Hormonal effects on glucose and ketone metabolism in a perfused liver of an elasmobranch, the North Pacific spiny dogfish, Squalus suckleyi.

Hormonal influence on hepatic function is a critical aspect of whole-body energy balance in vertebrates. Catecholamines and corticosteroids both influence hepatic energy balance via metabolite mobilization through glycogenolysis and gluconeogenesis. Elasmobranchs have a metabolic organization that appears to prioritize the mobilization of hepatic lipid as ketone bodies (e.g. 3-hydroxybutyrate [3-HB]), which adds complexity in determining the hormonal impact on hepatic energy balance in this taxon. Here, a liver perfusion was used to investigate catecholamine (epinephrine [E]) and corticosteroid (corticosterone [B] and 11-deoxycorticosterone [DOC]) effects on the regulation of hepatic glucose and 3-HB balance in the North Pacific Spiny dogfish, Squalus suckleyi. Further, hepatic enzyme activity involved in ketogenesis (3-hydroxybutyrate dehydrogenase), glycogenolysis (glycogen phosphorylase), and gluconeogenesis (phosphoenolpyruvate carboxykinase) were assessed in perfused liver tissue following hormonal application to discern effects on hepatic energy flux. mRNA transcript abundance key transporters of glucose (glut1 and glut4) and ketones (mct1 and mct2) and glucocorticoid function (gr, pepck, fkbp5, and 11ßhsd2) were also measured to investigate putative cellular components involved in hepatic responses. There were no changes in the arterial-venous difference of either metabolite in all hormone perfusions. However, perfusion with DOC increased gr transcript abundance and decreased flow rate of perfusions, suggesting a regulatory role for this corticosteroid. Phosphoenolpyruvate carboxykinase activity increased following all hormone treatments, which may suggest gluconeogenic function; E also increased 3-hydroxybutyrate dehydrogenase activity, suggesting a function in ketogenesis, and decreased pepck and fkbp5 transcript abundance, potentially showing some metabolic regulation. Overall, we demonstrate hormonal control of hepatic energy balance using liver perfusions at various levels of biological organization in an elasmobranch.

Molecular and pharmacological analysis of the melanocortin-2 receptor and its accessory proteins Mrap1 and Mrap2 in a Squalomorph shark, the Pacific spiny dogfish.

The hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is a conserved vertebrate neuroendocrine mechanism regulating the stress response. The penultimate step of the HPA/I axis is the exclusive activation of the melanocortin-2 receptor (Mc2r) by adrenocorticotropic hormone (ACTH), requiring an accessory protein, Mrap1 or Mrap2. Limited data for only three cartilaginous fishes support the hypothesis that Mc2r/Mrap1 function in bony vertebrates is a derived trait. Further, Mc2r/Mrap1 functional properties appear to contrast among cartilaginous fishes (i.e., the holocephalans and elasmobranchs). This study sought to determine whether functional properties of Mc2r/Mrap1 are conserved across elasmobranchs and in contrast to holocephalans. The deduced amino acid sequences of Pacific spiny dogfish (Squalus suckleyi; pd) pdMc2r, pdMrap1, and pdMrap2 were obtained from a de novo transcriptome of the interrenal gland and validated against the S. suckleyi genome. pdMc2r showed high primary sequence similarity with elasmobranch and holocephalan Mc2r except at extracellular domains 1 and 2, and transmembrane domain 5. pdMraps showed similarly high sequence similarity with holocephalan and other elasmobranch Mraps, with all cartilaginous fish Mrap1 orthologs lacking an activation motif. cAMP reporter gene assays demonstrated that pdMc2r requires an Mrap for activation, and can be activated by stingray (sr) ACTH(1-24), srACTH(1-13)NH2 (i.e., α-MSH), and γ-melanocyte-stimulating hormone at physiological concentrations. However, pdMc2r was three orders of magnitude more sensitive to srACTH(1-24) than srACTH(1-13)NH2. Further, pdMc2r was two orders of magnitude more sensitive to srACTH(1-24) when expressed with pdMrap1 than with pdMrap2. These data suggest that functional properties of pdMc2r/pdMrap1 reflect other elasmobranchs and contrast what is seen in holocephalans.

Energy and corticosteroid mobilization following an induced stress response in an elasmobranch fish, the North Pacific spiny dogfish (Squalus acanthias suckleyi).

