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.
Highlights
Skeletal muscle is an important tissue in the maintenance of postprandial glucose homeostasis [1] and a major site for insulin resistance in metabolic disease [2]
While the high abundance of intracellular proteins might initially be explained by potential apoptosis following serum-deprivation of cells, proteomic analysis revealed that cytosolic protein lactate dehydrogenase A (LDHA), a marker of membrane leakage/damage, was unchanged in the conditioned media from cultured myotubes
A third cluster contained proteins that were increased by insulin (421 proteins, including 323 secreted proteins; Figure 3F), and this last cluster was enriched by proteins involved in cytokine activity, and included members such as Tgf-β1/2
Summary
Skeletal muscle is an important tissue in the maintenance of postprandial glucose homeostasis [1] and a major site for insulin resistance in metabolic disease [2]. Insulin- and AMPK-dependent pathways are major regulators of skeletal muscle metabolism. The quantitative coverage of the secretome of the immortalized mouse myoblast C2C12 cell line is variable, and detailed comparisons of its composition following the activation of distinct signaling pathways are underexplored [13,14,15]. This is surprising given that differentiated C2C12 myotubes are widely used to garner mechanistic insight into skeletal muscle metabolism [16]. Given that AICAR and insulin initiate different signaling pathways, we hypothesized that the composition and dynamics of myokine secretion would be distinct
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