Polyunsaturated fatty acids modulate the properties of the sex steroid binding protein in goldfish

This study examines the effects of nonesterified fatty acids on the properties of the sex steroid binding protein (SSBP) in the plasma of goldfish (Carassius auratus). Scatchard analysis revealed a single class of high affinity (Kd 1.89±0.20 nM), low capacity (Bmax 302±17 nM) binding sites for [3H]17-estradiol in female goldfish plasma. The SSBP bound 17-estradiol and testosterone with similar affinity but had much lower affinity for estriol, 17,20-dihydroxy-4-pregnen-3-one and cholesterol. Nonesterified fatty acids inhibit the binding of [3H]17-estradiol to the SSBP as a function of dose, degree of unsaturation and fatty acid chain length. Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (C20:4), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) strongly inhibit the binding of [3H]17-estradiol to the SSBP. By comparison, saturated fatty acids such as heptadecanoic acid (C17:0), palmitic acid (C16:0) and stearic acid (C18:0) were without effect. Scatchard analysis and Lineweaver-Burk plots showed that PUFAs act through a competitive mechanism whereby they reduce the affinity but have no effect on the binding capacity of the SSBP. Collectively, these studies suggest that in addition to their roles as metabolic energy sources and as precursors to eicosanoids, PUFAs can be potent modulators of steroid hormone interactions with the SSBP in goldfish plasma.     

久效磷对金鱼精巢超显微结构和特征性酶活性的影响

用0.01、0.10、1.00 mg.L-1久效磷暴露金鱼21 d,测定了精巢乳酸脱氢酶(LDH)、酸性磷酸酶(ACP)、碱性磷酸酶(ALP)的活性,并观察了精巢显微和超微结构的变化。结果表明:久效磷暴露21 d,精巢小叶基膜断裂,Leydig氏细胞水肿,小叶间质扩大,支持细胞核膜溶解,胞质内脂滴和髓磷脂象数量增多。1.00 mg.L-1久效磷造成了生殖腺指数的显著下降,对精巢发育有一定程度的延迟作用。随着久效磷暴露浓度的升高,LDH,ACP,ALP的活性逐渐下降,0.10、1.00 mg.L-1久效磷暴露组精巢LDH,ACP的活性下降显著,而各暴露组的精巢ALP活性呈现不显著下降趋势。推测久效磷可能通过损伤精巢超显微结构和降低LDH,ACP和ALP活性,干扰精巢能量代谢,造成对雄性金鱼的生殖毒性。     

Intracellular mechanisms mediating gonadotropin and growth hormone release in the goldfish,Carassius auratus

Evidence for the involvement of Ca²⁺, protein kinase C, cAMP, and arachidonic acid metabolism in mediating gonadotropin (GTH) and growth hormone (GH) release in the goldfish is reviewed. Models for the signal transduction pathways mediating GTH-releasing hormone (GnRH) and dopamine actions on GTH and GH secretion are postulated. A novel hypothesis that two GnRHs which bind to the same receptor type activate different transduction cascade in two different cell types (GTH vs. GH) as well as within the same cell type (GTH) is presented.

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Résumé Cette revue présente les données expérimentales démontrant l'implication de Ca⁺⁺, de la protéine kinase C et du métabolismes de l'acide arachidonique dans les mécanismes régulant la sécrétion des hormones gonadotrope (GTH) et de croissance (GH). Des modèles de signaux de transduction de l'action de la gonadolibérine (GnRH) et de la dopamine sur la sécrétion de GTH et de GH sont proposés. Les deux GnRHs existant chez le poisson rouge pourraient se lier au même type de récepteur et activer différentes voies de transduction dans deux différents types cellulaires (GTH vs. GH) ou dans un seul type (GTH).

     

Organ and phospholipid class fatty acid specificity in response to dietary depletion of essential n‐3 fatty acids in Atlantic salmon (Salmo salar L.)

