Soluble adenylyl cyclase is an acid-base sensor in rainbow trout red blood cells that regulates intracellular pH and haemoglobin-oxygen binding.

AIM: To identify the physiological role of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout. METHODS: We used: (i) super-resolution microscopy to determine the subcellular location of sAC protein; (ii) live-cell imaging of RBC intracellular pH (pHi) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid-base regulation; (iii) spectrophotometric measurements of haemoglobin-oxygen (Hb-O2) binding in steady-state conditions; and (iv) during simulated arterial-venous transit, to determine the role of sAC in systemic O2 transport. RESULTS: Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pHi regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pHi recovery after an acid-base disturbance. RBC pHi recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO3 --dependent sAC signaling. Inhibition of sAC decreased Hb-O2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O2 binding. During in vitro simulations of arterial-venous transit, sAC inhibition decreased the amount of O2 that is unloaded by ~11%. CONCLUSION: sAC represents a novel acid-base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pHi and blood O2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

GPCR Signaling Measurement and Drug Profiling with an Automated Live-Cell Microscopy System.

A major limitation of time-lapse microscopy combined with fluorescent biosensors, a powerful tool for quantifying spatiotemporal dynamics of signaling in single living cells, is low-experimental throughput. To overcome this limitation, we created a highly customizable, MATLAB-based platform: flexible automated liquid-handling combined microscope (FALCOscope) that coordinates an OpenTrons liquid handler and a fluorescence microscope to automate drug treatments, fluorescence imaging, and single-cell analysis. To test the feasibility of the FALCOscope, we quantified G protein-coupled receptor (GPCR)-stimulated Protein Kinase A activity and cAMP responses to GPCR agonists and antagonists. We also characterized cAMP dynamics induced by GPR68/OGR1, a proton-sensing GPCR, in response to variable extracellular pH values. GPR68-induced cAMP responses were more transient in acidic than neutral pH values, suggesting a pH-dependence for signal attenuation. Ogerin, a GPR68 positive allosteric modulator, enhanced cAMP response most strongly at pH 7.0 and sustained cAMP response for acidic pH values, thereby demonstrating the capability of the FALCOscope to capture allosteric modulation. At a high concentration, ogerin increased cAMP signaling independent of GPR68, likely via phosphodiesterase inhibition. The FALCOscope system thus enables enhanced throughput single-cell dynamic measurements and is a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells and for drug profiling and screening.

PDE4B Is a Homeostatic Regulator of Cyclic AMP in Dendritic Cells.

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings, but how cells compensate for such decreases is unknown. We have used a unique system-murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein Gαs-to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in Gαs-depleted DCs. PDE4B expression is dynamic, falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations, respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations.

Detection of GPCR mRNA Expression in Primary Cells Via qPCR, Microarrays, and RNA-Sequencing.

A workflow is described for assaying the expression of G protein-coupled receptors (GPCRs) in cultured cells, using a combination of methods that assess GPCR mRNAs. Beginning from the isolation of cDNA and preparation of mRNA, we provide protocols for designing and testing qPCR primers, assaying mRNA expression using qPCR and high-throughput analysis of GPCR mRNA expression via TaqMan qPCR-based, GPCR-selective arrays. We also provide a workflow for analysis of expression from RNA-sequencing (RNA-seq) assays, which can be queried to yield expression of GPCRs and related genes in samples of interest, as well as to test changes in expression between groups, such as in cells treated with drugs or from healthy and diseased subjects. We place priority on optimized protocols that distinguish signal from noise, as GPCR mRNAs are typically present in low abundance, necessitating techniques that maximize sensitivity while minimizing noise. These methods may also be applicable for assessing the expression of members of families of other low abundance genes via high-throughput analyses of mRNAs, followed by independent confirmation and validation of results via qPCR.

Molecular and biochemical characterization of the bicarbonate-sensing soluble adenylyl cyclase from a bony fish, the rainbow trout Oncorhynchus mykiss.

Soluble adenylyl cyclase (sAC) is a HC O 3 - -stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid-base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by HC O 3 - at physiologically relevant levels for fish internal fluids (EC50 ∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid-base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.

