Skeletal muscle: from birth to old age, routes to mechanical and metabolic failure. / Músculo esquelético: del nacimiento a la vejez, rutas hacia la falla mecánica y metabólica.

Skeletal muscle (SM) is the most abundant tissue and the largest reservoir of protein in the body. It transports glucose in an insulin dependent manner by the glucose transporter type 4 (GLUT4) and contributes in the maintenance of serum amino acids concentration. By its mass and energetic requirements, it is fundamental for the systemic metabolic balance. In the present work, we present the effect of gestational undernourishment (GU) on the mechanical and metabolic properties of SM at birth and in old age in an animal model. Mechanical studies were performed on isolated muscles, while the GLUT4, amino acid transporters LAT2, SNAT2 and insulin receptors (IR) determination were performed on isolated transverse-tubule membranes (TT). The GU in offspring at birth, results in low muscle mass with increased contraction force and resistance to fatigue. However, in two-years old rats, there was muscle hypotrophy and sarcopenia, the force decreased between 50 and 70% in control rats and rats with GU respectively, accompanied by a lower expression of LAT2, SNAT2 and IR in TT. In conclusion, GU irreversibly affects the SM, an effect that could be similar in humans, which help us to understand the events that associate the GU with the metabolic debacle of SM and the metabolic diseases of human adulthood.

Synthesis and preliminary biological evaluation of O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET) as a potential cationic amino acid PET tracer for tumor imaging.

Amino acid transport is an attractive target for oncologic imaging. Despite a high demand of cancer cells for cationic amino acids, their potential as PET probes remains unexplored. Arginine, in particular, is involved in a number of biosynthetic pathways that significantly influence carcinogenesis and tumor biology. Cationic amino acids are transported by several cationic transport systems including, ATB(0,+) (SLC6A14), which is upregulated in certain human cancers including cervical, colorectal and estrogen receptor-positive breast cancer. In this work, we report the synthesis and preliminary biological evaluation of a new cationic analog of the clinically used PET tumor imaging agent O-(2-[(18)F]fluroethyl)-L-tyrosine ([(18)F]FET), namely O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET). Reference compound and precursor were prepared by multi-step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 16-20% decay-corrected yields with radiochemical purity >99%. The new tracer showed good stability in vitro and in vivo. Cell uptake assays demonstrated that FEMAET and [(18)F]FEMAET accumulate in prostate cancer (PC-3) and small cell lung cancer cells (NCI-H69), with an energy-dependent mechanism. Small animal PET imaging with NCI-H69 xenograft-bearing mice revealed good tumor visualization comparable to [(18)F]FET and low brain uptake, indicating negligible transport across the blood-brain barrier. In conclusion, the non-natural cationic amino acid PET probe [(18)F]FEMAET accumulates in cancer cells in vitro and in vivo with possible involvement of ATB(0,+).

Aminoácidos en líquido cefalorraquídeo y plasma: utilidad en el estudio de las enfermedades neuropediátricas / Amino acids in cerebrospinal fluid and plasma: its usefulness in the study of neuropaediatric diseases

Introducción. El estudio de aminoácidos en el líquido cefalorraquídeo (LCR) es imprescindible en el diagnóstico de algunas enfermedades neurológicas y apoya el diagnóstico en otras. No existen trabajos en la bibliografía que muestren la relación fisiológica entre los valores de aminoácidos en LCR y plasma en población pediátrica. Objetivo. Definir unas ratios de aminoácidos en plasma y LCR en población pediátrica que permitan su uso en la práctica clínica diaria. Pacientes y métodos. Se han recogido y analizado de forma retrospectiva los aminogramas en plasma y LCR de 105 pacientes de edades comprendidas entre 0 y 12 meses. Se han incluido aminogramas cuyos valores de aminoácidos son normales según los valores de referencia de nuestro laboratorio. El análisis cuantitativo de aminoácidos se realizó mediante cromatografía líquida de alta resolución, y el análisis estadístico, con el programa SPSS v. 19.0. Resultados. Se muestran los valores medios, rango y desviación estándar de las concentraciones de aminoácidos en plasma y LCR, así como de las ratios LCR/plasma. Se han encontrado correlaciones significativas a partir de 0,6 entre diferentes aminoácidos neutros, sobre todo los de peso molecular más pequeño (Thr, Ser, Gly y Ala). Conclusiones. La existencia de correlaciones significativas entre diferentes aminoácidos neutros apoya el hecho de que éstos compartan los mismos transportadores en la barrera hematoencefálica. La estandarización de ratios de aminoácidos permitirá aumentar la sensibilidad en la detección de valores patológicos en plasma y LCR, profundizar en la fisiopatología de enfermedades neurológicas y quizá describir nuevas aminoacidopatías (AU)

