Josep V. Planas

Somatolactin stimulates in vitro gonadal steroidogenesis in coho salmon, Oncorhynchus kisutch.

Somatolactin (SL), a newly discovered pituitary hormone of the teleost pars intermedia, is structurally similar to prolactin and growth hormone. The function(s) of SL are not yet established, although evidence suggests that it may play a role in reproduction. Possible steroidogenic activity of coho salmon SL was tested and compared with gonadotropin I (GTH I) in incubations of ovarian follicles or testicular fragments. SL stimulated production of 11-ketotestosterone and testosterone by testicular fragments, and production of estradiol by ovarian follicles in a concentration-dependent manner. However, the steroidogenic activity of SL was considerably less than that of GTH I. These results suggest that SL may play a role in regulation of gonadal function in salmon.

Regulation of Ovarian Steroidogenesis In Vitro by Follicle-Stimulating Hormone and Luteinizing Hormone During Sexual Maturation in Salmonid Fish

Abstract The regulation of ovarian steroidogenesis in vitro by coho salmon FSH and LH was investigated in intact coho salmon follicles and isolated follicular layers at various stages of oocyte maturation, from late vitellogenesis until the completion of germinal vesicle breakdown (GVBD). In granulosa layers from all stages, LH, but not FSH, stimulated 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) production. In theca-interstitial layers from all stages, FSH and LH stimulated steroid production, LH being more potent than FSH. The basal steroid output of intact follicles was significantly lower than that of isolated follicular layers, and their response to FSH and LH also differed. First, the intact follicles produced 17α-hydroxyprogesterone in response to FSH during the central germinal vesicle stage while theca-interstitial layers did not. Second, estradiol-17β production was not inhibited by LH during final oocyte maturation in intact follicles, as observed for granulosa layers. Our results indicate that LH is the determining factor regulating the production of the maturation-inducing steroid, 17,20β-P, and the induction of GVBD in the salmonid ovary. In summary, we have provided evidence for maturation-associated changes in the effects of FSH and LH in the salmonid ovary, which further supports the hypothesis that FSH and LH have distinct functions in the teleost ovary.

Mutation of the RIIβ Subunit of Protein Kinase A Differentially Affects Lipolysis but Not Gene Induction in White Adipose Tissue

Targeted disruption of the RIIβ subunit of protein kinase A (PKA) produces lean mice that resist diet-induced obesity. In this report we examine the effects of the RIIβ knockout on white adipose tissue physiology. Loss of RIIβ is compensated by an increase in the RIα isoform, generating an isoform switch from a type II to a type I PKA. Type I holoenzyme binds cAMP more avidly and is more easily activated than the type II enzyme. These alterations are associated with increases in both basal kinase activity and the basal rate of lipolysis, possibly contributing to the lean phenotype. However, the ability of both β3-selective and nonspecific β-adrenergic agonists to stimulate lipolysis is markedly compromised in mutant white adipose tissue. This defect was foundin vitro and in vivo and does not result from reduced expression of β-adrenergic receptor or hormone-sensitive lipase genes. In contrast, β-adrenergic stimulated gene transcription remains intact, and the expression of key genes involved in lipid metabolism is normal under both fasted and fed conditions. We suggest that the R subunit isoform switch disrupts the subcellular localization of PKA that is required for efficient transduction of signals that modulate lipolysis but not for those that mediate gene expression.

Expression of rainbow trout glucose transporters GLUT1 and GLUT4 during in vitro muscle cell differentiation and regulation by insulin and IGF-I

Insulin is an important factor for the maintenance of glucose homeostasis, enhancing glucose uptake in its target tissues in a process that has been conserved between fish and mammals. In fish skeletal muscle cells, like in mammals, insulin promotes GLUT4 translocation to the plasma membrane and, consequently, glucose uptake, but its role regulating the expression of glucose transporters in vitro has not been demonstrated to date. Thus, we investigated the expression of GLUT4 and GLUT1 throughout skeletal muscle cell differentiation and their regulation by insulin and IGF-I using a primary culture of trout muscle cells. GLUT4 expression gradually increased during the muscle cell differentiation process, whereas GLUT1 expression remained fairly constant. Insulin and IGF-I similarly increased the mRNA levels of GLUT4 in myoblasts and myotubes. On the other hand, IGF-I appeared to be more potent than insulin in stimulating GLUT1 expression, particularly at the myoblast stage. Therefore, this work provides the first demonstration in nonmammalian vertebrates that insulin and IGF-I may act directly on trout muscle cells to regulate the expression of GLUT4 and GLUT1.

Physiological regulation of glucose transporter (GLUT4) protein content in brown trout (Salmo trutta) skeletal muscle.

