| Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus L.) are marine carnivorous fish species cultured in northern China. A gas chromatography-mass spectrometry (GC-MS) metabolomic platform was used to analyze the effect of high glucose load on plasma metabolism in Japanese flounder. In addition, feeding trail was conducted to investigate effects of ratios of dietary carbohydrate to lipid on leptin, leptin receptor and adiponectin receptors gene expression in turbot. The experiments and results of the present study are summarized as follows:1. A metabolomics approach to elucidate effects of high glucose load on plasma metabolism in Japanese flounderGas chromatography-mass spectrometry-based metabolomics method was applied to determine the metabolites in the plasma of Japanese flounder (225±50g) after high glucose load. Intraperitoneal injection (IP,500mg/ml,1g/kg body weight) or oral administration (OR, 500mg/ml,1.65g/kg body weight) was used as two different methods of glucose tolerance test. After Intraperitoneal injection of glucose, glycemia of Japanese flounder peaked (20.06±1.92mM) at 5h, and resumed to the normal level (7.40±5.47mM) at 24h (P>0.05); the plasma insulin content reduced sharply, and after reached to the lowest point (1.58±0.21mIU/L) at 3h (P< 0.05), it restored at 24h (3.03±0.06mIU/L); campared with the Oh group with none operation, six metabolites were upregulated at 3h, including talose, xylose, isomaltose, galacturonic acid, hydroxyurea and conduritol β epoxide, and five metabolites were upregulated at 5h (P< 0.05), including talose, xylose, isomaltose, xanthine, lactic acid, aconitic acid, mehyl jasmonate, conduritol β epoxide and cortisone. After oral administration of glucose, glycemia of Japanese flounder peaked (1.90±0.23mM) at 3h, and resumed the normal level (0.89±0.04mM) at 7h; the plasma insulin content transiently increased to 3.37±0.34mIU/L at 3h, but resumed (2.56±0.41mIU/L) at 5h; campared with the Oh group, there were five metabolites rising at 3h (P<0.05), including talose, lactic acid, 3-hydroxyanthranilic acid, xanthine and hypoxanthine, but one metabolite (galactonic acid) was downregulated at 5h (P< 0.05). Additionally, the difference of plasma metabolites between two glucose tolerance tests with different method was also analyzed. The result showed that four metabolites in IP test were higher than OR test at 3h after glucose load, involving talose, isomaltose, conduritol β epoxide and 3(2-hydroxyphenyl)propionic acid. Six metabolites including talose, isomaltose, xylose, conduritol β epoxide, aconitic acid and galactonic acid, in IP test were higher, but one metabolite (N-carbamylglutamate) was lower than OR test at 5h. The different metabolites in Japanese flound revealed that the level of many glucose metabolic pathways, including glycolysis, Kreb’s cycle, glycogen synthesis, pentose and glucuronate interconversions and pentose phosphate metabolism, changed after glucose load. Meanwhile, the changing glycemia also influenced amino acid metabolism and enteric microorganism metabolism. Remarkably,3-(2-hydroxyphenyl)propiomic acid and N-carbamylglutanate were found to be relevant with some hormones, such as leptin, insulin, glucagon and somatostatin. The change of 3-(2-hydroxyphenyl) propiomic acid, N-carbamylglutanate and cortisone levels between different groups uncovered that cortisone, leptin, insulin, glucagon and somatostatin might regulate glucose metabolism in Japanese flounder.2. Cloning and characterization of leptin, leptin receptor and adiponectin receptors in turbot.In the present study, the full-length cDNA sequences of leptin (LEP), leptin receptor (LEPR) and adiponectin receptors (AdipoR1, AdipoR2) from turbot were cloned. The cDNA of tLEP was 1126bp in length encoding 157 amino acids. The amino acid sequence shared low identity with human LEP (18.8%), but the three-dimensional structures of these two LEPs were strongly conserved. The deduced 1173-amino acid sequence of tLEPR was 28% identical to human LEPR, and 82% to orange-spotted grouper LEPR, containing all functionally important domains conserved in vertebrate LEPR. Tissue distribution analysis showed that tLEP was abundantly expressed in brain, eyes and liver. The highest level of tLEPR mRNA was found in liver and kidney. The cDNA sequence of tAdipoRl was 1125bp, encoding 375 amino acids. The deduced amino acid sequence of tAdipoRl was conserved with all the vertebrate AdipoRl, such as human (80%) and tilapia (97%). The cDNA sequence of tAdipoR2 was 1940bp in length, encoding 380 amino acids. The amino acid sequence of tAdipoR2 was 78% identical to human and 92% to tilapia. Same with other vertebrate, both tAdipoR1 and tAdipoR2 had 7 transmembrane domains, and the N-terminal of the optimal topological structure was inside cells. Turbot AdipoR was widely expressed in all detected tissues. The highest mRNA level of tAdipoR1 was found in gill and kidney, and higher expression of tAdipoR2 was found in gill, stomach, intestine, kidney and pyloric caeca, but little in muscle.3. Effects of dietary carbohydrate-to-lipid ratio on the gene expression of leptin, leptin receptor and adiponectin receptors.After a 9-week feeding trial using diets with different ratios of carbohydrate to lipid (1:6, 1:2,2:1 and 14:1), it was found that the increase in dietary carbohydrate-to-lipid ratios from 1:6 to 2:1 did not significantly influence tLEP and tLEPR expression in turbot (P> 0.05). The hepatic tLEP expression was significantly elevated in treatment with 14:1 dietary carbohydrate-to-lipid ratio (P< 0.05). The hepatic tLEPR mRNA level in group with 14:1 dietary carbohydrate-to-lipid ratio was notably lower than that in 1:6 group (P< 0.05), but had no significant difference with the other two groups (P> 0.05). However, the tLEPR mRNA level in muscle in 14:1 group was the highest. Compared with the control group with 1:6 ratio, the expression of tAdipoRl in group with 1:2 dietary carbohydrate-to-lipid ratio was decreased remarkably (P< 0.05) in turbot muscle, but it had no difference with the other two group (P> 0.05). The changes of dietary carbohydrate-to-lipid ratio had no effect on the tAdipoR expression in liver and tAdipoR2 expression in muscle (P> 0.05). These results revealed the important correlation between dietary carbohydrate level and LEP and adiponectin in turbot. |
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