Temperature Affects Musculoskeletal Development and Muscle Lipid Metabolism of Gilthead Sea Bream (Sparus aurata).

World population is expected to increase to approximately 9 thousand million people by 2050 with a consequent food security decline. Besides, climate change is a major challenge that humanity is facing, with a predicted rise in mean sea surface temperature of more than 2°C during this century. This study aims to determine whether a rearing temperature of 19, 24, or 28°C may influence musculoskeletal development and muscle lipid metabolism in gilthead sea bream juveniles. The expression of growth hormone (GH)/insulin-like growth factors (IGFs) system-, osteogenic-, myogenic-, and lipid metabolism-related genes in bone and/or white muscle of treated fish, and the in vitro viability, mineralization, and osteogenic genes expression in primary cultured cells derived from bone of the same fish were analyzed. The highest temperature significantly down-regulated igf-1, igf-2, the receptor igf-1ra, and the binding proteins igfbp-4 and igfbp-5b in bone, and in muscle, igf-1 and igf-1ra, suggesting impaired musculoskeletal development. Concerning myogenic factors expression, contrary responses were observed, since the increase to 24°C significantly down-regulated myod1 and mrf4, while at 28°C myod2 and myogenin were significantly up-regulated. Moreover, in the muscle tissue, the expression of the fatty acid transporters cd36 and fabp11, and the lipases lipa and lpl-lk resulted significantly increased at elevated temperatures, whereas ß-oxidation markers cpt1a and cpt1b were significantly reduced. Regarding the primary cultured bone-derived cells, a significant up-regulation of the extracellular matrix proteins on, op, and ocn expression was found with increased temperatures, together with a gradual decrease in mineralization along with fish rearing temperature. Overall, these results suggest that increasing water temperature in this species appears to induce unfavorable growth and development of bone and muscle, through modulating the expression of different members of the GH/IGFs axis, myogenic and osteogenic genes, while accelerating the utilization of lipids as an energy source, although less efficiently than at optimal temperatures.

Effects of ß2-adrenoceptor agonists on gilthead sea bream (Sparus aurata) cultured muscle cells.

ß2-adrenoceptors are a subtype of G-protein coupled receptors whose activation leads to increased protein synthesis and decreased degradation in mammalian skeletal muscle, causing hypertrophy. In this study, we compared the effects of the classical ß2-agonist noradrenaline (NA) with two representatives of a new generation of agonists (formoterol, FOR and salmeterol, SALM) on growth and metabolism of primary cultured muscle cells of gilthead sea bream. Activation of signaling pathways, cell development and expression of relevant genes were analyzed in day 4 myocytes. The three agonists increased either cAMP levels or PKA phosphorylation, plus TOR phosphorylation, and the proportion of proliferating cell nuclear antigen (PCNA)-positive cells, in parallel with pcna mRNA levels. Thus, demonstrating that these cells are ß2-agonists-responsive, and supporting enhanced cell proliferation. The expression of the myogenic factor myf5 was significantly down-regulated, suggesting that the cells were already destined to the muscular linage; while insulin-like growth factors (igf-1 and igf-2) transcript levels were up-regulated, proposing an additional anabolic effect through their local production. Furthermore, SALM treatment up-regulated expression of the lipases (hsl and lipa) and the ß-oxidation marker cpt1a, and all three agonists increased mitochondrial dehydrogenase hadh mRNA levels. These data correspond with a situation of enhanced lipolytic and ß-oxidation capacity, a fact supported by the higher glycerol released into the media induced by the agonists. Overall, these results suggest a hyperplastic growth condition and a favorable protein/fat ratio profile upon these treatments; consequently, ß2-agonists (especially SALM) may be considered good candidates to optimize the growth in this aquaculture species.

Contribution of in vitro myocytes studies to understanding fish muscle physiology.