SUMMARY In brown trout, red and white skeletal muscle express the insulin-regulatable glucose transporter 4 (btGLUT4). We have previously shown that the mRNA expression of btGLUT4 in red muscle, but not white muscle, is altered under experimental conditions designed to cause changes in the plasma levels of insulin, such as fasting, insulin and arginine administration. In order to determine whether changes of btGLUT4 expression at the mRNA level are correlated with changes at the protein level, we performed in vivo experiments to alter blood insulin concentrations and determined the abundance of btGLUT4 protein in trout red and white skeletal muscle by immunoblotting using an antibody to salmon GLUT4. In the present study we show that btGLUT4 protein content in red muscle decreases after fasting and increases after insulin administration. By contrast, btGLUT4 protein content in white muscle decreases after fasting but is not affected by insulin treatment. Our results show a good correlation between the changes observed in btGLUT4 protein and the previously reported changes in mRNA levels in response to alterations in circulating insulin, indicating that the regulation of btGLUT4 in brown trout takes place predominantly in the red skeletal muscle.

Evolutionary structural and functional conservation of an ortholog of the GLUT2 glucose transporter gene (SLC2A2) in zebrafish

In mammals, GLUT2 plays an essential role in glucose homeostasis. From an evolutionary perspective, relatively little is known about the biology of GLUT2, or other GLUTs, in nonmammalian vertebrates. Here, we have conducted studies to functionally characterize GLUT2 in zebrafish. First, we cloned the zebrafish ortholog of GLUT2 (zfGLUT2) encoding a protein of 504 amino acids with high-sequence identity to other known vertebrate GLUT2 proteins. The zfGLUT2 gene consists of 11 exons and 10 introns, spanning 20 kb and mapping to a region of chromosome 2 that exhibits conserved synteny with human chromosome 3. When expressed in Xenopus oocytes, zfGLUT2 transported 2-deoxyglucose (2-DG) with similar affinity than mammalian GLUT2 (K(m) of 11 mM). Transport of 2-DG was competed mostly by D-fructose and D-mannose and was inhibited by cytochalasin B. During early development, zfGLUT2 expression was detected already at 10 h postfertilization and remained elevated in 5-day larvae, when it was clearly localized to the liver and intestinal bulb. In the adult, zfGLUT2 expression was highest in testis, brain, skin, kidney, and intestine, followed by liver and muscle. In the intestine, zfGLUT2 transcripts were detected in absorptive enterocytes, and its mRNA levels were altered by fasting and refeeding, suggesting that its expression in the intestine may be regulated by the nutritional status. These results indicate that the structure and function of GLUT2 has been remarkably well conserved during vertebrate evolution and open the way for the use of zebrafish as a model species in which to study the biology and pathophysiology of GLUT2.

Swimming physiology of fish : towards using exercise to farm a fit fish in sustainable aquaculture

Part I Extreme swimming in the natural environment 1 Salmonid reproductive migration and effects on sexual maturation Hayashida K, Fukaya K, Palstra AP, Ueda H 2 Extreme swimming: the oceanic migrations of anguillid eels Righton D, Aarestrup K, Jellyman D, Sebert P, van den Thillart GEEJM , Tsukamoto K 3 Physiology of swimming and migration in tunas Shadwick RE, Schiller L, Fudge D Part II Potential impacts on swimming fish in aquaculture 4 Forced and preferred swimming speeds of fish ? A methodological approach Tudorache C, de Boeck G, Claireaux G 5 Effects of turbulence on fish swimming in aquaculture Liao JC, Cotel A 6 The effect of hypoxia on fish swimming performance and behaviour Domenici P, Herbert, NA, Lefrancois C, Steffensen JF, McKenzie DJ 7 Exercise, stress and welfare Huntingford FA, Kadri S Part III Nutrition, energy metabolism and muscular growth in swimming fish 8 Swimming enhanced growth Davison W, Herbert NA 9 Metabolic fuel utilization during swimming: Optimizing nutritional requirements for enhanced performance Magnoni LJ, Felip O, Blasco J, Planas JV 10 Transcriptomic and proteomic response of skeletal muscle to swimming-induced exercise in fish Planas JV, Martin-Perez M, Magnoni LJ, Blasco J, Ibarz A, Fernandez-Borras J, Palstra AP 11 Molecular adaptive mechanisms in the cardiac muscle of exercised fish Takle H, Castro V 12 Exercise effects on fish quality and implications for consumer preferences Rasmussen RS, Lopez Albors O, Alfnes F 13 Swimming effects on developing zebrafish Kranenbarg S, Pelster B 14 Exercise physiology of zebrafish: Swimming effects on skeletal and cardiac muscle growth, on the immune system and the involvement of the stress axis Palstra AP, Schaaf M, Planas JV Part IV Novel technologies for studying fish swimming and aquaculture applications 15 Swimming flumes as a tool for studying swimming behavior and physiology: current applications and future developments Ellerby DJ, Herskin J 16 Practical aspects of induced exercise in finfish aquaculture Herbert NA 17 Robotic fish to lead the school Rossi C, Coral W, Barrientos A

