Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Sep 1;311(3):E628-37.
doi: 10.1152/ajpendo.00175.2016. Epub 2016 Jul 19.

Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice

Matthew M Robinson  1 , Surendra Dasari  2 , Helen Karakelides  1 , H Robert Bergen 3rd  3 , K Sreekumaran Nair  4
Affiliations

Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice

Matthew M Robinson et al. Am J Physiol Endocrinol Metab. .

Abstract

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 ∼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-γ (+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.

Keywords: autophagy; isotope tracer; low molecular weight; method; peptidomics.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Minimizing ex vivo generation of low-molecular weight (LMW) peptides. LMW peptides were extracted with and without protease inhibitors from quadriceps from 6 mice. More LMW peptides and mass spectra and were generated without protease inhibitors, thus demonstrating that protease inhibitors are necessary to avoid ex vivo generation of LMW fragments. Data displayed as means ± SD and P values are from unpaired t-test between treatment conditions with and without protease inhibitors.
Fig. 2.
Fig. 2.
Streptozotocin (STZ) mouse model. A: glucose dysregulation was confirmed in mice following STZ injection and normalized for 2 wk by subcutaneous insulin implants. Implants were removed from the mice for 6 (D-6) and 96 h (D-96) of untreated diabetes (n = 7 each) compared with nondiabetic controls (CON; n = 5). B and C: at euthanasia compared with control mice, D-6 and D-96 mice had higher blood glucose (B) and lower body weight (C). *P < 0.05 vs. baseline (A) or CON (B and C) for multiple comparisons following significant ANOVA. Data are displayed as means ± SD.
Fig. 3.
Fig. 3.
Activation of protein degradation in STZ mice. A and B: activation of protein degradation pathways was determined by real-time PCR for proteasome pathway (E3 ubiquitin ligase; A) and autophagy (beclin; B). Immunoblotting revealed insulin deprivation decreased accumulation of the autophagy receptor protein p62 in quadriceps lysates (C) and isolated mitochondria fractions (D), indicating activation of autophagy. Autophagy negative (HeLa cells untreated) and positive controls (HeLa cells + chloroquine) were included in lanes 1 and 2. Loading controls were vinculin (Vinc) for muscle lysates and complexes III and V for isolated mitochondria. Representative images are from a single blot, with vertical bars representing discontinuous lanes. Data are means ± SD with n = 5–7 mice/group. *P < 0.05 vs. control. AU, arbitrary units.
Fig. 4.
Fig. 4.
Mitochondrial protein degradation in STZ mice identified by LMW. Volcano plots represent total and mitochondrial proteins identified in LMW peptide fragments from quadriceps during 6 (A) and 96 h (B) of insulin deprivation in STZ diabetes mice compared with control mice. Data are presented with statistical significance on vertical axis and fold change on horizontal axis relative to control. The horizontal dashed line at y = 1.3 corresponds to significance at 0.05 for false discovery rate. Proteins with the greatest change in each condition are identified and circled.
Fig. 5.
Fig. 5.
Protein degradation increased with insulin deprivation in type 1 diabetic (T1D) humans. T1D humans (n = 7) were studied on 2 visits during insulin treatment (INS+) or insulin deprivation (INS−) with primed, continuous infusion of [ring-13C6]phenylalanine and biopsies of the vastus lateralis. A: plasma tracer enrichment in molar %excess (MPE) at steady state was lower during INS− study day. B: whole body protein breakdown was increased during insulin deprivation conditions and is displayed as the rate of appearance (Ra) of phenylalanine (Phe) into circulation. C: fractional synthesis rate (FSR) of mixed-muscle proteins was not different between insulin-treated and deprivation conditions. D: posttranslational modifications to proteins were determined by mass spectrometry and revealed that oxidative damage was increased, whereas deamidation was decreased during insulin deprivation conditions. Posttranslational modifications to proteins were determined by mass spectrometry and differential expression based on peptide intensities made by paired comparison and then presented as %difference between INS− and INS+. *P < 0.05 and **P < 0.01 for paired comparison between INS+ and INS−. Data are means ± SD. FFM, fat-free mass.
Fig. 6.
Fig. 6.
Proteins identified by LMW peptides released during INS+ and INS− in humans. LMW peptide fragments identified individual proteins that were differentially generated (P < 0.05) during insulin treatment (INS+) and insulin deprivation (INS−). Each original full-length protein is identified along with P value (paired t-test) and fold change between INS+ and INS−, with negative values indicating more fragments generated during INS+ and positive values indicating more fragments generated during INS−.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Asmann YW, Stump CS, Short KR, Coenen-Schimke JM, Guo Z, Bigelow ML, Nair KS. Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia. Diabetes 55: 3309–3319, 2006. - PubMed
    1. Barazzoni R, Short KR, Asmann Y, Coenen-Schimke JM, Robinson MM, Nair KS. Insulin fails to enhance mTOR phosphorylation, mitochondrial protein synthesis, and ATP production in human skeletal muscle without amino acid replacement. Am J Physiol Endocrinol Metab 303: E1117–E1125, 2012. - PMC - PubMed
    1. Barrett EJ, Revkin JH, Young LH, Zaret BL, Jacob R, Gelfand RA. An isotopic method for measurement of muscle protein synthesis and degradation in vivo. Biochem J 245: 223–228, 1987. - PMC - PubMed
    1. Befroy DE, Petersen KF, Dufour S, Mason GF, de Graaf RA, Rothman DL, Shulman GI. Impaired mitochondrial substrate oxidation in muscle of insulin-resistant offspring of type 2 diabetic patients. Diabetes 56: 1376–1381, 2007. - PMC - PubMed
    1. Biolo G, Fleming RY, Maggi SP, Wolfe RR. Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle. Am J Physiol Endocrinol Metab 268: E75–E84, 1995. - PubMed

Publication types