Proton-sensing G protein-coupled receptors: detectors of tumor acidosis and candidate drug targets.

Cells in tumor microenvironments (TMEs) use several mechanisms to sense their low pH (<7.0), including via proton-sensing G protein-coupled receptors (psGPCRs): GPR4, GPR65/TDAG8, GPR68/OGR1 and GPR132/G2A. Numerous cancers have increased expression of psGPCRs. The psGPCRs may contribute to features of the malignant phenotype via actions on specific cell-types in the TME and thereby promote tumor survival and growth. Here, we review data regarding psGPCR expression in tumors and cancer cells, impact of psGPCRs on survival in solid tumors and a bioinformatics approach to infer psGPCR expression in cell types in the TME. New tools are needed to help define contributions of psGPCRs in tumor biology and to identify potentially novel therapeutic agents for a variety of cancers.

GPCRs in pancreatic adenocarcinoma: Contributors to tumour biology and novel therapeutic targets.

Pancreatic cancer has one of the highest mortality rates (5-year survival ~9%) among cancers. Pancreatic adenocarcinoma (PAAD) is the most common (>80%) and the most lethal type of pancreatic cancer. A need exists for new approaches to treat pancreatic adenocarcinoma. GPCRs, the largest family of cell-surface receptors and drug targets, account for ~35% of approved drugs. Recent studies have revealed roles for GPCRs in PAAD cells and cells in the tumour micro-environment. This review assesses current information regarding GPCRs in PAAD by summarizing omics data for GPCRs expression in PAAD. The PAAD "GPCRome" includes GPCRs with approved agents, thereby offering potential for their repurposing/repositioning. We then reviewed the evidence for functional roles of specific GPCRs in PAAD. We also highlight gaps in understanding the contribution of GPCRs to PAAD biology and identify several GPCRs that may be novel therapeutic targets for future work in search of GPCR-targeted drugs to treat PAAD tumours.

Detection and Quantification of GPCR mRNA: An Assessment and Implications of Data from High-Content Methods.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and targets for approved drugs. The analysis of GPCR expression is, thus, important for drug discovery and typically involves messenger RNA (mRNA)-based methods. We compared transcriptomic complementary DNA (cDNA) (Affymetrix) microarrays, RNA sequencing (RNA-seq), and quantitative polymerase chain reaction (qPCR)-based TaqMan arrays for their ability to detect and quantify expression of endoGPCRs (nonchemosensory GPCRs with endogenous agonists). In human pancreatic cancer-associated fibroblasts, RNA-seq and TaqMan arrays yielded closely correlated values for GPCR number (∼100) and expression levels, as validated by independent qPCR. By contrast, the microarrays failed to identify ∼30 such GPCRs and generated data poorly correlated with results from those methods. RNA-seq and TaqMan arrays also yielded comparable results for GPCRs in human cardiac fibroblasts, pancreatic stellate cells, cancer cell lines, and pulmonary arterial smooth muscle cells. The magnitude of mRNA expression for several Gq/11-coupled GPCRs predicted cytosolic calcium increase and cell migration by cognate agonists. RNA-seq also revealed splice variants for endoGPCRs. Thus, RNA-seq and qPCR-based arrays are much better suited than transcriptomic cDNA microarrays for assessing GPCR expression and can yield results predictive of functional responses, findings that have implications for GPCR biology and drug discovery.

GPCRomics: An Approach to Discover GPCR Drug Targets.

G protein-coupled receptors (GPCRs) are targets for ∼35% of approved drugs but only ∼15% of the ∼800 human GPCRs are currently such targets. GPCRomics, the use of unbiased, hypothesis-generating methods [e.g., RNA-sequencing (RNA-seq)], with tissues and cell types to identify and quantify GPCR expression, has led to the discovery of previously unrecognized GPCRs that contribute to functional responses and pathophysiology and that may be therapeutic targets. The combination of GPCR expression data with validation studies (e.g., signaling and functional activities) provides opportunities for the discovery of disease-relevant GPCR targets and therapeutics. Here, we review insights from GPCRomic approaches, gaps in knowledge, and future directions by which GPCRomics can advance GPCR biology and the discovery of new GPCR-targeted drugs.

