Protein tyrosine phosphatases as novel therapeutic targets to overcome inflammation-induced insulin resistance and skeletal muscle atrophy

蛋白酪氨酸磷酸酶作为克服炎症引起的胰岛素抵抗和骨骼肌萎缩的新治疗靶点

• 批准号: 442364946
• 负责人: Professor Dr. Jens Fielitz
• 金额: --
• 依托单位: Center for Cardiovascular Research (CCM)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/442364946?language=en
• 关键词: Protein; tyrosine; phosphatases; novel; therapeutic

Critically ill patients frequently develop muscle wasting and atrophy leading to a pronounced loss of muscle mass and strength. This condition is known as intensive care unit (ICU)-acquired weakness (ICUAW) and is associated with high morbidity and mortality. A specific therapy is not available. Risk factors include systemic inflammation, sepsis and uncontrolled hyperglycemia. How these factors are interconnected and lead to ICUAW is unclear.In ICUAW patients, peripheral insulin resistance often occurs, which reduces muscular glucose uptake. Since energy production in muscle is mainly driven by glucose, the result is a reduced synthesis of adenosine triphosphate (ATP) and an increased protein breakdown, which leads to muscle atrophy and weakness. In addition, systemic inflammation occurs in critically ill patients, which aggravates insulin resistance. Clinical studies have shown that correcting high blood glucose levels by intensive insulin therapy reduces but does not prevent ICUAW. Insulin resistance develops at the level of the insulin receptor or its post-receptor signaling. Protein tyrosine phosphatases (PTPs) inhibit insulin receptor activity. Since our preliminary work implicates the phosphatases PTP1B and TC-PTP in inflammatory muscle weakness, we aim to test the hypothesis that these PTPs are therapeutic targets of inflammatory insulin resistance and muscle weakness.Using myocytes in culture and a mouse model of polymicrobial sepsis, we will evaluate PTP1B and TC-PTP as key molecules for inflammatory insulin resistance and muscle atrophy. For this approach, PTP activity will be measured by tyrosine dephosphorylation assays. Protein content and gene expression of components of insulin signaling, including PTPs, will be analyzed under basal and inflammatory conditions in myocytes and skeletal muscle. In addition, we investigate the insulin receptor phosphorylation, the activity of insulin signaling and the effect of pharmacological PTP1B and TC-PTP inhibition or knockdown / knockout by RNAi and CRISPR / Cas9 on inflammatory myocyte atrophy in vitro. Further, we will investigate whether specific PTP1B-inhibition prevents inflammatory insulin resistance and muscle atrophy in vivo as well. For this purpose, insulin sensitivity will be quantified by hyperinsulinemic/euglycemic clamp in vivo as well as glucose uptake assays in myocytes and muscles of myocyte-specific PTP1B- and TC-PTP knockout mice. The impact of PTPs on cell- and fiber-specific insulin receptor phosphorylation will be investigated by proximity ligation assays. The effect of a specific PTP deletion or PTP inhibition on inflammatory muscle weakness will be evaluated by muscle strength measurements.We aim to establish PTP1B and TC-PTP as therapeutic targets in ICUAW and to pave the way for a novel causally-oriented approach to overcome insulin resistance and to prevent inflammatory muscle weakness in critically ill patients.

重症患者经常出现肌肉萎缩和萎缩，导致肌肉质量和力量的明显损失。这种情况被称为重症监护室（ICU）获得性虚弱（ICUAW），并与高发病率和死亡率相关。没有特定的治疗方法。危险因素包括全身性炎症、败血症和不受控制的高血糖症。这些因素是如何相互联系并导致ICUAW的尚不清楚。在ICUAW患者中，外周胰岛素抵抗经常发生，这降低了肌肉葡萄糖摄取。由于肌肉中的能量产生主要由葡萄糖驱动，结果是三磷酸腺苷（ATP）的合成减少和蛋白质分解增加，从而导致肌肉萎缩和虚弱。此外，重症患者会发生全身性炎症，从而加重胰岛素抵抗。临床研究表明，通过强化胰岛素治疗纠正高血糖水平可以减少但不能预防ICUAW。胰岛素抵抗在胰岛素受体或其受体后信号传导水平上发展。蛋白酪氨酸磷酸酶（PTPs）抑制胰岛素受体活性。由于我们的初步工作涉及炎症性肌无力的磷酸酶PTP 1B和TC-PTP，我们的目标是测试的假设，这些PTP是炎症性胰岛素抵抗和muscle weaken.Using培养的心肌细胞和小鼠模型的多微生物脓毒症，我们将评估PTP 1B和TC-PTP作为炎症性胰岛素抵抗和肌肉萎缩的关键分子的治疗靶点。对于这种方法，将通过酪氨酸去磷酸化测定来测量PTP活性。将在肌细胞和骨骼肌的基础和炎症条件下分析胰岛素信号传导组分（包括PTP）的蛋白质含量和基因表达。此外，我们研究了胰岛素受体磷酸化，胰岛素信号传导的活性以及药理学PTP 1B和TC-PTP抑制或RNAi和CRISPR / Cas9敲除/敲除对体外炎性肌细胞萎缩的影响。此外，我们将研究是否特异性PTP 1B抑制预防炎症性胰岛素抵抗和肌肉萎缩以及在体内。为此，将通过体内高胰岛素/正葡萄糖钳夹以及肌细胞特异性PTP 1B和TC-PTP敲除小鼠的肌细胞和肌肉中的葡萄糖摄取测定来定量胰岛素敏感性。将通过邻位连接试验研究PTP对细胞和纤维特异性胰岛素受体磷酸化的影响。特定PTP缺失或PTP抑制对炎性肌无力的影响将通过肌力测量进行评估。我们的目标是将PTP 1B和TC-PTP确立为ICUAW的治疗靶点，并为克服胰岛素抵抗和预防重症患者炎性肌无力的新的因果导向方法铺平道路。