The cell membrane phospholipid composition is of major importance for normal cell functions. However, it is not known how complete depletion of both shorter and longer chain omega‐3 fatty acids in salmon diets influences fatty acid composition of phospholipid subclasses in different organs of Atlantic salmon. We describe here the fatty acid composition in phospholipid subclasses of liver, muscle, heart and intestine in Atlantic salmon after 18 months of dietary n‐3 essential fatty acids deprivation. The percentage of 22:6n‐3 was markedly reduced in almost all phospholipid subclasses, and except for muscle phosphatidylcholine, phosphatidylethanolamine (PE) and phosphatidylinositol (PI), phospholipids in deficient fish were totally devoid of 20:5n‐3. As compensation, we observed significant increases in 20:4n‐6, and especially in 20:3n‐9 (Mead acid) and 22:5n‐6, varying among phospholipids and organs. High amounts of 20:3n‐9 were found in liver and intestinal PE, little in PE from heart and muscle. For 22:5n‐6, we saw a small incorporation in PI in liver and intestine compared to heart and muscle. Generally in PI, the preference for 20:4n‐6 to 20:5n‐3 differed significantly between organs. Overall, changes upon n‐3 deprivation seemed to be strongest in liver and intestine, the lipid‐secreting organs, and less in muscle and heart.     

Polyunsaturated fatty acid metabolism in a cell culture model of essential fatty acid deficiency in a freshwater fish, carp (Cyprinus carpio)

Proliferation of an essential fatty acid deficient cell line from carp (EPC-EFAD; epithelioma papillosum carp-essential fatty acid deficient) is stimulated by supplementing the cells with C₂₀, but not C₁₈ polyunsaturated fatty acids (PUFA). It is hypothesized that the differential ability of the PUFA to stimulate proliferation of the EPC-EFAD cells may be related to the extent of the cells' ability to desaturate and elongate C₁₈ PUFA. In the present study, the metabolism of ¹⁴C-labeled C₁₈ and C₂₀ PUFA was investigated in EPC-EFAD cells in comparison with normal EPC cells. The incorporation of all the PUFA was significantly greater in EPC-EFAD cells but the rank order, 20:5n-3 > 18:3n-3 = 18:2n-6 >20:4n-6 was the same in both cell lines. The proportion of radioactivity from all labeled PUFA recovered in phosphatidylethanolamine and total polar lipids was significantly lower in EPC-EFAD cells compared to EPC cells, whereas the proportion of radioactivity recovered in all the other phospholipid classes and total neutral lipid was greater in EPC-EFAD cells. Both cell lines desaturated[1-¹⁴C]18:3n-3 and [1-¹⁴C]20:5n-3 to a greater extent than the corresponding (n-6) substrates but the desaturation of all the ¹⁴ C-labeled PUFA was significantly greater in EPC-EFAD cells compared to EPC cells. The results showed that, although essential fatty acid deficiency had several significant effects on PUFA metabolism in EPC cells, the fatty acid desaturation/elongation pathway was not impaired in EPC-EFAD cells and so they can desaturate 18:3n-3 to 20:5n-3 and 22:6n-3, and 18:2n-6 to 20:4n-6. However, 20:4n-3 and 20:3n-6, and not 20:4n-6 and 20:5n-3, were the predominant C₂₀ PUFA produced by the elongation and desaturation of [1-¹⁴C]18:3n-3 and [1-¹⁴C]18:2n-6, respectively. Therefore, the previously reported inability of 18:3n-3 and 18:2n-6, compared to 20:5n-3 and 20:4n-6, to stimulate proliferation of the cells is apparently not due to a general deficiency in the fatty acid desaturation pathway in EPC-EFAD cells but may be related to potential differences in eicosanoid profiles in cells supplemented with C₁₈ PUFA compared to C₂₀ PUFA.     