GPR68: An Emerging Drug Target in Cancer.

GPR68 (or ovarian cancer G protein-coupled receptor 1, OGR1) is a proton-sensing G-protein-coupled receptor (GPCR) that responds to extracellular acidity and regulates a variety of cellular functions. Acidosis is considered a defining hallmark of the tumor microenvironment (TME). GPR68 expression is highly upregulated in numerous types of cancer. Emerging evidence has revealed that GPR68 may play crucial roles in tumor biology, including tumorigenesis, tumor growth, and metastasis. This review summarizes current knowledge regarding GPR68-its expression, regulation, signaling pathways, physiological roles, and functions it regulates in human cancers (including prostate, colon and pancreatic cancer, melanoma, medulloblastoma, and myelodysplastic syndrome). The findings provide evidence for GPR68 as a potentially novel therapeutic target but in addition, we note challenges in developing drugs that target GPR68.

Molecular, Enzymatic, and Cellular Characterization of Soluble Adenylyl Cyclase From Aquatic Animals.

The enzyme soluble adenylyl cyclase (sAC) is the most recently identified source of the messenger molecule cyclic adenosine monophosphate. sAC is evolutionarily conserved from cyanobacteria to human, is directly stimulated by [Formula: see text] ions, and can act as a sensor of environmental and metabolic CO2, pH, and [Formula: see text] levels. sAC genes tend to have multiple alternative promoters, undergo extensive alternative splicing, be translated into low mRNA levels, and the numerous sAC protein isoforms may be present in various subcellular localizations. In aquatic organisms, sAC has been shown to mediate various functions including intracellular pH regulation in coral, blood acid/base regulation in shark, heart beat rate in hagfish, and NaCl absorption in fish intestine. Furthermore, sAC is present in multiple other species and tissues, and sAC protein and enzymatic activity have been reported in the cytoplasm, the nucleus, and other subcellular compartments, suggesting even more diverse physiological roles. Although the methods and experimental tools used to study sAC are conventional, the complexity of sAC genes and proteins requires special considerations that are discussed in this chapter.

Adipogenesis in fish.

White adipose tissue (AT) is the main lipid storage depot in vertebrates. Initially considered to be a simple lipid store, AT has recently been recognized as playing a role as an endocrine organ that is implicated in processes such as energy homeostasis and as a rich source of stem cells. Interest in adipogenesis has increased not only because of the prevalence of obesity, metabolic syndrome and type 2 diabetes in humans, but also in aquaculture because of the excessive fat deposition experienced in some cultured fish species, which may compromise both their welfare and their final product quality. Adipocyte development is well conserved among vertebrates, and this conservation has facilitated the rapid characterization of several adipogenesis models in fish. This Review presents the main findings of adipogenesis research based in primary cultures of the preadipocytes of farmed fish species. Zebrafish has emerged as an excellent model for studying the early stages of adipocyte fish development in vivo Nevertheless, larger fish species are more suitable for the isolation of preadipocytes from visceral AT and for studies in which preadipocytes are differentiated in vitro to form mature adipocytes. Differentiated adipocytes contain lipid droplets and express adipocyte marker genes such as those encoding the peroxisome proliferator activated receptor γ (pparγ), CCAAT-enhancer-binding protein α (c/ebpα), lipoprotein lipase (lpl), fatty acid synthase (fas), fatty acid binding protein 11 (fabp11), fatty acid transporter protein1 (fatp1), adiponectin and leptin. Differentiated adipocytes also have elevated glycerol 3-phosphate (G3P) dehydrogenase (GPDH) activity. To better understand fish adipocyte development and regulation, different adipokines, fatty acids, growth factors and PPAR agonists have been studied, providing relevant insights into which factors affect these processes and counterbalance AT dysregulation.

Proteolytic systems' expression during myogenesis and transcriptional regulation by amino acids in gilthead sea bream cultured muscle cells.

