Carp Skeletal Muscle Research Articles

Determining the Potential Roles of Branched-Chain Amino Acids in the Regulation of Muscle Growth in Common Carp (Cyprinus carpio) Based on Transcriptome and MicroRNA Sequencing.

Branched-chain amino acids (BCAAs) can be critically involved in skeletal muscle growth and body energy homeostasis. Skeletal muscle growth is a complex process; some muscle-specific microRNAs (miRNAs) are involved in the regulation of muscle thickening and muscle mass. Additionally, the regulatory network between miRNA and messenger RNA (mRNA) in the modulation of the role of BCAAs on skeletal muscle growth in fish has not been studied. In this study, common carp was starved for 14 days, followed by a 14-day gavage therapy with BCAAs, to investigate some of the miRNAs and genes that contribute to the regulation of normal growth and maintenance of skeletal muscle in response to short-term BCAA starvation stress. Subsequently, the transcriptome and small RNAome sequencing of carp skeletal muscle were performed. A total of 43,414 known and 1,112 novel genes were identified, in addition to 142 known and 654 novel miRNAs targeting 22,008 and 33,824 targets, respectively. Based on their expression profiles, 2,146 differentially expressed genes (DEGs) and 84 differentially expressed miRNA (DEMs) were evaluated. Kyoto Encyclopedia of Genes and Genome pathways, including the proteasome, phagosome, autophagy in animals, proteasome activator complex, and ubiquitin-dependent protein catabolic process, were enriched for these DEGs and DEMs. Our findings revealed the role of atg5, map1lc3c, ctsl, cdc53, psma6, psme2, myl9, and mylk in skeletal muscle growth, protein synthesis, and catabolic metabolism. Furthermore, miR-135c, miR-192, miR-194, and miR-203a may play key roles in maintaining the normal activities of the organism by regulating genes related to muscle growth, protein synthesis, and catabolism. This study on transcriptome and miRNA reveals the potential molecular mechanisms underlying the regulation of muscle protein deposition and provides new insights into genetic engineering techniques to improve common carp muscle development.

The lipid metabolism in carp during invasion by the tapeworn Bothriocephalus acheilognathi

The changes in total lipids, their fatty acid composition and the ratio of individual classes were established in tissues of the intestine, hepatopancreas and skeletal muscles of carp (Cyprinus carpio Linnaeus, 1758), with body weight 14.5–20.5 g, at different rates of invasion by Bothriocephalus acheilognathi (Yamaguti, 1934) helminth, which belongs to the family Bothriocephalidae, of the Pseudophyllidae order, of the Cestoda class. The examined carp was divided into three groups: first group of fish was free from intestinal helminths of B. acheilognathi (control); second group of fish was weakly infected with helminths (intensity of invasion is 1–3 helminths per fish); the third group of fish was highly infected (the invasion intensity is 4 worms and more per fish). Our results showed that fish infected with helminth B. acheilognathi compared to uninfected fish had reduced total lipid level in the gut due to phospholipids, triacylglycerols, and also lipids were characterized by lower content of linoleic, linolenic, arachidonic, penta- and hexanoic fatty acids; decrease in the level of unsaturated and increase in the content of saturated fatty acids, which leads to an increase of the saturation factor. During the infection of carp with B. acheilognathi in the hepatopancreas, the content of total lipids, structural lipids – phospholipids and reserve energy sources – triacylglycerols is probably reduced, and lipids are characterized by a high content of saturated fatty acids (С14:0, С16:0, С18:0) and lower content of unsaturated: arachidonic (С20:4), linolenic (С18:3) and linoleic acid (С18:2), which is associated with a decrease in the source for the synthesis of a number of polyunsaturated fatty acids, especially docosahexaenoic (С22:6). The total content of lipids, triacylglycerols, free fatty acids and phospholipids in skeletal muscle of carp during the Bothriocephalus invasion decreased and the content of free cholesterol, mono- and triacylglycerols increased. Helminth B. acheilognathi has the effect of reducing the total lipids of the skeletal muscle content of С18-, С20-, С22-polyunsaturated fatty acids and increasing the content of saturated (С14:0, С16:0, С18:0) and monounsaturated (С16:1, С18:1) fatty acids. The obtained results prove that the parasite B. acheilognathi in the intestine of the carp significantly affects the nutrition processes of the host depending on the intensity of the damage by helminths, which is accompanied by impaired lipid metabolism.

