Identification of mRNAs Responsive to Leucine in Skeletal Muscle of Rats–A Deep‐Sequencing Approach
Authors: Silvestre João Guilherme , Cruz André , Bento Mirella Ribeiro , Graça Flávia Aparecida , Moriscot Anselmo Sigari 
Published: November 2023

Our laboratory has been focusing on the anti‐catabolic effects of Leucine (Leu) related to the control of skeletal muscle mass. Leu, is as a potent pharmacological agent, preventing catabolism in muscular atrophy and previous studies have demonstrated that rats supplemented daily with Leu showed reduced weight loss of soleus muscle and also reduced loss of cross‐sectional area after 7 days of immobilization. Next generation sequencing (NGS) technologies represents a helpful tool to accurately identify modulated genes in large scale, therefore can be useful to approach the effects of nutrients on metabolic and catabolic pathways. In the current study we have used mRNA NGS to identify transcripts, which are responsive to Leu in soleus muscle.

The protocols used in this study submitted and approved by the Institute of Biomedical Sciences/University of São Paulo ‐ Ethical Committee for Animal Research (# 151/12), which is in agreement with the international guidelines for animal research. Male rats (n=32 ~2 months‐old weighing 260 ± 30g) received dietary Leu supplementation for 3 days and 7 days. The soleus muscles were stored at −70°C and RNA was isolated by Trizol. For deep‐sequencing analysis, the cDNA libraries were mounted by using the Illumina TruSeq Stranded mRNA Sample kit and then sequenced by HiSeq SBS v4 High Output. Subsequently the reads were trimmed, sorted, aligned, mapped and counted at Illumina Basespace® platform using Tuxedo's based apps. The differential expression output through DeSeq2 was downstream analyzed by matplotlib and bioconductor's pathview Python and R packages respectively, gene ontology Enrichment was done at PANTHER tools.

Initially, we applied a filter to display the deferentially expressed transcripts (cut off: log2>=1), up‐regulated (UR) or down‐regulated (DR) by 3 and 7 days of Leu supplementation in soleus muscle. Three days of Leu showed 54 transcripts deferentially expressed (70.37% UR and 29.69% DR) and 7 days of Leu showed 987 transcripts deferentially expressed (18.14% UR and 81.86% DR). To approach the dynamics among the regulated genes we used the Venn diagram and found 26 and as much as 167 transcripts exclusively UR by 3 and 7 days of Leu respectively. Accordingly, only 13 and as much as 805 transcripts were exclusively DR by 3 and 7 days of Leu respectively. Out of those only 12 and 3 transcripts were UR and DR in both time points respectively. Subsequently we focused our enrichment analysis on the 805 transcripts DR by 7 days of Leu and identified that 28% belonged to the metabolic process category. Interestingly we have found that components of the FoxO signaling pathway IRS1, PIK3r, Pten and Prkaa2 were DR by 7 days of leu treatment. In addition the E3 ligases Xiap and Mid1 were DR by 7 days of leu treatment.

Our results showed that Leu supplementation initially (3 days of treatment) regulate a restrict set of genes and subsequently (7 days of treatment) generate massive numbers of regulated transcripts, especially DR. In addition we have identified potential targets related to regulation of skeletal muscle mass responsive to Leu.

Support or Funding Information

FAPESP: 2015/04090‐0; FAPESP: 2016/12941‐2