Proteolytic systems exert an important role in vertebrate muscle controlling protein turnover, recycling of amino acids (AA) or its use for energy production, as well as other functions like myogenesis. In fish, proteolytic systems are crucial for the relatively high muscle somatic index they possess, and because protein is the most important dietary component. Thus in this study, the molecular profile of proteolytic markers (calpains, cathepsins and ubiquitin-proteasome system (UbP) members) were analyzed during gilthead sea bream (Sparus aurata) myogenesis in vitro and under different AA treatments. The gene expression of calpains (capn1, capn3 and capns1b) decreased progressively during myogenesis together with the proteasome member n3; whereas capn2, capns1a, capns1b and ubiquitin (ub) remained stable. Contrarily, the cathepsin D (ctsd) paralogs and E3 ubiquitin ligases mafbx and murf1, showed a significant peak in gene expression at day 8 of culture that slightly decreased afterwards. Moreover, the protein expression analyzed for selected molecules presented in general the same profile of the mRNA levels, which was confirmed by correlation analysis. These data suggest that calpains seem to be more important during proliferation, while cathepsins and the UbP system appear to be required for myogenic differentiation. Concerning the transcriptional regulation by AA, the recovery of their levels after a short starvation period did not show effects on cathepsins expression, whereas it down-regulated the expression of capn3, capns1b, mafbx, murf1 and up-regulated n3. With regards to AA deficiencies, the major changes occurred at day 2, when leucine limitation suppressed ctsb and ctsl expression. Besides at the same time, both leucine and lysine deficiencies increased the expression of mafbx and murf1 and decreased that of n3. Overall, the opposite nutritional regulation observed, especially for the UbP members, points out an efficient and complementary role of these factors that could be useful in gilthead sea bream diets optimization.

Adipogenic Gene Expression in Gilthead Sea Bream Mesenchymal Stem Cells from Different Origin.

During the last decades, adipogenesis has become an emerging field of study in aquaculture due to the relevance of the adipose tissue in many physiological processes and its connection with the endocrine system. In this sense, recent studies have translated into the establishment of preadipocyte culture models from several fish species, sometimes lacking information on the mRNA levels of adipogenic genes. Thus, the aim of this study was to determine the gene expression profile of gilthead sea bream (Sparus aurata) primary cultured mesenchymal stem cells (MSCs) from different origin (adipose tissue and vertebra bone) during adipogenesis. Both cell types differentiated into adipocyte-like cells, accumulating lipids inside their cytoplasm. Adipocyte differentiation of MSCs from adipose tissue resulted in downregulation of several adipocyte-related genes (such as lpl, hsl, pparα, pparγ and gapdh2) at day 4, gapdh1 at day 8, and fas and pparß at day 12. In contrast, differences in lxrα mRNA expression were not observed, while g6pdh levels increased during adipocyte maturation. Gapdh and Pparγ protein levels were also detected in preadipocyte cultures; however, only the former increased its expression during adipogenesis. Moreover, differentiation of bone-derived cells into adipocytes also resulted in the downregulation of several adipocyte gene markers, such as fas and g6pdh at day 10 and hsl, pparß, and lxrα at day 15. On the other hand, the osteogenic genes fib1a, mgp, and op remained stable, but an increase in runx2 expression at day 20 was observed. In summary, the present study demonstrates that gilthead sea bream MSCs, from both adipose tissue and bone, differentiate into adipocyte-like cells, although revealed some kind of species- and cell lineage-specific regulation with regards to gene expression. Present data also provide novel insights into some of the potential key genes controlling adipogenesis in gilthead sea bream that can help to better understand the regulation of lipid storage in fish.

Characterization data of gilthead sea bream (Sparus aurata) IGF-I receptors (IGF-IRa/Rb).

In this data article we describe the coding sequence of two IGF-IR paralogues (IGF-IRa and IGF-IRb) obtained from gilthead sea bream embryos. The putative protein architecture (domains and other important motifs) was determined and, amino acid sequences alignment and phylogenetic analysis of both receptors together with IGF-IR orthologues from different vertebrates was performed. Additionally, a semi-quantitative conventional PCR was done to analyze the mRNA expression of both receptors in different tissues of gilthead sea bream. These data will assist in further physiological studies in this species. In this sense, the expression of both receptors during ontogeny in muscle as well as the differential effects of IGF-I and IGF-II on their regulation during in vitro myogenesis has been recently studied (doi: 10.1016/j.ygcen.2015.11.011; [1]).