Purification, characterization, and cDNA cloning of a matrix metalloproteinase from the skeletal muscle of silver carp (Hypophthalmichthys molitrix) with collagen degradation activity

A 72-kDa matrix metalloproteinase (MMP; sMP) was purified from silver carp skeletal muscle by 30–70% ammonium sulfate fractionation and chromatography. The sMP revealed high gelatinolytic activity from 30 to 60°C and showed an optimal pH of 8.0. The gelatin-hydrolyzing activity of sMP was inhibited by ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), l-Cys, and l-His. Ca2+ enhanced the proteinase activity, while a higher concentration of Zn2+ inhibited this activity. Peptide mass fingerprinting revealed that 14 fragments were identical to MMP-2 from grass carp (Ctenopharyngodon idella). The full length of the cDNA sequence encoding sMP was 3005bps, with an open reading frame (ORF) of 1977 bps encoding 658 amino acid residues. Further, sMP could effectively hydrolyze type I collagen even at 4°C; its activity remained steady during the entire storage period at 4°C. These results indicated that sMP is MMP-2 and may have been involved in the protein degradation of fish muscle during the postmortem stage.

Hyperplasia and Cellularity Changes in IGF-1-Overexpressing Skeletal Muscle of Crucian Carp

The zebrafish skeletal muscle-specific promoter mylz2 was used to cause crucian carp overexpression of the zebrafish IGF-1 cDNA. In stable transgenic germline F1 progenies, a 5-fold increase in the level of IGF-1 in skeletal muscle was observed. Evident skeletal muscle hyperplasia was observed in the transgenic fish through histologic analysis. By analyzing the RNA sequencing transcriptome of the skeletal muscle of IGF-1 transgenic fish and nontransgenic control fish at 15 months of age, 10 966 transcripts with significant expression levels were identified with definite gene descriptions based on the corresponding zebrafish genome information. Based on the results of our RNA sequencing transcriptome profiling analysis and the results of the real-time quantitative PCR analysis performed to confirm the skeletal muscle transcriptomics analysis, several pathways, including IGF-1 signaling, aerobic metabolism, and protein degradation, were found to be activated in the IGF-1-overexpressing transgenic fish. Intriguingly, our transcriptional expression and protein assays indicated that the overexpression of IGF-1 stimulated a significant shift in the myofiber type toward a more oxidative slow muscle type. Although the body weight was surprisingly decreased by IGF-1 transgenic expression, significantly higher oxygen consumption rates were measured in IGF-1-overexpressing transgenic fish compared with their nontransgenic control fish. These results indicate that the sustained overexpression of IGF-1 in crucian carp skeletal muscle promotes myofiber hyperplasia and cellularity changes, which elicit alterations in the body energy metabolism and skeletal muscle growth.

Expression and characterization of common carp (Cyprinus carpio) matrix metalloproteinase-2 and its activity against type I collagen