Direct involvement of tumor necrosis factor α in the regulation of glucose uptake in rainbow trout muscle cells

The proinflammatory cytokine TNF-α is known to have a direct action on skeletal muscle in mammals. However, little is known regarding the potential effects of cytokines on nonimmune tissues, particularly in skeletal muscle, in fish. The aim of this study was to investigate the effects of recombinant trout TNF-α (rtTNF-α) on skeletal muscle carbohydrate metabolism in rainbow trout (Oncorhynchus mykiss). We used a primary cell culture of muscle cells from rainbow trout to show that rtTNF-α stimulates glucose uptake in myoblasts and myotubes at concentrations that do not affect the viability of the cells, requiring de novo protein synthesis as shown by the impairment of rtTNF-α-stimulated glucose uptake by cycloheximide. With the use of specific inhibitors, we show that rtTNF-α-stimulated glucose uptake is mediated by the p38MAPK, NF-κB, and JNK pathways. Additionally, we provide evidence that the stimulatory effects of rtTNF-α on glucose uptake in trout skeletal muscle cells may be caused, at least in part, by an increase in the amount of GLUT4 at the plasma membrane. Incubation of trout muscle cells with conditioned medium from LPS-stimulated trout macrophages, enriched in TNF-α, increased glucose uptake. Our results indicate that recombinant, as well as native trout TNF-α, directly stimulates glucose uptake in trout muscle cells and provide evidence, for the first time in nonmammalian vertebrates, for a potential regulatory role of TNF-α in skeletal muscle metabolism.

Effects of Insulin-Like Growth Factor I (Igf-I) On Steroid Production By Isolated Ovarian Theca and Granulosa Layers of Preovulatory Coho Salmon

The effects of recombinant human and salmon insulin-like growth factor-I (IGF-I) on in vitro steroid production by isolated theca and granulosa cell-layers of the preovulatory coho salmon ovary were examined. Prior to germinal vesicle breakdown (GVBD), IGF-I decreased basal theca cell-layer production of testosterone (T) and 17α-hydroxy-progesterone (17OH-P) and also inhibited the stimulatory effects of gonadotropin II (GTH II) on T production. After GVBD, IGF-I lowered the GTH II-induced production of both steroids by theca cells. In contrast, IGF-I enhanced the stimulatory effects of GTH II on conversion of 17OH-P to 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) by granulosa cells during final oocyte maturation.

Saving energy to fuel exercise: swimming suppresses oocyte development and downregulates ovarian transcriptomic response of rainbow trout Oncorhynchus mykiss

Metabolic processes and sexual maturation closely interact during the long-distance reproductive migration of many fish species to their spawning grounds. In the present study, we have used exercise experimentally to investigate the effects on sexual maturation in rainbow trout. Pubertal autumn-spawning seawater-raised female rainbow trout Oncorhynchus mykiss (n = 26; 50 cm, 1.5 kg) were rested or swum at a near optimal speed of 0.75 body lengths per second in a 6,000-liter swim flume under natural reproductive conditions (16 degrees C fresh-water, starvation, 8:16-h light-dark photoperiod). Fish were sampled after arrival and subsequently after 10 days (resting or swimming 307 km) and 20 days (resting or swimming 636 km). Ovarian development was significantly reduced in the swimmers. Analysis of the expression of key factors in the reproductive axis included pituitary kiss1-receptor, lh, and fsh and ovarian lh-receptor, fsh-receptor, aromatase, and vitellogenin-receptor (vtgr). Swimmers had lower pituitary lh and ovarian vtgr expression than resters. Furthermore, the number of late vitellogenic oocytes was lower in swimmers than in resters, probably resulting from the lower vtgr expression, and vitellogenin plasma levels were higher. Therefore, swimming exercise suppresses oocyte development possibly by inhibiting vitellogenin uptake. Transcriptomic changes that occurred in the ovary of exercised fish were investigated using a salmonid cDNA microarray platform. Protein biosynthesis and energy provision were among the 16 functional categories that were all downregulated in the ovary. Downregulation of the transcriptomic response in the ovary illustrates the priority of energy reallocation and will save energy to fuel exercise. A swimming-induced ovarian developmental suppression at the start of vitellogenesis during long-term reproductive migration may be a strategy to avoid precocious muscle atrophy.