IGF-I and IGF-II effects on local IGF system and signaling pathways in gilthead sea bream (Sparus aurata) cultured myocytes.

The insulin-like growth factors (IGFs) have a fundamental role in a vast range of functions acting through a tyrosine-kinase receptor (IGF-IR). IGFs in muscle can affect the expression of components of the local IGF system, myogenic regulatory factors (MRFs), proliferating (proliferating cell nuclear antigen, PCNA) or differentiating molecules (myosin heavy chain, MHC) and, lead to the activation of different signaling pathways. The response of all these genes to IGFs incubation at two different times in day 4 cultured myocytes of gilthead sea bream was analyzed. Both IGFs increased the expression of IGF-I and IGFBP-5, but showed different effects on the receptors, with IGF-I suppressing the expression of both isoforms (IGF-IRa and IGF-IRb) and IGF-II up-regulating only IGF-IRb. Moreover, the protein levels of PCNA and target of rapamycin (TOR) increased after IGF-II incubation, although a decline in Myf5 and a rise in MHC gene expression was caused by IGF-I. Taken together, these results provide evidence for the importance of IGFs on controlling muscle development and growth in gilthead sea bream and suggest that each IGF may be preferentially acting through a specific IGF-IR. Moreover, the data support the hypothesis that IGF-II has a more important role during proliferation, whereas IGF-I seems to be relevant for the differentiation phase of myogenesis.

Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro.

Leptin and ghrelin are important regulators of energy homeostasis in mammals, whereas their physiological roles in fish have not been fully elucidated. In the present study, the effects of leptin and ghrelin on adipogenesis, lipolysis and on expression of lipid metabolism-related genes were examined in rainbow trout adipocytes in vitro. Leptin expression and release increased from preadipocytes to mature adipocytes in culture, but did not affect the process of adipogenesis. While ghrelin and its receptor were identified in cultured differentiated adipocytes, ghrelin did not influence either preadipocyte proliferation or differentiation, indicating that it may have other adipose-related roles. Leptin and ghrelin increased lipolysis in mature freshly isolated adipocytes, but mRNA expression of lipolysis markers was not significantly modified. Leptin significantly suppressed the fatty acid transporter-1 expression, suggesting a decrease in fatty acid uptake and storage, but did not affect expression of any of the lipogenesis or ß-oxidation genes studied. Ghrelin significantly increased the mRNA levels of lipoprotein lipase, fatty acid synthase and peroxisome proliferator-activated receptor-ß, and thus appears to stimulate synthesis of triglycerides as well as their mobilization. Overall, the study indicates that ghrelin, but not leptin seems to be an enhancer of lipid turn-over in adipose tissue of rainbow trout, and this regulation may at least partly be mediated through autocrine/paracrine mechanisms. The mode of action of both hormones needs to be further explored to better understand their roles in regulating adiposity in fish.

Characterisation and expression analysis of cathepsins and ubiquitin-proteasome genes in gilthead sea bream (Sparus aurata) skeletal muscle.

BACKGROUND: The proteolytic enzymes involved in normal protein turnover in fish muscle are also responsible for post-mortem softening of the flesh and are therefore potential determinants of product quality. The main enzyme systems involved are calpains, cathepsins, and the ubiquitin-proteasome (UbP). In this study on Sparus aurata (Sa), the coding sequences of cathepsins (SaCTSB and SaCTSDb) and UbP family members (SaN3 and SaUb) were cloned from fast skeletal muscle, and their expression patterns were examined during ontogeny and in a fasting/re-feeding experiment. RESULTS: The amino acid sequences identified shared 66-100% overall identity with their orthologues in other vertebrates, with well conserved characteristic functional domains and catalytic residues. SaCTSDb showed phylogenetic, sequence and tissue distribution differences with respect to its paralogue SaCTSDa, previously identified in the ovary. Expression of gilthead sea bream cathepsins (B, L, Da, Db) and UbP members (N3, Ub, MuRF1 and MAFbx) in fast skeletal muscle was determined at three different life-history stages and in response to fasting and re-feeding in juveniles. Most of the proteolytic genes analysed were significantly up-regulated during fasting, and down-regulated with re-feeding and, between the fingerling (15 g) and juvenile/adult stages (~50/500 g), consistent with a decrease in muscle proteolysis in both later contexts. In contrast, SaCTSDa and SaMuRF1 expression was relatively stable with ontogeny and SaUb had higher expression in fingerlings and adults than juveniles. CONCLUSIONS: The data obtained in the present study suggest that cathepsins and UbP genes in gilthead sea bream are co-ordinately regulated during ontogeny to control muscle growth, and indicate that feeding regimes can modulate their expression, providing a potential dietary method of influencing post-mortem fillet tenderisation, and hence, product quality.

