SummaryThe present study was conducted to identify reliable gene biomarkers for the adverse effects of excessive leucine (Leu) in Sprague-Dawley rats by DNA microarray. It has long been known that the adverse effects of excessive amino acid intake depend on dietary protein levels. Male rats were divided into 12 groups (n56) and fed for 1 wk a diet containing low (6%), moderate (12%) or high (40%) protein. Different levels of Leu (0, 2, 4, and 8%) were added to the diets. Consumption of diets containing more than 4% Leu in 6% protein resulted in growth retardation and reduced liver weight, whereas the administration of the same dose of Leu with 12% or 40% protein did not affect them. By a process of systematic data extraction, 6 candidate gene markers were identified. The liver gene expression data obtained from another experiment with 0, 2, 3, 4, and 8% Leu in a low-protein diet was used to examine the validity of these biomarker candidates with receiver operating characteristic (ROC) curve analysis. All of AUC values of the biomarker candidates were more than 0.700, suggesting the effectiveness of the marker candidates as the indices of Leu excess. The cut-off value for the ROC curve of the gene-marker panel, which was obtained by multiple regression analysis of gene markers, indicated that Leu levels higher than 3% have adverse effects. In conclusion, the gene-marker panel suggested that for male rats dietary Leu supplementation of 2% is the NOAEL dose in low-protein (6%) diets. Key Words leucine, transcriptome, biomarker, DNA microarray, dietary protein * To whom correspondence should be addressed. E-mail: akatoq@mail.ecc.u-tokyo.ac.jp Abbreviations: Actb, actin, beta; Ccnb2, cyclin B2; Ccnd1, cyclin D1; Cdkn1a, cyclin-dependent kinase inhibitor 1A; Fgf21, fibroblast growth factor 21; Igf1, insulin-like growth factor 1; Igfals, insulin-like growth factor binding protein, acid labile subunit; Igfbp1, insulin-like growth factor binding protein 1; Inhbe, inhibin beta E; Leu, leucine; NOAEL, no observable adverse effect level; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROC curve, receiver operating characteristic curve; SD rat, Sprague-Dawley rat; Stat5a, signal transducer and activator of transcription 5A; Stat5b, signal transducer and activator of transcription 5B; UL, tolerable upper intake level.

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Summary Asparagine synthetase (ASNS), 3-phosphoglycerate dehydrogenase (PHGDH) and serine dehydratase (SDS) in rat liver are expressed in response to protein and amino acid intake. In the present study, we examined the expression of these enzymes in relation to amino acid imbalance caused by leucine. Rats were subjected to leucine administration in the diet or orally between meals. Consumption of more than 2% leucine in a 6% casein diet suppressed food intake and caused growth retardation in a dose-dependent manner, but this was not seen in a 12% or 40% casein diet. ASNS and PHGDH expression in the liver was significantly induced by the 6% casein diet and was suppressed by leucine in a dose-dependent manner, whereas the SDS expression was induced. These effects were leucine specific and not seen with ingestion of isoleucine or valine. However, leucine orally administered between meals did not change the food intake or growth of rats fed a 6% casein die, though it similarly affected the expression of ASNS, PHGDH and SDS in the liver. These results suggest that the growth retardation caused by leucine imbalance was mainly because of the suppression of food intake, and demonstrated that there are no causal relationships between ASNS, PHGDH and SDS expression and amino acid imbalance caused by leucine.

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