Journal
AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM
Volume 311, Issue 3, Pages E628-E637Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpendo.00175.2016
Keywords
peptidomics; isotope tracer; low molecular weight; method; autophagy
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Funding
- National Center for Advancing Translational Sciences, a component of the National Institutes of Health (NIH) [5-R01-DK-041973, UL1-TR-000135]
- NIH Director's Common Fund [U24-DK100469]
- Center for Individualized Medicine at the Mayo Clinic
- [T32-D-K-007352]
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Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that similar to 80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-gamma (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria.
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