Matrix metalloproteinases (MMPs) play essential roles in the metabolism of animal collagen while few reports are available for MMPs in aquatic animals. In this study, we report the complete sequence of matrix metalloproteinase-2 (MMP-2) gene from common carp (Cyprinus carpio) skeletal muscle. The full-length cDNA of MMP-2 was 2792bp which contains an open reading frame of 1974bp, corresponding to a protein of 657 amino acid residues. Based on the structural feature of MMP-2, the gene of the catalytic domain containing 351 amino acid residues was cloned and expressed in Escherichia coli. SDS-PAGE showed that the truncated recombinant MMP-2 (trMMP-2) with molecular mass of approximately 38kDa was in the form of inclusion body. The trMMP-2 was further purified by immobilized metal ion affinity chromatography. After renaturation, similar to native MMP-2, the trMMP-2 exhibited high hydrolyzing activity toward gelatin as appeared on gelatin zymography and optimal activity was at pH 8.0 and 40°C. The activity of the trMMP-2 was completely suppressed by metalloproteinase inhibitors, including EDTA, EGTA and 1,10-phenanthroline while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca2+ was necessary for the gelatinolytic activity, suggesting it is a calcium-dependent metalloproteinase. Moreover, the trMMP-2 effectively hydrolyzed native type I collagen at 37°C and even at 4°C, implying its potential application value as a collagenase for preparation of biologically active oligopeptides.

Purification and characterization of a gelatinolytic matrix metalloproteinase from the skeletal muscle of grass carp (Ctenopharyngodon idellus)

A gelatinolytic matrix metalloproteinase (gMMP) from grass carp skeletal muscle was purified by 30–70% ammonium sulphate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, and affinity on gelatin–sepharose. The molecular weight of the proteinase as estimated by SDS–PAGE was 70kDa under non-reducing conditions. The enzyme revealed high activity from 30 to 50°C, and the gelatin hydrolysing activity was investigated at a slightly alkaline pH range using gelatin as substrate. Metalloproteinase inhibitor EDTA completely suppressed the gelatinolytic activity, while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca2+ was essential for the gelatinolytic activity. Further, peptide mass fingerprinting obtained four fragments with 45 amino acid residues, which were highly identical to MMP-2 from fish species. The gMMP could effectively hydrolyse type I collagen even at 4°C, suggesting its involvement in the texture softening of fish muscle during the post-mortem stage.

Identification and Profiling of MicroRNAs from Skeletal Muscle of the Common Carp

The common carp is one of the most important cultivated species in the world of freshwater aquaculture. The cultivation of this species is particularly productive due to its high skeletal muscle mass; however, the molecular mechanisms of skeletal muscle development in the common carp remain unknown. It has been shown that a class of non-coding ∼22 nucleotide RNAs called microRNAs (miRNAs) play important roles in vertebrate development. They regulate gene expression through sequence-specific interactions with the 3′ untranslated regions (UTRs) of target mRNAs and thereby cause translational repression or mRNA destabilization. Intriguingly, the role of miRNAs in the skeletal muscle development of the common carp remains unknown. In this study, a small-RNA cDNA library was constructed from the skeletal muscle of the common carp, and Solexa sequencing technology was used to perform high throughput sequencing of the library. Subsequent bioinformatics analysis identified 188 conserved miRNAs and 7 novel miRNAs in the carp skeletal muscle. The miRNA expression profiling showed that, miR-1, miR-133a-3p, and miR-206 were specifically expressed in muscle-containing organs, and that miR-1, miR-21, miR-26a, miR-27a, miR-133a-3p, miR-206, miR-214 and miR-222 were differentially expressed in the process of skeletal muscle development of the common carp. This study provides a first identification and profiling of miRNAs related to the muscle biology of the common carp. Their identification could provide clues leading towards a better understanding of the molecular mechanisms of carp skeletal muscle development.

Structural differences in the motor domain of temperature-associated myosin heavy chain isoforms from grass carp fast skeletal muscle

