Adverse Effects of Excessive Leucine Intake Depend on Dietary Protein Intake: A Transcriptomic Analysis to Identify Useful Biomarkers

Abstract

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.

Maternal high-protein diet during pregnancy, but not during suckling, induced altered expression of an increasing number of hepatic genes in adult mouse offspring

“…The genes Igfbp1 and Cdkn1a were up-regulated and Ccnb2, Inhbe, Fgf21, and Ccnd1 down-regulated [33]. Interestingly, four of these genes were also identified in the current study; however, their regulation was in the opposite direction.…”

The Use of Transcriptomics to Unveil the Role of Nutrients in Mammalian Liver

“…Similar findings of amino acid impact were observed when studying that consumption of diets containing more than 4% Leu in 6% protein content 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 Sprague-Dawley rats. Using this approach, 6 candidate gene markers were identified in liver [115]. …”

Down Regulation of Asparagine Synthetase and 3-Phosphoglycerate Dehydrogenase, and the Up-Regulation of Serine Dehydratase in Rat Liver from Intake of Excess Amount of Leucine Are Not Related to Leucine-Caused Amino Acid Imbalance

“…They were housed individually in stainless-steel cages in an air-conditioned room at 2361˚C with a 12-h light/dark cycle (lights on from 08:00 to 20:00). Rats were acclimated for 3 or 4 d and provided ad libitum access to water and a 20% casein diet based on AIN-93G (10). Then, the rats were assigned to the experimental groups described below.…”

“…The composition of the experimental diets is described elsewhere (10). The rats were allowed free access to food and water during the experimental period.…”

“…Recently, using an animal model, we assessed the safe level of leucine intake from the viewpoint of gene expression (10). The results demonstrated that the tolerable upper intake level of supplemental leucine is 2% of the diet of rats maintained on 6% casein as the sole protein source.…”