Use of biomass of the marine microalga Isochrysis galbana in the nutrition of goldfish (Carassius auratus) larvae as source of protein and vitamins

The effect of the replacement of fish protein hidrolizate and vitamin premix by freeze‐dried biomass of the marine microalga Isochrysis galbana in the feed for goldfish (Carassius auratus) larvae was tested. Larvae (3.4±0.7 mg) were fed with three experimental microparticulated diets that differ from each other in the percentage of replacement of fish protein hidrolizate (25% or 100%) or vitamin premix by I. galbana biomass. The control diet and the diet containing microalgae biomass as a substitute of 25% of fish protein hidrolizate (MP₂₅) presented the highest survival, being almost 100%, with no significant differences between them. Survival in diets in which 100% of fish protein hidrolizate (MP₁₀₀) or vitamin premix (MV) had been substituted by microalgal biomass was 78% and 66% respectively. Growth, measured as weight, was lower than with the control diet in all treatments in which microalgal biomass was included, with lowest results being obtained with the MP₁₀₀ diet. Differences between treatments and control were lower when growth was measured as length. The harvesting and processing microalgae biomass is crucial to maintain the nutritive value and could be the cause for the obtained results.     

Metabolic fate of <Superscript>14</Superscript>C-labelled chlorinated and non-chlorinated fatty acids in goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

In order to study the metabolic fate of chlorinated fatty acids in fish, goldfish were fed either 9,10-dichlorostearic acid or oleic acid, chosen as the unchlorinated analogue, both radiolabelled at either the carboxyl (1st) or the terminal (18th) carbon of the fatty acid chain. By keeping the fish in hermetically closed aquaria, all the respired, assimilated and excreted radioactivity could be accounted for. Fish fed 9,10-dichlorostearic acid labelled in the terminal end respired radioactive CO₂ to a much lower degree than fish fed the other test compounds. As a consequence, the radioactivity bound in lipids was higher in the group of fish fed dichlorostearic acid labelled in the terminal end. It is suggested that the chlorine atoms in the middle of the carbon chain obstruct the metabolic turn-over of 9,10-dichlorostearic acid, which may have an impact on the residence time of these compounds in the ecosystem.     

The effect of time‐dependent protein restriction on growth factors,nonspecific immunity,body composition,fatty acids and amino acids in the Siberian sturgeon (Acipenser baerii)

The effect of time‐dependent protein restriction (PR) and refeeding were investigated on growth, body composition, fatty acids (FA), amino acids (AA) and nonspecific immune functions in the juvenile Siberian sturgeon (Acipenser baeri; 75 ± 5 g). Test diets included: T1 (control) and T2 fish fed diets containing 400 and 300 g/kg protein respectively during the whole experimental period, T3 (every other day) and T4 (every other week) fish were fed diets containing 300 g/kg protein (PR) and refeeding with diet containing 400 g/kg protein respectively, and T5 fish fed a diet containing 300 g/kg protein for 3 weeks and a diet containing 400 g/kg protein for 5 weeks, were fed to the fish for 56 days by visual satiation. Unlike ration T2 and T5, feeding treatments of T3 and T4 showed an increase in fish growth and body composition (p < 0.05) and they were very close to the control group (p > 0.05). Regarding the fatty acid profile, although the percentage of highly unsaturated fatty acids (HUFA) and polyunsaturated fatty acids (PUFA) declined significantly in the T2 and T5 groups, the saturated fatty acid (SFA) and monounsaturated fatty acids (MUFA) indicated no change among the treatments. In addition, the T2 and T5 groups demonstrated a reduction in essential amino acids (EAA) with a simultaneous increase in nonessential amino acids (NEAAs), which was significant from that of controls and other groups (p < 0.05). In terms of nonspecific immune parameters (serum lysozyme activity and alternate complement activity [ACH50]), treatment 3 has an appropriate result that did not have a significant difference with the control group (p > 0.05), but there was significant difference with other groups (p < 0.05). As a result, the T3 treatment can be used in sturgeon aquaculture practically without any negative impacts on biological or physiological indices.     