We determined coding sequences for three types of grass carp myosin subfragment-1 (S1) heavy chain by extending 5'-regions of the three known genes encoding light meromyosin isoforms (10 degrees C, intermediate and 30 degrees C types). The primary structures of these three S1 heavy chain isoforms showed 81.4%, 81.2%, and 97.8% identities between the 10 degrees C and intermediate types, between the 10 degrees C and 30 degrees C types, and between the intermediate and 30 degrees C types, respectively. Isoform-specific differences were clearly observed between the 10 degrees C type and the other two types in 97 amino acid residues. Furthermore, among these amino acid mutations, 51 mutations occurred at the conserved residue sites of S1 heavy chain from fish and homoiotherm. Additionally, the 10 degrees C type showed striking differences compared with the other two types in the two surface loops, loop 1 located near the ATP-binding pocket and loop 2, which is one of the actin-binding sites, suggesting that such structural differences possibly affect their motor functions. Interestingly, this 10 degrees C-type myosin heavy chain isolated from adult grass carp skeletal muscle was surprisingly similar to the embryonic fast-type myosin heavy chain from juvenile silver carp in the structure of S1 heavy chain, indicating that it may also function as embryonic fast-type myosin heavy chain in juvenile stage.

Regional variation in parvalbumin isoform expression correlates with muscle performance in common carp (Cyprinus carpio)

The mechanical properties of the axial muscles vary along the length of a fish's body. This variation in performance correlates with the expression of certain muscle proteins. Parvalbumin (PARV) is an important calcium binding protein that helps modulate intracellular calcium levels which set the size and shape of the muscle calcium transient. It therefore has a central role in determining the functional properties of the muscle. Transcript data revealed eight specific isoforms of PARV in common carp (Cyprinus carpio) skeletal muscle which we classified as alpha1 and beta1-7. This study is the first to show expression of all eight skeletal muscle PARV isoforms in carp at the protein level and relate regional differences in expression to performance. All of the PARV isoforms were characterised at the protein level using 2D-PAGE and tandem mass spectrometry. Comparison of carp muscle from different regions of the fish revealed a higher level of expression of PARV isoforms beta4 and beta5 in the anterior region, which was accompanied by an increase in the rate of relaxation. We postulate that changes in specific PARV isoform expression are an important part of the adaptive change in muscle mechanical properties in response to varying functional demands and environmental change.

Global cooling: Cold acclimation and the expression of soluble proteins in carp skeletal muscle

The common carp (Cyprinus carpio) has a well-developed capacity to modify muscle properties in response to changes in temperature. Understanding the mechanisms underpinning this phenotypic response at the protein level may provide fundamental insights into the molecular basis of adaptive processes in skeletal muscle. In this study, common carp were subjected to a cooling regimen and soluble extracts of muscle homogenates were separated by 1-D SDS-PAGE and 2-DE. Proteins were identified using MALDI-TOF-MS and de novo peptide sequencing using LC-MS/MS. The 2-D gel was populated with numerous protein spots that were fragments of all three muscle isoforms (M1, M2 and M3) of carp creatine kinase (CK). The accumulation of the CK fragments was enhanced when the carp were cooled to 10 degrees C. The protein changes observed in the skeletal muscle of carp subjected to cold acclimation were compared to changes described in a previous transcript analysis study. Genes encoding CK isoforms were downregulated and the genes encoding key proteins of the ubiquitin-proteasome pathway were upregulated. These findings are consistent with a specific cold-induced enhancement of proteolysis of CK.

C-protein (MyBP-C) isoforms from carp ordinary and dark muscles and muscle type-specific binding to myosin

C-protein is a myosin-associated protein of vertebrate striated muscle, and its function and properties have been extensively examined. However, there has been no report of C-protein of fish skeletal muscle so far. C-protein was identified in carp skeletal muscle by immunoassay using antibody against chicken C-protein, and the muscle-type specific C-protein was purified from carp ordinary and dark muscles for the first time. Although C-protein could be prepared from crude myosin by the reported procedure, C-protein degraded appreciably during the purification steps. Accordingly, C-protein was selectively extracted from the muscle with 0.15 M K-phosphate buffer (pH 5.8), and purified by ammonium sulfate fractionation, followed by AF-blue chromatography. Myosin free from the accessory proteins was obtained by diethylaminoethyl (DEAE) chromatography and used to assay the binding of C-protein with myosin. Ordinary muscle C-protein bound to ordinary muscle myosin in a saturable manner, but its maximum amount of binding was approximately twice that of dark muscle myosin. Similarly, dark muscle C-protein bound to dark muscle myosin much more than to ordinary muscle myosin. These results suggest that C-protein isoforms specifically bound with myosin isoforms originated from the same type of muscle.