The dominant corticosteroid in elasmobranchs, 1α-hydroxycorticosterone (1α-OHB), has a described role in mineral regulation but a presumptive role in energy balance. Energy demand in vertebrates following exposure to a stressor typically involves an immediate but transient release of glucocorticoids as a means of mobilizing available energy stores, usually in the form of glucose. Although a glucocorticoid role for 1α-OHB would be expected, direct glucocorticoid function of this steroid has yet to be reported in any elasmobranch. In addition, elasmobranchs also utilize the metabolite ß-hydroxybutyrate (ß-HB), which is thought to replace the role fatty acids play in most vertebrates as a predominant fuel source in extrahepatic tissues. To determine the mobilization of metabolites and corticosteroids during a stress event, North Pacific spiny dogfish, Squalus acanthias suckleyi, were cannulated and held in a darkened isolation box to recover (24-48 h) before being subjected to an acute air exposure or corticosterone injection. Dogfish were then serially blood sampled at nine timepoints over 48 h. Glucose, ß-HB, 1α-OHB, corticosterone, as well as lactate, pH, and osmolality were quantified in plasma samples. All measured variables increased in control and treatment groups within 48 h from the start of experimentation, and ß-HB and 1α-OHB remained elevated for the duration of the experiment. There was no linear correlation between glucose and 1α-OHB, but there was a weak (R2 = 0.230) although significant (p = 0.001), positive correlation between ß-HB and 1α-OHB. Interestingly, there were also significant correlations between increasing circulating glucose and corticosterone (R2 = 0.349; p < 0.001), and decreasing ß-HB and corticosterone concentrations (R2 = 0.180; p = 0.008). Our data suggest that following successive stressors of capture, surgery, and confinement, 1α-OHB was not correlated with circulating glucose, only weakly correlated with circulating ß-HB concentrations (R2 = 0.230; p = 0.001), and that corticosterone may also serve a role in energy mobilization in this species.

Survival and gene expression responses in immune challenged larval lake sturgeon.

Larval lake sturgeon, Acipenser fulvescens, reared in hatcheries for stock enhancement of wild populations may be susceptible to early opportunistic bacterial infection. Thus, we examined survival and whole-body mRNA expression of both stress- and immune-related genes (MyD88, IL-1ß, StAR, GR1, and HSP70) in 30 days post fertilization larval lake sturgeon following immune challenge with lipopolysaccharides (LPS). Larval sturgeon were exposed to 0, 25, 50, 100, 150, and 200 µg ml-1 LPS and sampled after 30 min, 4 h, and 48 h. Mortality was zero in 0 and 25 µg ml-1 LPS; 37.5% in 50 µg ml-1 LPS and 100% in the higher concentrations. Expression of MyD88 and StAR mRNA were positively correlated and increased with time in the 50 µg ml-1 LPS treatment. There was an influence of both treatment and time on IL-1ß mRNA, with expression 10-fold higher than controls after 4 h. Expression of HSP70 mRNA was suppressed within 30 min of 50 µg ml-1 LPS exposure and remained so throughout the time course. Correlated mRNA expression of GR1 with MyD88, StAR and IL-1ß suggests a potential relationship between the innate immune and glucocorticoid responses of larval lake sturgeon during this early developmental stage. Data presented suggest that larval lake sturgeon largely responded with predicted changes in gene expression of immune related and stress response genes following LPS challenge. This study provides a foundation for future research examining the effects of hatchery and naturally occurring stressors on the immune responses of larval lake sturgeon.

Effects of glucose and insulin administration on glucose transporter expression in the North Pacific spiny dogfish (Squalus suckleyi).

Elasmobranchs (sharks, skates, and rays) are a primarily carnivorous group of fish, consuming few carbohydrates. Further, they tend to exhibit delayed responses to glucose and insulin administration in vivo relative to mammals, leading to a presumption of glucose-intolerance. To investigate the glucoregulatory capabilities of the spiny dogfish (Squalus suckleyi), plasma glucose concentration, muscle and liver glycogen content, and glucose transporter (glut1 and 4) mRNA levels were measured following intra-arterial administration of bovine insulin (10ngkg-1) or an approximate doubling of fasting plasma glucose concentration. Within 6h, following glucose administration, approximately half of the introduced glucose load had been cleared, with control levels being restored by 24h post-injection. It was determined that plasma clearance was due in part to increased uptake by the tissues as muscle and liver glycogen content increased significantly, correlating with an upregulation of glut mRNA levels. Following administration of bovine insulin, plasma glucose steadily decreased through 18h before returning toward control levels. Observed decreases in plasma glucose following insulin injection were, however, relatively minor, and no increases in tissue glycogen content were observed. glut4 and glycogen synthase mRNA levels did significantly increase in the muscle in response to insulin, but no changes occurred in the liver. The responses observed mimic what occurs in mammals and teleosts, thus suggesting a conserved mechanism for glucose homeostasis in vertebrates and a high degree of glucose tolerance in these predominantly carnivorous fish.

Effects of chlorpyrifos on in vitro sex steroid production and thyroid follicular development in adult and larval Lake Sturgeon, Acipenser fulvescens.

