Endocrine Abstracts (2015) 38 P210 | DOI: 10.1530/endoabs.38.P210

Proteomic detection of extracellular matrix proteins in insulin-resistant skeletal muscle

Li Kang, De Lin & Claire Sneddon

University of Dundee, Dundee, UK.

Increased deposition of extracellular matrix (ECM) components such as collagens and hyaluronan is associated with diet-induced insulin resistance (IR) in skeletal muscle. Studies of the role of specific ECM components in muscle IR have been pivotal but incomplete. Here we used an approach (i.e. ECM-specific mass spectrometry-based proteomics) that gives a more complete view of ECM protein abundance with muscle IR. Male C57BL/6 mice were fed with either a chow or high fat (HF) diet, which contains 60% calories as fat for 20 weeks before gastrocnemius muscle was collected. Muscle samples were incubated in 1% sodium dodecyl sulfate for 3 days to remove cytosolic proteins and the remaining extracellular and fibril compartments were frozen in liquid nitrogen, grounded, and applied to quantitative proteomics using isobaric tags for relative and absolute quantitation (iTRAQ) technique. Over 80 proteins were detected by proteomics and >90% of these proteins were ECM proteins or contained an extracellular domain, of which collagen (COL) XXIV was the most elevated (10.1±0.80) ECM protein in muscle from HF-fed mice compared to chow-fed mice. In addition, protein expression of COLIα1, COLIα2, and COLIIIα1 were increased by HF feeding by 2.86±0.80, 2.86±0.79, and 2.56±0.64 fold respectively. We further verified these proteomic results in gastrocnemius homogenates by real-time PCR and immunohisochemistry. We showed that mRNA levels of COLXXIV (2.15±0.44 fold), COLIα1 (2.20±0.53), COLIα2 (1.88±0.24) and COLIIIα1 (1.97±0.17) were increased in muscle of HF-fed mice and protein staining of COLIII was increased by 2.08±0.54 fold in HF-fed mice. These results were consistent with results from proteomics and suggest that the ECM-specific proteomics is a powerful tool to identify new ECM targets for muscle IR. COLXXIV was shown for the first time to be increased in insulin-resistant skeletal muscle and may represent a new ECM target for treating muscle IR.

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