Insulin and 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Differentially Regulate the Skeletal Muscle Cell Secretome

Abstract

Skeletal muscle is a major contributor to whole-body glucose homeostasis and is an important endocrine organ. To date, few studies have undertaken the large-scale identification of skeletal muscle-derived secreted proteins (myokines), particularly in response to stimuli that activate pathways governing energy metabolism in health and disease. Whereas the AMP-activated protein kinase (AMPK) and insulin-signaling pathways have received notable attention for their ability to independently regulate skeletal muscle substrate metabolism, little work has examined their ability to re-pattern the secretome. The present study coupled the use of high-resolution MS-based proteomics and bioinformatics analysis of conditioned media derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR—an AMPK activator)- and insulin-treated differentiated C2C12 myotubes. We quantified 858 secreted proteins, including cytokines and growth factors such as fibroblast growth factor-21 (Fgf21). We identified 377 and 118 proteins that were significantly altered by insulin and AICAR treatment, respectively. Notably, the family of insulin growth factor binding-proteins (Igfbp) was differentially regulated by each treatment. Insulin- but not AICAR-induced conditioned media increased the mitochondrial respiratory capacity of myotubes, potentially via secreted factors. These findings may serve as an important resource to elucidate secondary metabolic effects of insulin and AICAR stimulation in skeletal muscle.

Mass spectrometry‐based proteomics approaches to interrogate skeletal muscle adaptations to exercise

“…Gaining a functional understanding of the molecular transducers of exercise and training may require new and complementary tools, beyond transcriptomic analysis. The robust, high‐throughput and unbiased nature of liquid chromatography‐based tandem MS (LC–MS/MS) provides an ideal candidate to spearhead the large‐scale identification of proteins from biological samples, an exciting scientific venture termed “proteomics.” 23,24 Until recently, the majority of proteomics‐based investigations in skeletal muscle have focused their efforts on characterizing global changes to protein abundance in response to exercise and training, termed “expression proteomics.” For example, by comparing prolonged resistance and endurance training, expression proteomics in human skeletal muscle has validated, to a larger scale, the long‐held contention that these modalities differentially regulate skeletal muscle proteins, while concomitantly providing deeper insights into the endurance and resistance training‐mediated regulation of oxidative metabolism and hypertrophy, respectively 25–27 . Importantly, major advantages to proteomics analyses are the detection of secreted proteins, PTMs (e.g., phosphorylation, acetylation, and ubiquitination), and protein–protein interactions, which are key exercise‐responsive elements to muscle plasticity that remain undetectable using conventional transcriptomic approaches 23,28 .…”

“…The robust, high-throughput and unbiased nature of liquid chromatography-based tandem MS (LC-MS/ MS) provides an ideal candidate to spearhead the largescale identification of proteins from biological samples, an exciting scientific venture termed "proteomics." 23,24 Until recently, the majority of proteomics-based investigations in skeletal muscle have focused their efforts on characterizing global changes to protein abundance in response to exercise and training, termed "expression proteomics." For example, by comparing prolonged resistance and endurance training, expression proteomics in human skeletal muscle has validated, to a larger scale, the long-held contention that these modalities differentially regulate skeletal muscle proteins, while concomitantly providing deeper insights into the endurance and resistance training-mediated regulation of oxidative metabolism and hypertrophy, respectively.…”

The Candidate Schizophrenia Risk Gene Tmem108 Regulates Glucose Metabolism Homeostasis

“…The blood glucose is affected in the Tmem108 mutant mice. From the expression of glycolytic and lipogenic genes, muscle is one of the biggest organs in the body (39), and it is a major contributor to glucose homeostasis (40,41). Though the brain constitutes no more than 5% of the body weight, the brain's energy consumption accounts for 20%-30% of the total glucose utilization.…”

Quantification of AICAR and study of metabolic markers after administration

“…10 Various studies on the mechanism conrmed that the transcription of such genes has a signicant effect on the endurance enhancement by increasing the activity of transcriptional regulators at a genetic level and enhancing the expression of genes in skeletal muscle. 11,12 As a result, doping with the substance AICAR was prohibited by the World Anti-Doping Agency (WADA) 13 in 2009, and its misuse was classied via metabolic regulators aer three years.…”