Chlorpyrifos is a widely used organophosphate pesticide that has previously been shown to enter waterways in biologically relevant concentrations and has the potential to disrupt both thyroid hormone and sex steroid biosynthesis in vertebrates. Because gonadal maturation and larval development in Lake Sturgeon, Acipenser fulvescens, potentially coincide with the application of chlorpyrifos we examined the effects of chlorpyrifos on both thyroid follicular development in larval Lake Sturgeon, and sex hormone synthesis in adult Lake Sturgeon. For the first time, the present study reports steroidogenesis from testicular and ovarian tissue in Lake Sturgeon using an established in vitro bioassay. Furthermore, incubating gonad tissue with 5, 500 or 2000ngmL(-1) chlorpyrifos revealed an inhibitory effect on testosterone synthesis in both testicular (control, 40.29pgmg(-1) tissue wet weight(-1)h(-1) compared to experimental, 21.84pgmg(-1) tissue wet weight(-1)h(-1)) and ovarian (control, 33.83pgmg(-1) tissue wet weight(-1)h(-1) compared to experimental, 15.19pgmg(-1) tissue wet weight(-1)h(-1)) tissue. In a second series of experiments, larval Lake Sturgeon were exposed to equivalent concentrations of chlorpyrifos as above for 10days (d) between hatch and the onset of exogenous feeding. Larvae from each treatment group were raised until 67days post hatch (dph) and growth rates were compared alongside key indicators of thyroid follicle growth. Chlorpyrifos treatment had no effect on the measured indicators of thyroid follicular development.

Examining urea flux across the intestine of the spiny dogfish, Squalus acanthias.

Recent examination of urea flux in the intestine of the spiny dogfish shark, Squalus acanthias, has shown that feeding significantly enhances urea uptake across the intestine, and this was significantly inhibited following mucosal addition of phloretin. The present study examined potential mechanisms of urea uptake across the dogfish intestine in starved and fed dogfish. Unidirectional flux chambers were used to examine the kinetics of urea uptake, and to determine the influence of sodium, ouabain, competitive urea analogues, and phloretin on urea uptake across the gut of fed dogfish. Intestinal epithelial preparations from starved and fed dogfish were mounted in Ussing chambers to examine the effect of phloretin on bidirectional solute transport across the intestine. In the unidirectional studies, the maximum uptake rate of urea was found to be 35.3±6.9 µmol.cm(-2).h(-1) and Km was found to be 291.8±9.6 mM in fed fish, and there was a mild inhibition of urea uptake following mucosal addition of competitive agonists. Addition of phloretin, Na-free Ringers and ouabain to the mucosal side of intestinal epithelia also led to a significant reduction in urea uptake in fed fish. In the Ussing chamber studies there was a net influx of urea in fed fish and a small insignificant efflux in starved fish. Addition of phloretin blocked urea uptake in fed fish when added to the mucosal side. Furthermore, phloretin had no effect on ion transport across the intestinal epithelia with the exception of the divalent cations, magnesium and calcium.

Sequence, circulating levels, and expression of C-type natriuretic peptide in a euryhaline elasmobranch, Carcharhinus leucas.

The present study has examined expression and circulating levels of C-type natriuretic peptide (CNP) in the euryhaline bull shark, Carcharhinus leucas. Complementary DNA and deduced amino acid sequence for CNP in C. leucas were determined by RACE methods. Homology of CNP amino acid sequence in C. leucas was high both for proCNP and for mature CNP when compared with previously identified elasmobranch CNPs. Mature CNP sequence in C. leucas was identical to that in Triakis scyllia and Scyliorhinus canicula. Levels of expression of CNP mRNA were significantly decreased in the atrium but did not change in either the brain or ventricle following acclimation to a SW environment. However, circulating levels of CNP significantly increased from 86.0+/-7.9 fmol ml(-1) in FW to 144.9+/-19.5 fmol ml(-1) in SW. The results presented demonstrate that changes in environmental salinity influences both synthesis of CNP from the heart and also circulating levels in C. leucas. Potential stimulus for release and modes of action are discussed.

Urea based osmoregulation and endocrine control in elasmobranch fish with special reference to euryhalinity.

Since the landmark contributions of Homer Smith and co-workers in the 1930s there has been a considerable advance in our knowledge regarding the osmoregulatory strategy of elasmobranch fish. Smith recognised that urea was retained in the body fluids as part of the 'osmoregulatory ballast' of elasmobranch fish so that body fluid osmolality is raised to a level that is iso- or slightly hyper-osmotic to that of the surrounding medium. From studies at that time he also postulated that many marine dwelling elasmobranchs were not capable of adaptation to dilute environments. However, more recent investigations have demonstrated that, at least in some species, this may not be the case. Gradual acclimation of marine dwelling elasmobranchs to varying environmental salinities under laboratory conditions has demonstrated that these fish do have the capacity to acclimate to changes in salinity through independent regulation of Na(+), Cl(-) and urea levels. This suggests that many of the presumed stenohaline marine elasmobranchs could in fact be described as partially euryhaline. The contributions of Thomas Thorson in the 1970s demonstrated the osmoregulatory strategy of a fully euryhaline elasmobranch, the bull shark, Carcharhinus leucas, and more recent investigations have examined the mechanisms behind this strategy in the euryhaline elasmobranch, Dasyatis sabina. Both partially euryhaline and fully euryhaline species utilise the same physiological processes to control urea, Na(+) and Cl(-) levels within the body fluids. The role of the gills, kidney, liver, rectal gland and drinking process is discussed in relation to the endocrine control of urea, Na(+) and Cl(-) levels as elasmobranchs acclimate to different environmental salinities.