Comparative proteomic analysis of the insulin‐induced L6 myotube secretome

Abstract

Emerging evidence has revealed an endocrine function for skeletal muscle; in fact, certain anti-inflammatory cytokines are secreted only from contractile skeletal muscle. However, the skeletal muscle secretome as a whole is poorly characterized, as is how it changes in response to extracellular stimuli. Herein, we sought to identify and characterize the members of the skeletal muscle secretome, and to determine which protein secretion levels were modulated in response to insulin stimulation. To conduct these studies, we treated differentiated L6 rat skeletal muscle cells with insulin or left them untreated, and we comparatively analyzed the proteins secreted into the media. We fractionated this conditioned media using offline RP HPLC, digested the fractionated proteins, and analyzed the resulting peptides with LC-ESI-MS/MS. We identified a total of 254 proteins, and by using three different filtering methods, we identified 153 of these as secretory proteins. Fourteen proteins were secreted at higher levels under insulin stimulation, including several proteins known to be highly secreted in metabolic diseases; 19 proteins were secreted at lower levels under insulin stimulation. These result not only pinpointed several previously unknown, insulin induced, secretory proteins of skeletal muscle, it also described a novel approach for conditioned secretome analysis.

Reduction of Oxalate Levels in Tomato Fruit and Consequent Metabolic Remodeling Following Overexpression of a Fungal Oxalate Decarboxylase

“…In cases where more than one protein was indicated with a significant score for the MS/MS-derived peptide sequence, the match was considered in terms of the highest ranked hit, molecular mass matches, and emPAI, an approach that has been routinely used for proteomics studies (Perkins et al, 1999;Poetsch and Wolters, 2008;Yoon et al, 2009). The details of the MS/MS analyses, including the protein identification, score of the identified protein, threshold score, number of matched peptides, percentage of sequence coverage, Sol Genomics Network (SGN) database accession number, theoretical and experimental M r , and pI values for each protein, peptide sequence, and corresponding peptide score are provided in Supplemental Table S4.…”

Myokines: Do they really exist?

“…Recently, not only IL-6, but also some humoral factors already known as cytokines, such as IL-8, IL-15, brain-derived neurotrophic factor (BDNF), and fibroblast growth factor-21 (FGF-21) 7,8) , have been considered myokines. In addition, recent proteomic analysis indicated that a few hundred proteins are potential myokines [9][10][11] . These accumulating data led to the gradual wide acceptance of the myokine concept.…”

“…However, it is also true that a few hundred proteins are detected in the medium of noncontracted skeletal muscle cells and insulin-stimulated cells [9][10][11] . Therefore, the definition of myokines needs to be expanded.…”

“…Serum FGF-21 levels also increase in Akt1-overexpressing mice, suggesting that activation of the PI-3-Akt signaling pathway is related to FGF-21 secretion. From proteomic analysis, Yoon et al found 14 proteins that increased in L6 cell culture medium following insulin stimulation 10) . To verify the proteomic results, they performed Western blotting and found that matrix metalloproteinase-2 and plasminogen activator inhibitor-1 were actually released from skeletal muscle in response to insulin stimulation 10) .…”

Quantitative Proteomics for Investigation of Secreted Factors: Focus on Muscle Secretome

“…Interestingly, the secretion of myostatin, a negative regulator of skeletal muscle growth and development but also implicated in metabolic homeostasis, was found to be markedly upregulated in extreme obesity cases. Subsequently, Yoon and colleagues presented a study investigating the effects of insulin on the secretory profile of differentiated myotubes (Yoon et al, 2009). The authors combined off-line reverse-phased HPLC fractionation with LC-MS/MS and identified 153 secreted proteins from rat L6 myotubes.…”