Effects of dietary arachidonic and eicosapentaenoic acids on common dentex (Dentex dentex Linnaeus 1758) larval performance

Oily emulsions containing constant levels of total fatty acids (FAs) and varying eicosapentaenoic acid (EPA) and arachidonic acid (ARA) levels were used to enrich rotifers. Common dentex larval survival and growth were compared between groups fed different enriched live prey. Growth, survival rate, and lipid composition of larvae suggest that feeding common dentex in the first 15 days posthatching with 2.5–3% EPA, 6–8% docosahexaenoic acid (DHA), and DHA/EPA ratio of 2.0–2.5 is sufficient to fulfill their EPA requirements. Higher amounts of dietary EPA did not result in any significant improvement in growth or survival. EPA requirement during this period of larval development does not seem to be as critical as other FAs during the first 15 days of common dentex larval development, but it does not exclude its essentiality later in development. In the case of ARA, nutritional requirements are low compared to other marine finfish species, with the upper limit of this essential FA being around 2% of total FAs provided in the live prey composition.     

Effects of short‐term starvation on the rhythmic expression of microRNAs in skeletal muscle of goldfish (Carassius auratus)

Molecular oscillators exist in peripheral tissues such as in skeletal muscles and diet is a dominant factor to affect Zeitgeber for peripheral clocks. MicroRNAs (miRNAs) play an important role in muscle cell proliferation and differentiation. However, the knowledge of starvation effects on miRNA rhythmic expression remains limited in teleost. In this study, the circadian expression pattern of miRNAs was investigated in goldfish muscle upon restricting feeding treatment. The data showed that 15 miRNAs exhibited a daily rhythmicity among the 70 miRNAs assayed in muscles of normally fed goldfish. While after 7‐day and 15‐day fasting treatment, 23 and 18 miRNAs showed circadian rhythmicity in goldfish muscles respectively. Only 4 miRNAs (miR‐23a, miR‐29, miR‐199a‐3p and miR‐455) exhibited a daily rhythmicity in all three groups of different nutrient treatments. Correlation analysis of the circadian‐miRNAs indicates different feeding stimulation could alter the circadian‐miRNA components and their expression profile in skeletal muscle upon short‐term feed deprivation. These miRNAs may play important roles in regulating circadian expression of genes involved in muscle cell differentiation and growth by nutritional alteration, thus having a potential application in fish aquaculture.     

Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured turbot (Scophthalmus maximus) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to the culture medium was investigated in a turbot cell line (TF). The distribution, and the occurrence and extent of further metabolism of incorporated PUFA via desaturation/elongation mechanisms in specific phospholipid classes was determined from the different fatty acid compositions. The cells contained Δ6 and Δ4 desaturase activities but were generally deficient in C18–20 elongase activity. Δ5 Desaturase activity was generally masked by this deficiency but was present. The compositional data indicated that there was a high degree of specificity between individual phospholipid classes and particular fatty acids probably driven by the specificities of the acylating enzymes. The highest percentages of the supplemented acids were generally observed in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for PA in the incorporation of the supplemented acids into the phospholipid pool. PI had a characteristic composition consistent with a putative role as a pool of precursor fatty acid for eicosanoid synthesis. Mechanisms were evident for generating and/or maintaining this composition.     

The conversion of linoleic acid and linolenic acid to longer chain polyunsaturated fatty acids by Tilapia (Oreochromis) nilotica in vivo

Tilapia (Oreochromis) nilotica were fed either a commercial diet containing 2.2% (n-3) and 0.5% (n-6) polyunsaturated fatty acids (PUFA), or a diet containing 1.0% methyl linoleate as the only PUFA. The fatty acid composition of tissue lipids generally reflected that of the diet. Fish from both dietary groups were injected intraperitoneally with ¹⁴C-labelled linoleic acid, 18:2 (n-6), or linolenic acid, 18:3 (n-3), and the distribution of radioactivity in tissue lipids examined. The conversion of both 18:2 (n-6) and 18:3 (n-3) to longer chain PUFA was lower in fish fed the commercial diet than in those fed the diet containing only 18:2 (n-6). Half of the radioactivity from both substrates recovered in liver polar lipids was present in C20 and C22 PUFA with fish maintained on the experimental diet. It is concluded that T. nilotica is capable of elongating and desaturating both 18:2 (n-6) and 18:3 (n-3), but that this conversion is suppressed by dietary longer chain PUFA. NERC Unit of Aquatic Biochemistry     

