Abstract
The expression and regulation of intestinal oligopeptide transporter (PepT)-1 when vegetable sources are used as a substitute for fish meal in the diet of marine fish has not yet been explored. In the present study, as part of our ongoing work on elucidating PepT1 gene expression in relation to different dietary treatments, we have now isolated and deposited in Genbank database (accession no. GU733710) a cDNA sequence representing the PepT1 in the sea bream (Sparus aurata). The “de novo” prediction of the three-dimensional structure of PepT1 protein is presented.We also analyzed diet-induced changes in the expression of PepT1 mRNA via real-time RT-PCR using the standard curve method. Sea bream were fed for 140 days with one of the following four diet formulations (43% protein/21% lipid): a control fast growth-promoting diet (C), and three diets with the same formulation but in which 15% of the fish meal was substituted by protein concentrates either from lupine (LPC), chick pea (CPC), or green pea (PPC). Fish fed PPC had significantly (p < 0.05) lower levels of PepT1 transcripts in the proximal intestine than the controls, whereas PepT1 transcript levels in fish fed LPC or CPC were not significantly different from the controls. Although growth was similar between fish fed with different diets during the first 72 days of feeding, growth of the fish fed with PPC was reduced during the second part of the trial and was significantly (p < 0.05) lower than fish fed LPC and CPC diets by the end of the experiment. Correlation between these results and fish growth performances highlights that the intestinal PepT1 mRNA level may serve as a useful marker of the dietary protein quality and absorption efficiency.
Highlights
The end products of intestinal protein digestion in both teleosts and mammals constitute a mixture of free amino acids and small peptides that are efficiently absorbed across the small intestinal epithelium (Clements & Raubenheimer 2006)
Preparation of total RNA, cDNA synthesis, sea bream PepT1 cloning and sequencing Total RNA was extracted from sea bream proximal intestine using PureYield RNA Midiprep System (Promega, Italy), following the protocol described in PureYieldTM RNA Midiprep System Technical Manual #TM279, available online at: www.promega.com/tbs
Sea bream PepT1 cDNA sequence Sea bream PepT1 primer design was based on the alignment of four fish PepT1 coding sequences available on the NCBI Genbank database: Dicentrarchus labrax
Summary
The end products of intestinal protein digestion in both teleosts and mammals constitute a mixture of free amino acids and small peptides that are efficiently absorbed across the small intestinal epithelium (Clements & Raubenheimer 2006). Cellular uptake of small peptides is mediated by H +/coupled peptide transporter (PepT1), located at the brush border membrane of intestinal epithelial cells (Yuen et al 2007). The affinity of PepT1 and its stereoselective capacity differ for different types of peptides, suggesting that its activity and expression may be modulated through the diet, when different protein sources are utilized (Ostaszewska et al 2010a; Ostaszewska et al 2010b). In the present study we first cloned a cDNA encoding PepT1 in gilthead sea bream and assessed the impact of different feeds in which various vegetable sources substituted the fish meal on PepT1 mRNA levels in the proximal intestine of sea bream (Sparus aurata) with the aim to relate these expression levels to fish performance during the feeding trial
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