Introduction. Studying the amino acids in cerebrospinal fluid (CSF) is essential in the diagnosis of some neurological diseases and is an important aid in the diagnosis of others. No research has been published in the literature to prove the physiological relationship between the values of amino acids in CSF and plasma in the paediatric population. Aim. To define a set of ratios for amino acids in plasma and CSF in the paediatric population that can be used in daily clinical practice. Patients and methods. The aminograms in plasma and CSF of 105 patients with ages between 0 and 12 months were collected and analysed retrospectively. Aminograms with amino acid values that are considered to be normal according to the reference values of our laboratory were included in the sample. The quantitative analysis of amino acids was performed using high-resolution liquid chromatography and statistical analysis with the software application SPSS 19.0. Results. The mean values, range and standard deviation of the amino acid concentrations in plasma and CSF, together with the CSF/plasma ratios, are reported. Significant correlations were found from 0.6 onwards between different neutral amino acids, above all in those with smaller molecular weights (Thr, Ser, Gly and Ala). Conclusions. The existence of significant correlations between the different neutral amino acids supports the idea that they share the same transporters in the blood-brain barrier. Standardising the amino acid ratios will make it possible to increase sensitivity in the detection of pathological values in plasma and CSF, to further knowledge of the pathophysiology of neurological diseases and perhaps to describe new aminoacidopathies (AU)

Isolation of plasma membrane vesicles from mouse placenta at term and measurement of system A and system beta amino acid transporter activity.

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and beta, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl(2) precipitation and centrifugation. Vesicles were enriched 11.3+/-0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system beta activity in mouse placental vesicles, measured as Na(+)-dependent uptake of (14)C-methylaminoisobutyric acid (MeAIB) and (3)H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system beta activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Growing steers grazing high versus low endophyte (Neotyphodium coenophialum)-infected tall fescue have reduced serum enzymes, increased hepatic glucogenic enzymes, and reduced liver and carcass mass.

It is well established that grazing Neotyphodium coenophialum-infected forages results in reduced BW gain and serum prolactin concentrations of cattle. The objective of this study was to determine the potential effects of toxic endophyte-infected tall fescue consumption on blood metabolites, carcass characteristics, and content of proteins critical for AA metabolism in the liver, kidney, and LM tissue of growing steers. Steers grazed a low toxic endophyte (LE; 0.023 microg/g ergot alkaloids) tall fescue-mixed grass pasture (n = 9; BW = 266 +/- 10.9 kg; 5.7 ha) or a high toxic endophyte (HE; 0.746 microg/g of ergot alkaloids) tall fescue pasture (n = 10; BW = 267 +/- 14.5 kg; 5.7 ha) from June 14 through at least September 11 (> or =89 d). No difference was observed for BW (P < 0.10) for the overall 85-d growth period. Also, no differences were observed for ribeye area/100 kg of HCW (P > 0.91), backfat (P > 0.95), or backfat/100 kg of HCW (P > 0.67). However, ADG (P < 0.01), final BW (P < 0.05), HCW (P < 0.01), dressing percentage (P < 0.01), ribeye area (P < 0.01), whole liver wet weight (P < 0.01), and whole liver wet weight/100 kg of end BW (P < 0.01) were greater for LE steers than HE steers. After 85 d of grazing, serum concentrations of alkaline phosphatase (P < 0.05), alanine aminotransferase (P < 0.01), aspartate aminotransferase (P < 0.03), cholesterol (P < 0.01), lactate dehydrogenase (P < 0.01), and prolactin (P < 0.01) were less for HE than LE steers. At slaughter, hepatic content of cytosolic phosphoenolpyruvate carboxykinase (P < 0.01) was greater in HE steers than LE steers. Hepatic content of aspartate aminotransferase (P < 0.01) also was greater, whereas renal and LM content were not (P > or = 0.42). No differences (P > or = 0.15) were observed for hepatic, renal, and LM content of alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase, and 3 glutamate transport proteins. These data indicate that the HE steers displayed classic endophyte toxicity symptoms for growth and blood variables, classic symptoms that were concomitant with novelly identified altered glucogenic capacity of the liver and decreases in carcass characteristics.

Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe.

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31(+) or erg32(+) did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in ergDelta mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6Delta but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the ergDelta mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.

ABC-type amino acid uptake transporters Bgt and N-II of Anabaena sp. strain PCC 7120 share an ATPase subunit and are expressed in vegetative cells and heterocysts.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N(2) in differentiated cells called heterocysts. Anabaena open reading frames alr4167 and alr3187 encode, respectively, an ATPase subunit, BgtA, and a composite protein bearing periplasmic substrate-binding and transmembrane domains, BgtB, of an ABC-type high-affinity basic amino acid uptake transporter (Bgt). Open reading frame alr4167 is clustered with open reading frames alr4164, alr4165 and alr4166 that encode a periplasmic substrate-binding protein, NatF, and transmembrane proteins NatG and NatH respectively. The NatF, NatG, NatH and BgtA proteins constitute an ABC-type uptake transporter for acidic and neutral polar amino acids (N-II). The Bgt and N-II transport systems thus share the ATPase subunit, BgtA. These transporters together with the previously characterized ABC-type uptake transporter for proline and hydrophobic amino acids (N-I) account for more than 98% of the amino acid transport activity exhibited by Anabaena sp. strain PCC 7120. In contrast to N-I that is expressed only in vegetative cells, the Bgt and N-II systems are present in both vegetative cells and heterocysts. Whereas Bgt is dispensable for diazotrophic growth, N-II appears to contribute together with N-I to the diazotrophic physiology of this cyanobacterium.

Comparative gene expression profiles of intestinal transporters in mice, rats and humans.

We have studied gene expression profiles of intestinal transporters in model animals and humans. Total RNA was isolated from duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mice, rats, and humans were about 60% of 22,690 sequences, 40% of 8739, and 47% of 12,559, respectively. A total of 86 genes involving transporters expressed in mice, 50 genes in rats, and 61 genes in humans were detected. Mice exhibited abundant mRNA expressions for peptide transporter HPT1, amino acid transporters CSNU3, CT1 and ASC1, nucleoside transporter CNT2, organic cation transporter SFXN1, organic anion transporter NBC3, glucose transporter SGLT1, and fatty acid transporters FABP1 and FABP2. Rats showed high expression profiles of peptide transporter PEPT1, amino acid transporters CSNU1 and 4F2HC, nucleoside transporter CNT2, organic cation transporter OCT5, organic anion transporter SDCT1, glucose transporter GLUT2 and GLUT5, and folate carrier FOLT. In humans, the highly expressed genes were peptide transporter HPT1, amino acid transporters LAT3, 4F2HC and PROT, nucleoside transporter CNT2, organic cation transporter OCTN2, organic anion transporters NADC1, NBC1 and SBC2, glucose transporters SGLT1 and GLUT5, multidrug resistance-associated protein RHO12, fatty acid transporters FABP1 and FABP2, and phosphate carrier PHC. Overall these data reveal diverse transcriptomic profiles for intestinal transporters among these species. Therefore, this transcriptional data may lead to more effective use of the laboratory animals as a model for oral drug development.

Amino acids regulate retrieval of the yeast general amino acid permease from the vacuolar targeting pathway.

Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.

Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes.

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.