Research on the regulation of fish muscle physiology and growth was addressed originally by classical in vivo approaches; however, systemic interactions resulted in many questions that could be better considered through in vitro myocyte studies. The first paper published by our group in this field was with Tom Moon on brown trout cardiomyocytes, where the insulin and IGF-I receptors were characterized and the down-regulatory effects of an excess of peptides demonstrated. We followed the research on cultured skeletal muscle cells through the collaboration with INRA focused on the characterization of IGF-I receptors and its signaling pathways through in vitro development. Later on, we showed the important metabolic role of IGFs, although these studies were only the first stage of a prolific area of work that has offered a useful tool to advance in our knowledge of the endocrine and nutritional regulation of fish growth and metabolism. Obviously, the findings obtained in vitro serve the purpose to propose the scenario that will need confirmation in vivo, but this technique has made possible many different, easy, fast and better controlled studies. In this review, we have summarized the main advances that the use of cultured muscle cells has permitted, focusing mainly in the role of IGFs regulating fish metabolism and growth. Although many articles have already appeared using this model system in salmonids, gilthead sea bream or zebrafish, it is reasonable to expect new studies with cultured cells using innovative approaches that will help to understand fish physiology and its regulation.

Memory Improvement in the AßPP/PS1 Mouse Model of Familial Alzheimer's Disease Induced by Carbamylated-Erythropoietin is Accompanied by Modulation of Synaptic Genes.

Neuroprotection of erythropoietin (EPO) following long-term administration is hampered by the associated undesirable effects on hematopoiesis and body weight. For this reason, we tested carbamylated-EPO (CEPO), which has no effect on erythropoiesis, and compared it with EPO in the AßPP/PS1 mouse model of familial Alzheimer's disease. Groups of 5-month old wild type (WT) and transgenic mice received chronic treatment consisting of CEPO (2,500 or 5,000 UI/kg) or EPO (2,500 U I/kg) 3 days/week for 4 weeks. Memory at the end of treatment was assessed with the object recognition test. Microarray analysis and quantitative-PCR were used for gene expression studies. No alterations in erythropoiesis were observed in CEPO-treated WT and AßPP/PS1 transgenic mice. EPO and CEPO improved memory in AßPP/PS1 animals. However, only EPO decreased amyloid-ß (Aß)plaque burden and soluble Aß(40). Microarray analysis of gene expression revealed a limited number of common genes modulated by EPO and CEPO. CEPO but not EPO significantly increased gene expression of dopamine receptors 1 and 2, and adenosine receptor 2a, and significantly down-regulated adrenergic receptor 1D and gastrin releasing peptide. CEPO treatment resulted in higher protein levels of dopamine receptors 1 and 2 in WT and AßPP/PS1 animals, whereas the adenosine receptor 2a was reduced in WT animals. The present results suggest that the improved behavior observed in AßPP/PS1 transgenic mice after CEPO treatment may be mediated, at least in part, by the observed modulation of the expression of molecules involved in neurotransmission.

IGF-I and amino acids effects through TOR signaling on proliferation and differentiation of gilthead sea bream cultured myocytes.

Skeletal muscle growth and development is controlled by nutritional (amino acids, AA) as well as hormonal factors (insulin-like growth factor, IGF-I); however, how its interaction modulates muscle mass in fish is not clearly elucidated. The purpose of this study was to analyze the development of gilthead sea bream cultured myocytes to describe the effects of AA and IGF-I on proliferating cell nuclear antigen (PCNA) and myogenic regulatory factors (MRFs) expression, as well as on the transduction pathways involved in its signaling (TOR/AKT). Our results showed that AA and IGF-I separately increased the number of PCNA-positive cells and, together produced a synergistic effect. Furthermore, AA and IGF-I, combined or separately, increased significantly Myogenin protein expression, whereas MyoD was not affected. These results indicate a role for these factors in myocyte proliferation and differentiation. At the mRNA level, AA significantly enhanced PCNA expression, but no effects were observed on the expression of the MRFs or AKT2 and FOXO3 upon treatment. Nonetheless, we demonstrated for the first time in gilthead sea bream that AA significantly increased the gene expression of TOR and its downstream effectors 4EBP1 and 70S6K, with IGF-I having a supporting role on 4EBP1 up-regulation. Moreover, AA and IGF-I also activated TOR and AKT by phosphorylation, respectively, being this activation decreased by specific inhibitors. In summary, the present study demonstrates the importance of TOR signaling on the stimulatory role of AA and IGF-I in gilthead sea bream myogenesis and contributes to better understand the potential regulation of muscle growth and development in fish.