Involvement of protein kinase C in the modulation of gonadotropin and growth hormone secretion from dispersed goldfish pituitary cells

It has been established that secretion of gonadotropin (GtH) and growth hormone (GH) release in goldfish are both stimulated by GtH-releasing hormone (GnRH); in addition GtH secretion is inhibited by dopamine D₂ mechanisms. In the present study, depletion of protein kinase C (PKC) in goldfish pituitary cells reduced the GtH and GH responses to GnRH and an activator of PKC in static culture. In perifusion studies, GtH released in response to sGnRH analog was greatly attenuated in PKC-depleted cells, however, hormone responses to forskolin were enhanced. Stimulation of dopamine D₂ receptors reduced the GtH, but not the GH, responses elicited by PKC activators. These results indicate that PKC participates in the GtH and GH responses to natural neuroendocrine regulators in the goldfish.

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Résumé Il a été établi que chez le poisson rouge, les sécrétions de gonadotropine (GtH) et d'hormone de croissance (GH) sont toutes les deux stimulées par la gonadolibérine (GnRH); de plus, la sécrétion de GtH est inhibée par des mécanismes dopaminergiques de type D₂. Dans le présent travail, la déplétion de la teneur en protéine kinase C (PKC) dans des cellules hypophysaires de poisson rouge réduit les résponses en GtH et GH au GnRH et à un activateur de la PKC de cellules maintenues en incubation statique. Dans des cellules maintenues en périfusion et soumises à une déplétion en PKC, la GtH libérée en réponse à un analogue du sGnRH est fortement diminuée, cependent les réponses hormonales à la forskoline sont augmentées. La stimulation des récepteurs dopaminergiques D₂ réduit, dans le cas d'action d'activateur de la PKC, la réponse en GtH mais pas en GH. Ces résultats indiquent que la PKC est impliquée dans les mécanismes de régulation de GtH et GH par des facteurs neuroendocriniens naturels.

     

Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured rainbow trout (Salmo gairdneri) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids supplemented to the culture medium was investigated in the rainbow trout cell line, RTG-2. The distribution, and the occurrence and relative extent of further desaturation and elongation of the incorporated acids was determined in individual phospholipid classes by analysis of the fatty acid compositions. RTG-2 cells exhibited Δ6 and Δ5 desaturase activities whereas Δ4 desaturase activity was almost totally absent. The percentage of precursor acids was greatest in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for possibly PA in the initial incorporation of these acids into the phospholipid pool. The compositional data indicated that individual intermediates and products of the desaturation pathways were associated with specific phospholipid classes probably via mechanisms depending upon the specificities of the acylating enzymes. The composition of phosphatidylinositol (PI) and the tightly controlled mechanisms for generating/maintaining it are consistent with a role for this phospholipid in providing precursor fatty acid for eicosanoid synthesis.     

The incorporation and metabolism of polyunsaturated fatty acids in phospholipids of cultured cells from chum salmon (Oncorhynchus keta)

The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids were studied in a cell line derived from chum salmon heart (CHH-1). Supplementing media with 25 M fatty acid considerably altered the cellular fatty acid composition but did not affect the lipid class composition or cause the appearance of cytoplasmic lipid droplets. CHH-1 cells exhibited considerable -6-desaturase activity but showed no preference between (n-3) and (n-6)PUFA substrates. CHH-1 cells also possess -5-desaturase activity which showed preference towards (n-3)PUFA, but -4-desaturase activity was totally absent. Elongation of 20-carbon PUFA was especially active in CHH-1 cells with 22-carbon PUFA being specifically incorporated into PE and PS lipid classes. The fatty acid composition of PI indicated specific incorporation of 20-carbon PUFA into this lipid class. Supplementation with 22:6(n-3) generated fatty acid compositions more closely resembling those of intact salmonid hearts. Substantial chain shortening of 22:6(n-3) to 20:5(n-3) occurred.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL cardiolipin - FCS fetal calf serum - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid - SM sphingomyelin     

