Effects Of Dietary Arginine Level And Carbohydrate-to-lipid Ratio On MRNA Expression Of Growth-related Hormones In Largemouth Bass

In the present study, insulin full-length cDNA of largemouth bass (Micropterus salmoides) was colned. Bsides, effects of dietary arginine (Arg) level or carbohydrate-to-lipid (CHO/LIP) ratio on pituitary growth hormone (GH), hepatic insulin-like growth factor-Ⅰ(IGF-Ⅰ) and insulin mRNA expression were investigated in largemouth bass. Results of this study are presented as follows:1. Full-length cDNA cloning of insulin gene and its encoding protein structure analysis in largemouth bassThe complete cDNA of largemouth bass insulin gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, and its encoding amino acid sequence as well as the protein structure was analyzed. The insulin full-length cDNA sequence consists of 655 bp, comprising a 101 bp 5′-untranslated region, a 213 bp 3′-untranslated region and an open reading frame (ORF) of 351 bp in length. The ORF encodes a preproinsulin of 116 amino acids with a molecular weight of about 12.7 kDa and a theoretical isoelectric point of 5.44, from which a signal peptide, a B-chain, a C-peptide and an A-chain could be derived. Online analysis suggests that the deduced preproinsulin contains an obvious hydrophobic domain and a transmembrane region, and that it has a signal peptide whose cleavage site occurs between Alanine24 and Phenylalanine25. Comparative analysis of preproinsulin amino acid sequences related indicates that A-chain and B-chain sequences of propreinsulins are highly conserved between teleost and other vertebrate species, and that the deduced preproinsulin amino acid sequence of largemouth bass shares high similarity of 75-94% to those of other teleosts, but low similarity of 59-62% to those of other vertebrates. The phylogenetic tree by the Neighbor-joining method shows that the preproinsulin of largemouth bass has highly homology with those of some of the other Perciformes fishes, such as rock bass, greater amberjack and tilapia.2. Effects of dietary arginine levels on GH, IGF-Ⅰand insulin mRNA experession in largemouth bassEffects of different dietary Arg levels on growth and expression of pituitary GH, hepatic IGF-Ⅰand insulin mRNA of largemouth bass were evaluated by a real-time fluorescence quantitative PCR (FQ-PCR) method. The results showed that with an increase in dietary Arg levels, specific growth rate (SGR) gradually increased, and reached the highest value at dietary Arg level of 3.01% (P < 0.05); while SGR of other groups didn't show any significant differences. When dietary Arg levels were 1.70-2.52%, pituitary GH mRNA levels didn't change; until dietary Arg level of 2.76%, GH mRNA expression was significantly up-regulated (P < 0.05). With the elevated Arg levels from 1.70% to 2.23%, hepatic IGF-ⅠmRNA expression were significantly up-regulated (P < 0.05); while higher than 2.23%, IGF-ⅠmRNA levels presented inregular fluctuations. However, no effect of Arg intake on hepatic insulin mRNA expression was found. The analysis of the data by the Pearson correlation test revealed that hepatic IGF-ⅠmRNA expression had a significant and positive relation to SGR (P=0.017, r=0.892). Results of this trial indicated that dietary Arg levels affected growth and the hepatic IGF-ⅠmRNA expression as well as pituitary GH mRNA expression in largemouth bass.3. Effects of dietary CHO/LIP ratio on GH, IGF-Ⅰand insulin mRNA expression in largemouth bassEffects of dietary CHO/LIP ratio on growth and expression of pituitary GH, hepatic IGF-Ⅰand insulin mRNA of largemouth bass were evaluated by a FQ-PCR method. The results showed that at dietary CHO/LIP ratio of 0.32, SGR reached a higher value (P < 0.05), while SGR of other groups had no significant differences. When dietary CHO/LIP ratios were not higher than 2.36, pituitary GH and hepatic insulin mRNA levels showed significant upward trends (P < 0.05), but hepatic IGF-ⅠmRNA expression showed a linear downward trend (P < 0.05); until higher than 2.36, they had no significant changes. The analysis of the data by the Pearson correlation test revealed that CHO/LIP ratio (0.32-2.36) had significant and linearly positive correlations to insulin mRNA expression (P=0.038, r=0.962), and that highly negative relations of IGF-ⅠmRNA expression to GH mRNA expression (P=0.000, r= -0.984) occurred, and that SGR had negative relations to GH (P=0.041, r= -0.829), but positive relations to IGF-ⅠmRNA expression (P=0.019, r=0.885). These results indicated that dietary CHO/LIP ratio indeed affected growth and the hepatic IGF-Ⅰand insulin mRNA expression as well as pituitary GH mRNA expression in largemouth bass, and that there was positive or negative correlations of SGR to hepatic IGF-ⅠmRNA expression or pituitary GH mRNA expression respectively, and that the nutritional factors would affect endocrine regulation of growth in largemouth bass through regulating related mRNA expression.