背景:胰岛素信号调节心脏底物的利用,并与心脏的生理适应有关。心脏内信号应答的改变被认为导致病理状况,例如2型糖尿病和心力衰竭。虽然使用磷酸蛋白质组学策略在几个代谢器官中进行了广泛的研究,一般在心脏组织中引起的信号反应,特别是在专门的心肌细胞中,尚未进行相同程度的调查。
方法:通过静脉内注射将胰岛素或载体给予C57BL6/JRj雄性小鼠。提取心室组织并进行定量磷酸蛋白质组学分析以评估胰岛素信号传导反应。为了描述心肌细胞特异性反应并研究Tbc1d4在胰岛素信号转导中的作用,来自心脏和骨骼肌特异性Tbc1d4基因敲除小鼠心脏的心肌细胞,以及野生同窝,被研究过。磷酸蛋白质组学研究涉及用串联质量标签(TMT)标记的同量异序肽,富集磷酸化肽,通过微流反相液相色谱进行分馏,和高分辨率质谱测量。
结果:我们定量了来自心室组织的10,399个磷酸化肽和来自分离心肌细胞的12,739个磷酸化肽,定位到3,232和3,128个独特的蛋白质,分别。在心脏组织中,我们确定了84个胰岛素调节的磷酸化事件,包括胰岛素受体(InsrY1351,Y1175,Y1179,Y1180)本身的位点以及胰岛素受体底物蛋白1(Irs1S522,S526)。响应于胰岛素刺激具有增加的活性的预测激酶包括Rps6kb1、Akt1和Mtor。Tbc1d4在心肌细胞中涌现为主要的磷酸化目标。尽管对全球磷酸化格局的影响有限,心肌细胞中的Tbc1d4缺乏减弱胰岛素诱导的Glut4易位和诱导的蛋白质重塑。我们观察到15种蛋白质在敲除Tbc1d4后被显着调节。虽然Glut4由于Tbc1d4缺乏而表现出蛋白质丰度降低,Txnip水平显著增加。用胰岛素刺激野生型心肌细胞导致262个显著的磷酸化事件的调节,预测受Akt1、Mtor、Akt2和Insr。在心肌细胞中,除了调节专门的心肌细胞蛋白外,还引发了典型的胰岛素信号应答,例如Kcnj11Y12和DspS2597。提供了所有磷酸化位点的细节。
结论:我们首次概述了心脏组织和分离的成年心肌细胞中胰岛素诱导的磷酸化信号反应,详述具有改变的磷酸化丰度的特定残基。我们的研究标志着了解胰岛素信号在与胰岛素抵抗相关的心脏病中的作用的重要一步。
BACKGROUND: Insulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent.
METHODS: Insulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements.
RESULTS: We quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided.
CONCLUSIONS: We present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.