Effects of nutritional status on plasma leptin levels and in vitro regulation of adipocyte leptin expression and secretion in rainbow trout.

As leptin has a key role on appetite, knowledge about leptin regulation is important in order to understand the control of energy balance. We aimed to explore the modulatory effects of adiposity on plasma leptin levels in vivo and the role of potential regulators on leptin expression and secretion in rainbow trout adipocytes in vitro. Fish were fed a regular diet twice daily ad libitum or a high-energy diet once daily at two ration levels; satiation (SA group) or restricted (RE group) to 25% of satiation, for 8weeks. RE fish had significantly reduced growth (p<0.001) and adipose tissue weight (p<0.001), and higher plasma leptin levels (p=0.022) compared with SA fish. Moreover, plasma leptin levels negatively correlated with mesenteric fat index (p=0.009). Adipocytes isolated from the different fish were treated with insulin, ghrelin, leucine, eicosapentaenoic acid or left untreated (control). In adipocytes from fish fed regular diet, insulin and ghrelin increased leptin secretion dose-dependently (p=0.002; p=0.033, respectively). Leptin secretion in control adipocytes was significantly higher in RE than in SA fish (p=0.022) in agreement with the in vivo findings, indicating that adipose tissue may contribute to the circulating leptin levels. No treatment effects were observed in adipocytes from the high-energy diet groups, neither in leptin expression nor secretion, except that leptin secretion was significantly reduced by leucine in RE fish adipocytes (p=0.025). Overall, these data show that the regulation of leptin in rainbow trout adipocytes by hormones and nutrients seems to be on secretion, rather than at the transcriptional level.

Characterisation and expression of calpain family members in relation to nutritional status, diet composition and flesh texture in gilthead sea bream (Sparus aurata).

Calpains are non-lysosomal calcium-activated neutral proteases involved in a wide range of cellular processes including muscle proteolysis linked to post-mortem flesh softening. The aims of this study were (a) to characterise several members of the calpain system in gilthead sea bream and (b) to examine their expression in relation to nutritional status and muscle tenderisation. We identified the complete open reading frame of gilthead sea bream calpains1-3, sacapn1, sacapn2, sacapn3, and two paralogs of the calpain small subunit1, sacapns1a and sacapns1b. Proteins showed 63-90% sequence identity compared with sequences from mammals and other teleost fishes, and the characteristic domain structure of vertebrate calpains. Transcripts of sacapn1, sacapn2, sacapns1a and sacapns1b had a wide tissue distribution, whereas sacapn3 was almost exclusively detected in skeletal muscle. Next, we assessed transcript expression in skeletal muscle following alteration of nutritional status by (a) fasting and re-feeding or (b) feeding four experimental diets with different carbohydrate-to-protein ratios. Fasting significantly reduced plasma glucose and increased free fatty acids and triglycerides, together with a significant increase in sacapns1b expression. Following 7 days of re-feeding, plasma parameters returned to fed values and sacapn1, sacapn2, sacapns1a and sacapns1b expression was significantly reduced. Furthermore, an increase in dietary carbohydrate content (11 to 39%) diminished growth but increased muscle texture, which showed a significant correlation with decreased sacapn1 and sacapns1a expression, whilst the other calpains remained unaffected. This study has demonstrated that calpain expression is modulated by nutritional status and diet composition in gilthead sea bream, and that the expression of several calpain members is correlated with muscle texture, indicating their potential use as molecular markers for flesh quality in aquaculture production.