Robinson Matthew M, Dasari Surendra, Karakelides Helen, Bergen H Robert, Nair K Sreekumaran
Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota;
Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota; and.
Am J Physiol Endocrinol Metab. 2016 Sep 1;311(3):E628-37. doi: 10.1152/ajpendo.00175.2016. Epub 2016 Jul 19.
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.
胰岛素调节骨骼肌蛋白质降解，但由于方法学限制，体内被降解蛋白质的类型仍有待确定。我们提出了一种方法，通过从肌肉样本中提取低分子量（LMW）肽形式的降解产物来评估被降解的骨骼肌蛋白质类型。利用高分辨率质谱鉴定产生LMW肽的原始完整蛋白质，我们在啮齿动物中验证了该方法，然后将其应用于人类。我们使胰岛素治疗的链脲佐菌素（STZ）糖尿病小鼠禁食胰岛素6小时和96小时，并使1型糖尿病患者（T1D）禁食胰岛素8小时，以与胰岛素治疗条件进行比较。使用自噬和蛋白酶体途径的激活、稳定同位素示踪剂和LMW方法来测量蛋白质降解。在小鼠中，胰岛素剥夺激活了肌肉匀浆和分离线粒体中的蛋白酶体途径和自噬。LMW提取物的重现性分析表明，约80%的蛋白质被一致检测到。正如预期的那样，胰岛素剥夺增加了T1D患者的全身蛋白质周转率。个体蛋白质降解随着胰岛素剥夺而增加，包括参与线粒体功能、蛋白质组稳态、核DNA支持以及收缩/细胞骨架的蛋白质。胰岛素剥夺时产生更多LMW片段的个体线粒体蛋白质包括ATP合酶亚基γ（增加0.5倍，P = 0.007）和细胞色素c氧化酶亚基6（增加0.305倍，P = 0.03）。总之，鉴定LMW肽片段提供了一种确定个体蛋白质降解的方法。胰岛素剥夺增加了特定蛋白质的降解，并为胰岛素在维持蛋白质组稳态，尤其是线粒体蛋白质组稳态中的调节作用提供了见解。