Biochemical Properties of Ordinary and Dark Muscle Myosin from Carp Skeletal Muscle

Two types of myosin isolated from ordinary (fast) and dark (slow) muscles of carp were examined by ATPase and in vitro motility assays. Vmax of the ATPase activity and sliding velocity of ordinary myosin showed 1.6 and 1.5 times higher activities than those of dark myosin, whereas those of mammalian fast myosin were much higher, 3 to 10 times, than those of slow myosin. Although ordinary myosin had almost identical activities to those of mammalian fast myosin, activities of dark myosin was twice of those of mammalian slow myosin. This high motile activity of dark myosin can account for the physiological role of dark muscle in cruising of fish. By comparing Km of the actin-activated ATPase activity, ordinary myosin was appeared to have higher affinity to F-actin than dark myosin, and this was confirmed by the binding assay of HMM or S-1 of carp myosin to F-actin. Investigation of myosin assembly by electron microscopy and the centrifugation assay revealed that ordinary myosin assembled much poorly than dark myosin or mammalian fast myosin. This phenomenon may reflect characteristic cellular function of fish skeletal muscle.

Uncoupling protein 1 in fish uncovers an ancient evolutionary history of mammalian nonshivering thermogenesis

Uncoupling proteins (UCPs) increase proton leakage across the inner mitochondrial membrane. Thereby, UCP1 in brown adipose tissue dissipates proton motive force as heat. This mechanism of nonshivering thermogenesis is considered as a monophyletic trait of endothermic placental mammals that emerged about 140 million years ago and provided a crucial advantage for life in the cold. The paralogues UCP2 and UCP3 are probably not thermogenic proteins but convey mild uncoupling, which may serve to reduce the rate of mitochondrial reactive oxygen species production. Both are present in endotherms (mammals and birds), but so far only UCP2 has been identified in ectothermic vertebrates (fish and amphibia). The evolution of UCPs is of general interest in the search for the origin of mammalian UCP1-mediated nonshivering thermogenesis. We here show the presence of UCP1 and UCP3 in ectothermic teleost fish species using comparative genomics, phylogenetic inference, and gene expression analysis. In the common carp (Cyprinus carpio), UCP1 is predominantly expressed in the liver and strongly diminished in response to cold exposure, thus contrasting the cold-induced expression of mammalian UCP1 in brown adipose tissue. UCP3 mRNA is only found in carp skeletal muscle with expression levels increased fivefold in response to fasting. Our findings disprove the monophyletic nature of UCP1 in placental mammals and demonstrate that all three members of the core UCP family were already present before the divergence of ray-finned and lobe-finned vertebrate lineages about 420 million years ago.

1P130 コイ普通筋(速筋)および血合筋(遅筋)ミオシンの生化学的性質について(筋肉(筋蛋白・収縮)))

1P130 コイ普通筋(速筋)および血合筋(遅筋)ミオシンの生化学的性質について(筋肉(筋蛋白・収縮)))

Sequence Differences between α1C and α1S Ca2+ Channel Subunits Reveal Structural Determinants of a Guarded and Modulated Benzothiazepine Receptor

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.

