Abstract
As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders.
Highlights
Skeletal muscle is considered as the primary tissue for insulinstimulated glucose uptake, accounting for up to 80% of the insulin-dependent glucose disposal in whole body glucose homeostasis [1]
Considering skeletal muscle tissues as an important secretory organ, which communicate with other organs though the secreted proteins, miRNAs, metabolites and others, we were interested in identifying peptides secreted from skeletal muscle tissues under the pathophysiology of metabolic diseases such as diabetes and obesity
Lactate dehydrogenase (LDH) and IL-6 release were evaluated in the supernatant from skeletal muscle explants isolated from the control mice, which partly reflected the signs of tissue damage along the incubation period
Summary
Skeletal muscle is considered as the primary tissue for insulinstimulated glucose uptake, accounting for up to 80% of the insulin-dependent glucose disposal in whole body glucose homeostasis [1]. IL-6 could be released from contracting muscle, exerting endocrine effects on peripherally insulin sensitive tissues [17,18,19]. Another known contraction-induced myokines including IL-15, IL-8, Irisin, and Myonectin showed potential metabolic function for preventing and treating T2DM [20]. These accumulating evidence of myokine from skeletal muscle secretome are central to our understanding of the cross talk between skeletal muscle and other organs during exercise. Identification of more types of muscle-secreted factors and exploration of the potential regulatory mechanisms by which they act remain to be established
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