Neuronal hemoglobin is reduced in Alzheimer's disease, argyrophilic grain disease, Parkinson's disease, and dementia with Lewy bodies.

Previous studies have demonstrated the presence of hemoglobin α-chain and ß-chain in neurons of the rodent and human brain thus indicating that hemoglobin is a normal component of nerve cells and that hemoglobin may play a role in intraneuronal oxygen homeostasis. Progressing with these studies, hemoglobin expression has been examined in selected cell population in the brains of Alzheimer's disease (AD), argyrophilic grain disease (AGD), Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Double labeling immunofluorescence and confocal microscopy revealed reduced hemoglobin α-chain and ß-chain in practically all neurons with small amounts of granular or punctuate hyperphosphorylated tau deposits and in neurons with tangles in the hippocampus and frontal cortex in AD and in the hippocampus in AGD; in ballooned neurons containing αB-crystallin in the amygdala in AD and AGD; and in about 80% of neurons with punctuate α-synuclein deposits and in neurons with Lewy bodies in the substantia nigra pars compacta and in vulnerable neurons of the medulla oblongata in PD and DLB; and in neurons with Lewy bodies in the frontal cortex in DLB. Hemoglobin immunoreactivity was also observed in the core of neuritic plaques and in diffuse plaques, but not in dystrophic neurites. Loss of hemoglobin was specific as neuroglobin was present equally in neurons with and without abnormal protein inclusions, and erythropoietin receptor was expressed equally in neurons without and in neurons with abnormal protein aggregates in AD, AGD, PD, and DLB.

Analysis of highly phosphorylated inositols in avian and crocodilian erythrocytes.

Both morphological and paleontological characteristics support the hypothesis of a monophyletic origin of crocodilian and avian groups. However, while the erythrocytes of all birds studied to date are reported to contain high levels of inositol pentakisphosphate (InsP(5)), which acts as an allosteric effector of hemoglobin, this molecule has not been reported in crocodilian erythrocytes. In this study we compare the highly phosphorylated inositols in crocodilian and avian erythrocytes using a particularly sensitive analytical procedure. Our aim was to obtain new data which might provide further evidence for the monophyletic origin, or otherwise, of crocodiles and birds. We studied three avian and three crocodilian species. The erythrocytes of the three bird species contained low levels of inositol-3,4,5,6-tetrakisphosphate and inositol-1,3,4,6-tetrakisphosphate, thought to be precursors of Ins(1,3,4,5,6)P(5). The crocodilian erythrocytes studied contained Ins(1,3,4,5,6)P(5) and InsP(6) in higher concentrations than those found in mammal erythrocytes and in other more active cells such as macrophages. Our data provide further evidence of the similarity between crocodilian and avian groups and agree with the hypothesis that both groups evolved from a common ancestor. The process by which the function of inositol phosphates changed from that of intracellular signaling to hemoglobin allosteric effector is discussed.

A straightforward method for analysis of highly phosphorylated inositols in blood cells by high-performance liquid chromatography.

Soluble inositol polyphosphates are found in many cells. The trisphosphate isomers, mainly inositol-1,4,5-trisphosphate, have been extensively studied because of their involvement in signal transduction. However, higher phosphorylated inositols are less frequently studied and their physiological role is poorly understood. Among these, only the myo-inositol-1,3,4,5,6-pentakisphosphate (Ins1,3,4,5,6P5), an important component of bird erythrocytes, has been intensively studied in comparative studies because it is a potent allosteric effector of hemoglobin and decreases its affinity to oxygen. We have developed a procedure for the analysis of inositol polyphosphates and other phosphate compounds in vertebrate blood cells based on a quick and accurate HPLC separation coupled to metal-dye detection. The procedure includes acid extraction of cellular phosphates, acid elimination and concentration of the extract, HPLC separation of phosphate compounds, and quantification by coupled highly sensitive metal-dye detection. The method is especially useful for analyses of highly phosphorylated inositols and for red cell comparative studies. Using the described method we have quantified Ins1,3,4,5,6P5 and the low quantities of InsP6 found in bird erythrocytes. We also identified traces of Ins3,4,5,6P4 and Ins1,3,4,6P4. Moreover, by applying the method in cultured murine macrophages, we have found changes of highly phosphorylated inositols when these cells are activated by lipopolysaccharide.