Two Amino Acid Residues in the IIIS5 Segment of L-Type Calcium Channels Differentially Contribute to 1,4-Dihydropyridine Sensitivity

The transmembrane segment IIIS5 of the L-type calcium channel alpha1 subunit participates in the formation of the 1,4-dihydropyridine (DHP) interaction domain (Grabner, M., Wang, Z., Hering, S., Striessnig, J., and Glossmann, H. (1996) Neuron 16, 207-218). We applied mutational analysis to identify amino acid residues within this segment that contribute to DHP sensitivity. DHP agonist and antagonist modulation of Ba2+ inward currents was assessed after coexpression of chimeric and mutant calcium channel alpha1 subunits with alpha2delta and beta1a subunits in Xenopus oocytes. Whereas DHP antagonists required Thr-1066, DHP agonist modulation crucially depended on the additional presence of Gln-1070 (numbering according to alpha1C-a), which also further increased the sensitivity to DHP antagonists. Asp-955, which is found at the corresponding position in the calcium channel alpha1S subunit from carp skeletal muscle, displayed functional similarity to Gln-1070 with respect to DHP interaction. We conclude that these residues (Thr-1066 plus Gln-1070 or Asp-955), which are located in close vicinity on the same side of the putative alpha-helix of transmembrane segment IIIS5, form a crucial DHP binding motif.

Identification of PK-A phosphorylation sites in the carboxyl terminus of L-type calcium channel alpha 1 subunits.

Full length L-type calcium channel alpha 1 subunits are rapidly phosphorylated by protein kinase A (PK-A) in vitro and in vivo at sites located in their long carboxyl terminal tails. In skeletal muscle, heart, and brain the majority of biochemically isolated alpha 1 subunits lacks these phosphorylation sites due to posttranslational proteolytic processing. Truncation may therefore modify the regulation of channel activity by PK-A. We combined site-directed mutagenesis and heterologous expression to investigate the extent to which putative cAMP-dependent phosphorylation sites in the C-terminus of alpha 1 subunits from skeletal muscle, heart, and brain are phosphorylated in vitro. The full length size form of wild-type and mutant calcium channel alpha 1 subunits was obtained at high yield after heterologous expression in Saccharomyces cerevisiae. Like in fetal rabbit myotubes [Rotman, E.I., et al. (1995) J. Biol. Chem. 270, 16371-16377], the rabbit skeletal muscle alpha 1 C-terminus was phosphorylated at serine residues 1757 and 1854. In the carboxyl terminus of alpha 1S from carp skeletal muscle and alpha 1C from rabbit heart a single serine residue was phosphorylated by PK-A in vitro. The C-terminus of alpha 1D was phosphorylated at more than one site. Employing deletion mutants, most of the phosphorylation ( > 70%) was found to occur between amino acid residues 1805 and 2072. Serine 1743 was identified as additional phosphorylation site in alpha 1D. We conclude that in class S and C calcium channels the most C-terminal phosphorylation sites are substrate for PK-A in vitro, whereas in class D calcium channels phosphorylation also occurs at a site which is likely to be retained even after posttranslational truncation.

Chimeric L‐type Ca2+ channels expressed in Xenopus laevis oocytes reveal role of repeats III and IV in activation gating.

1. Chimeric alpha 1 subunits consisting of repeat I and II from the rabbit cardiac (alpha 1C-a) and repeat III and IV from the carp skeletal muscle Ca2+ channel (alpha 1S) were constructed and expressed in Xenopus laevis oocytes without co-expressing other channel subunits. Ba2+-current kinetics of five chimeric channel constructs were studied in Xenopus oocytes using the two-microelectrode technique. 2. Exchange of repeats III and IV of alpha 1C-a with sequences of alpha 1S results in a significantly slower and biexponential activation (apparent activation time constants tau 1act = 19.8 +/- 1.8 ms and tau 2act = 214 +/- 28.7 ms, n = 7) of expressed Ca2+ channel currents; no current inactivation was observable during an 800 ms test pulse to 0 mV. 3. Activation of a chimera consisting of repeats I, II and IV from the alpha 1C-a subunit and repeat III from alpha 1S was fast and monoexponential (tau 1act = 6.33 +/- 1.7 ms, n = 5) and the current inactivated during a 350 ms test pulse to 0 mV (tau inact = 175 +/- 22 ms, n = 5). The current kinetics of this construct did not significantly differ from kinetics of a construct consisting of repeats I to IV from alpha 1C-a (tau 1act = 6.6 +/- 2.1 ms; tau inact = 198 +/- 14 ms; n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Changes Caused by Methidathion in Activity and Distribution of Molecular Forms of Carp ( Cyprinus carpio L.) AChE