Ceramide down-regulates System A amino acid transport and protein synthesis in rat skeletal muscle cells.

Skeletal muscle is a major insulin target tissue and has a prominent role in the control of body amino acid economy, being the principal store of free and protein-bound amino acids and a dominant locus for amino acid metabolism. Interplay between diverse stimuli (e.g., hormonal/nutritional/mechanical) modulates muscle insulin action to serve physiological need through the action of factors such as intramuscular signaling molecules. Ceramide, a product of sphingolipid metabolism and cytokine signaling, has a potent contra-insulin action with respect to the transport and deposition of glucose in skeletal muscle, although ceramide effects on muscle amino acid turnover have not previously been documented. Here, membrane permeant C2-ceramide is shown to attenuate the basal and insulin-stimulated activity of the Na+-dependent System A amino acid transporter in rat muscle cells (L6 myotubes) by depletion of the plasma membrane abundance of SNAT2 (a System A isoform). Concomitant with transporter down-regulation, ceramide diminished both intramyocellular amino acid abundance and the phosphorylation of translation regulators lying downstream of mTOR. The physiological outcome of ceramide signaling in this instance is a marked reduction in cellular protein synthesis, a result that is likely to represent an important component of the processes leading to muscle wasting in catabolic conditions.

Dual-phenotype GABA/glutamate neurons in adult preoptic area: sexual dimorphism and function.

It is generally assumed that the inhibitory neurotransmitter GABA and the stimulatory neurotransmitter glutamate are released from different neurons in adults. However, this tenet has made it difficult to explain how the same afferent signals can cause opposite changes in GABA and glutamate release. Such reciprocal release is a central mechanism in the neural control of many physiological processes including activation of gonadotropin-releasing hormone (GnRH) neurons, the neural signal for ovulation. Activation of GnRH neurons requires simultaneous suppression of GABA and stimulation of glutamate release, each of which occurs in response to a daily photoperiodic signal, but only in the presence of estradiol (E2). In rodents, E2 and photoperiodic signals converge in the anteroventral periventricular nucleus (AVPV), but it is unclear how these signals differentially regulate GABA and glutamate secretion. We now report that nearly all neurons in the AVPV of female rats express both vesicular glutamate transporter 2 (VGLUT2), a marker of hypothalamic glutamatergic neurons, as well as glutamic acid decarboxylase and vesicular GABA transporter (VGAT), markers of GABAergic neurons. These dual-phenotype neurons are the main targets of E2 in the region and are more than twice as numerous in females as in males. Moreover, dual-phenotype synaptic terminals contact GnRH neurons, and at the time of the surge, VGAT-containing vesicles decrease and VGLUT2-containing vesicles increase in these terminals. Thus, we propose a new model for ovulation that includes dual-phenotype GABA/glutamate neurons as central transducers of hormonal and neural signals to GnRH neurons.

The H+-coupled electrogenic lysosomal amino acid transporter LYAAT1 localizes to the axon and plasma membrane of hippocampal neurons.

Recent work has identified a lysosomal protein that transports neutral amino acids (LYAAT1). We now show that LYAAT1 mediates H+ cotransport with a stoichiometry of 1 H+/1 amino acid, consistent with a role in the active efflux of amino acids from lysosomes. In neurons, however, LYAAT1 localizes to axonal processes as well as lysosomes. In axons LYAAT1 fails to colocalize with synaptic markers. Rather, axonal LYAAT1 colocalizes with the exocyst, suggesting a role for membranes expressing LYAAT1 in specifying sites for exocytosis. A protease protection assay and measurements of intracellular pH further indicate abundant expression at the plasma membrane, raising the possibility of physiological roles for LYAAT1 on the cell surface as well as in lysosomes.

Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications.

Transport processes play a pivotal role in cellular metabolism, e.g. for the uptake of nutrients or the excretion of metabolic waste products. Moreover, they are also important in biotechnological processes such as the production of various amino acids by the use of microorganisms. The focus of this review is on bacterial amino acid transport systems, in particular those of Corynebacterium glutamicum and Escherichia coli, with respect to their function and biotechnological significance.