividad y oligomerización, observándose que los mutantes K8A, K10A y K8A-K10A presentan ~35% de la actividad BHMT wild type, sin afectarse su estado de asociación. Otros mutantes en residuos conservados en esa zona, reducen su actividad (70-50%) y provocan grandes cambios en afinidad, aumentando la de Hcy y en algún caso disminuyendo la de betaina. De ello, podemos concluir que el N-terminal tiene un papel importante en la unión de sustratos.Financiado por BFU2009-08977.Peer Reviewe

Folate deficiency alters homocysteine cycle in the cochlea and causes premature hearing loss in mice

Póster presentado en el 50th Inner Ear Biology Workshop (IEB), celebrado en Alcalá de Henares del 10 al 13 de septiembre de 2013.The methionine/homocysteine cycle is modulated by nutritional factors and its alterations have been associated to several pathologies including deafness (1). We have studied the impact of a dietary-induced folic acid deficiency on cochlear methionine metabolism and in hearing. C57BL/6J mice were fed with normal diet or folate deficient (FD) for 8 weeks. Hearing was evaluated by ABR threshold analyses and cochlear morphology was evaluated by hematoxylin-eosin staining and immunohistochemistry. RT-qPCR and Western blotting were used to determine cochlear levels of the methionine cycle enzymes, peptide mass fingerprint was carried out for protein identification, and plasma Hcy (pHcy) levels were determined by HPLC. The control group showed normal ABR thresholds (8 to 28 kHz, 27-48 dB SPL) whereas the FD group presented moderate to profound hearing loss (8 to 28 kHz, 52-85 dB SPL). Folic acid deficiency caused a reduction in plasma folate levels whilst pHcy levels were increased. All Hcy cycle enzymes studied were expressed in the cochlea. But some of them showed altered mobility in Western blotting as compared to the reference liver mobility patterns, suggesting post-translational modifications in the cochlea. In summary our data indicate that: i) the main enzymes of Hcy metabolism are expressed in the cochlea; ii) alterations in the methionine cycle secondary to folic acid deficit caused hearing loss; and iii) hearing loss correlated with alterations in the expression of Hcy cycle enzymes, and elevations of systemic pHcy.[1] Cohen-Salmon, M. et al. “Connexin30 deficiency causes instrastrial fluid-blood barrier disruption within the cochlear stria vascularis”, Proc Natl Acad Sci U S A 104: 6229-6234, 2007.RMV holds a JAE-CSIC fellowship. Ministerio de Economía y competitividad (SAF2011-24391, BFU2009-08977) and PULEVA.Peer reviewe

Cochlear Homocysteine Metabolism at the Crossroad of Nutrition and Sensorineural Hearing Loss

Hearing loss (HL) is one of the most common causes of disability, affecting 360 million people according to the World Health Organization (WHO). HL is most frequently of sensorineural origin, being caused by the irreversible loss of hair cells and/or spiralmganglion neurons. The etiology of sensorineural HL (SNHL) is multifactorial, with genetic and environmental factors such as noise, ototoxic substances and aging playing a role. The nutritional status is central in aging disability, but the interplay between nutrition and SNHL has only recently gained attention. Dietary supplementation could therefore constitute the first step for the prevention and potential repair of hearing damage before it reaches irreversibility. In this context, different epidemiological studies have shown correlations among the nutritional condition, increased total plasma homocysteine (tHcy) and SNHL. Several human genetic rare diseases are also associated with homocysteine (Hcy) metabolism and SNHL confirming this potential link. Accordingly, rodent experimental models have provided the molecular basis to understand the observed effects. Thus, increased tHcy levels and vitamin deficiencies, such as folic acid (FA), have been linked with SNHL, whereas long-term dietary supplementation with omega-3 fatty acids improved Hcy metabolism, cell survival and hearing acuity. Furthermore, pharmacological supplementations with the anti-oxidant fumaric acid that targets Hcy metabolism also improved SNHL. Overall these results strongly suggest that cochlear Hcy metabolism is a key player in the onset and progression of SNHL, opening the way for the design of prospective nutritional therapies.This work was supported by the European Commission FP7-PEOPLE-2013-IAPP TARGEAR and Spanish Ministerio de Economía y Competitividad (MINECO)/FEDER SAF2014- 53979-R to IV-N. NV holds a CSIC predoctoral contract associated to FP7-AFHELO.Peer reviewe

Amelioration of hearing loss by long-term omega-3 supplementation involves homocysteine metabolism

Póster presentado a la 10th International Conference one Carbon Metabolism, Vitamins B and Homocysteine, celebrada en Nancy (Francia) del 7 al 11 de julio de 2015.[Background]: Beneficial effects of omega-3 (w3) polyunsaturated fatty acids (PUFAs) in cognitive function, inflammation and oxidative stress have been described. Additionally, an association between hearing loss and low plasmatic levels of PUFAs and serum folate, as well as high total homocysteine (tHcy) has been reported. [Objective]: The evaluation of the potential effects of long-term w3-supplementation in prevention and/or delay of the onset of hearing loss. [Design]: Two-month-old C57BL/6J mice were fed control or w3-supplemented diets for 8 months. A set of 4 month-old control mice was used for comparative purposes. [Results]: Auditory brainstem response (ABR) thresholds in control mice increased with age, whereas distortion product otoacustic emissions (DPOAE) thresholds showed lower amplitudes in mid-high frequencies. These changes in hearing parameters were partially ameliorated by long-term w3-supplementation. No gross histological differences were detected between control and w3 cochleae by Cresyl violet staining and immunohistochemistry, neither significant changes in tHcy or serum folate levels were measured by HPLC and microbiological methods, respectively. Ageing in the control group was associated with imbalanced cochlear cytokine expression toward increased pro-inflammatory cytokines as determined by RT-qPCR, an effect prevented by w3-supplementation. Decreased expression of most genes of cochlear Hcy metabolism was shown during aging of the control group, but only alterations in Bhmt and Cbs were significantly precluded by w3-feeding. However, changes in protein levels by western blotting indicated increased cystathione b-synthase levels in the 10 month-old controls that were prevented by w3, although increasing betaine homocysteine methyltransferase protein. Moreover, no significant variations in global homocysteinylation levels were detected by western blotting between groups. [Conclusions]: Ageing induces inflammation and hearing loss together with increased transsulfuration in control mice. Long-term w3-supplementation improved signs of inflammation and hearing loss, simultaneously promoting Hcy remethylation and reducing transulfuration.FP7-AFHELO, FP7-TARGEAR, Ministerio de Economía y Competitividad and Puleva-LACTALIS.Peer Reviewe