Polyunsaturated fatty acid metabolism in cultured fish cells: Incorporation and metabolism of (n-3) and (n-6) series acids by Atlantic salmon (Salmo salar) cells

The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to growing cultures were studied in Atlantic salmon (AS) cells. A fatty acid concentration of 25 μM considerably altered the fatty acid composition of AS cells without increasing the neutral lipid content of the cells or inducing the production of cytoplasmic lipid droplets. Whereas Δ6 and Δ5 desaturase activities were significantly expressed in AS cells, Δ4 desaturase activity was very low. Both the Δ6 desaturase activity and the Δ5 desaturase activity showed some preference for (n-3) PUFA.     

Effects of dietary (n-3) and (n-6) polyunsaturated fatty acid ratio on the immune response of Atlantic salmon, Salmo salar L.

To examine the influence of the dietary ratio of (n-3) to (n-6) polyunsaturated fatty acids (PUFAs) on the immune system of Atlantic salmon, Salmo salar L., two dietary trials were carried out in which parr were maintained on diets containing either fish oil [(n-3)/(n-6) PUFA = 5.2] or sunflower oil [(n-3)/(n-6) PUFA = 0.3] and assessed for differences in immunological parameters. There were no significant differences in blood cell counts, differential leucocyte counts or haematocrit values between dietary groups, and while no apparent differences were observed in the non-specific immune parameters measured, there was a significantly higher number of B cells responding to Aeromonas salmonicida, in the kidney and spleen of vaccinated fish maintained on high (n-3)/(n-6) PUFAs diets. There was also a significant difference (P≤ 0.01) between the dietary groups in trial 1 and trial 2 when non-vaccinated fish were challenged with Aeromonas salmonicida and Vibrio anguillarum, respectively, with the (n-6) group succumbing to the bacterium before the (n-3) group. The results suggest that Atlantic salmon fed diets with a low ratio of (n-3)/(n-6) PUFA may be less resistant to infection than those fed diets containing lipid with a high (n-3)/(n-6) PUFA ratio.     

Exogenous polyunsaturated fatty acids (PUFAs) influence permeability,antimicrobial peptide resistance,biofilm formation and membrane phospholipid structure in an A-layer and non-A-layer strain of Aeromonas salmonicida

Aeromonas salmonicida is a Gram-negative bacterium that can infect a wide host range of fish populations, including salmonids and non-salmonids as well as freshwater and marine life. Some strains of A. salmonicida cause the disease furunculosis, which can cause lethargy, intestinal inflammation, ulcers, haemorrhaging and death. The infection is spread through fish-to-fish contact, and the presence of infection can have devastating effects on cultivated fish populations. The purpose of this study was to explore the ability of non-A-layer and A-layer A. salmonicida strains to incorporate polyunsaturated fatty acids (PUFAs) into their lipid profile and test the phenotypic effects thereof. Lipids were extracted from PUFA-exposed cultures and analysed for lipid modification by thin-layer chromatography and ultraperformance liquid chromatography-mass spectrometry, showing A. salmonicida, regardless of A-layer, capable of incorporating all seven of the PUFAs studied. Phenotypic effects were determined through the use of assays that tested for biofilm formation, membrane permeability and cyclic peptide susceptibility. Temperature-dependent effects on biofilm formation were observed, and PUFA exposure showed significant (p < .001) increases in membrane permeability as tested by the uptake of the hydrophobic compounds crystal violet and ethidium bromide. Additionally, some PUFAs elicited modest protection and vulnerability against the membrane-targeting cyclic peptides polymyxin B (PMB) and colistin. The diverse, strain-specific responses to exogenous PUFAs may allude to evolved adaptive strategies that enhance survival, persistence and virulence of non-pathogenic and pathogenic members of bacteria that oscillate between environmental and fish host niches.