The activity and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) were characterized in the heart and skeletal muscle of carp ( Cyprinus carpio L.) exposed to 2 mg/liter of the insecticide methidathion (MD; S-2,3-dihydro-5-methoxy-2-oxo-1,3,4-thiodiazol-3-yl-methyl- O, O-dimethyl phosphodithioate) for 5 days at a water temperature of 12 ± 1°C. The specific activity of the heart AChE (4.78 ± 2.14 × 10 −2 U/mg protein) was higher than that of the muscle AChE (2.53 ± 0.75 × 10 −2 U/mg protein) in control carp. The solubility properties and molecular forms of the AChE in the two tissues were studied by extraction in high-salt medium (1 and 0.4 M NaCl) with and without Triton X-100. The ratio of membrane-bound to salt-soluble forms of AChE was higher in the muscle than in the heart. Velocity sedimentation centrifugation revealed that the skeletal muscle and the heart contained three molecular forms: G 4, A 4, and A 12. After the in vivo exposure of fish to MD, the AChE activity decreased significantly (77-92%) in the tissues investigated. Furthermore, the relative distribution of the AChE molecular forms differed somewhat from that in the control fish. Form G l became detectable after the treatment, but could not be measured in the control tissues. The proportions of the other forms decreased by a few percentages. The accumulation of MD was studied in seven organs: the brain, heart, intestine, kidney, liver, muscle, and roe. The liver and roe had the highest MD contents, and the heart and muscle had the lowest. This study shows the MD not only blocks the activity of AChE, but also modifies the distribution of its molecular forms.

Up-regulation of insulin binding in fish skeletal muscle by high insulin levels

The effects of insulin titres on regulation of receptor binding were studied in several fish species. Insulin receptors were semi-purified by affinity chromatography (WGA-agarose) from skeletal muscle of carp, brown trout and rainbow trout that had been subjected to increases in insulinemia produced either by arginine injection, food administration, or adaptation to an experimental diet (extruded diet with high-digestibility carbohydrates). Arginine injection provoked acute hyper-insulinemia in both carp and trout. Specific binding of insulin to the skeletal muscle was significantly increased 3 h after injection (from 5.8 ± 0.3 to 9.6 ± 0.9%/10 μ g protein in carp and from 0.8 ± 0.2 to 1.5 ± 0.4%/10 μ g in trout). The same effect was observed in carp liver preparations (from 6.0 ± 0.75 to 9.9 ± 1.25%/10 μ g ). No alterations in tyrosine kinase activity of the receptors were detected in either carp or trout preparations: basal activities of the receptors were maintained ( 3100 ± 200 fmol P/fmol receptors/30 min and 3700 ± 400 fmol P/fmol receptors/30 min, in carp and trout, respectively), as were the percentage of stimulation over basal levels obtained by incubation with insulin ( 227 ± 25% and 160 ± 10% respectively). Food ingestion raised plasma insulin levels more steadily. Specific binding also increased in skeletal muscle preparations, especially in carp (from 5.7 ± 0.3 to 11 ± 1.7%/10 μ g at 4 h and 10 ± 0.7%/10 μ g at 8 h). Tyrosine kinase activity was maintained without significant changes. Rainbow trout adapted for 2 months to an extruded diet presented higher insulin titres and higher glycogen reserves in liver and muscle. Insulin binding to skeletal muscle preparations was also significantly increased (from 0.36 ± 0.02 to 0.77 ± 0.1%/10 μ g ), as was tyrosine kinase activity (from 132 ± 4% to 156 ± 6%, without alterations in the basal activity). Results showed that fish can respond to both acute and maintained increases in insulinemia by increasing the number of insulin receptors. Tyrosine kinase activity, in contrast, is only modified after long-term adaptation.