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

危重病人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病房（ICU）获得性虚弱（ICUAW），与高发病率和死亡率相关。目前还没有具体的治疗方法。危险因素包括全身性炎症、败血症和不受控制的高血糖。这些因素是如何相互关联并导致ICUAW的尚不清楚。在ICUAW患者中，经常发生外周胰岛素抵抗，这减少了肌肉葡萄糖摄取。由于肌肉中的能量生产主要由葡萄糖驱动，其结果是三磷酸腺苷（ATP）的合成减少，蛋白质分解增加，从而导致肌肉萎缩和无力。此外，危重病人会出现全身性炎症，这加重了胰岛素抵抗。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。胰岛素抵抗发生在胰岛素受体或其受体后信号水平。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暗示了磷酸酶PTP1B和TC-PTP与炎症性肌无力有关，我们的目的是验证这些ptp是炎症性胰岛素抵抗和肌无力的治疗靶点的假设。通过培养肌细胞和小鼠多微生物脓毒症模型，我们将评估PTP1B和TC-PTP作为炎症性胰岛素抵抗和肌肉萎缩的关键分子。对于这种方法，PTP活性将通过酪氨酸去磷酸化测定来测量。在肌细胞和骨骼肌的基础和炎症条件下，将分析胰岛素信号成分（包括ptp）的蛋白质含量和基因表达。此外，我们还研究了胰岛素受体磷酸化、胰岛素信号活性以及RNAi和CRISPR / Cas9抑制或敲低/敲除PTP1B和TC-PTP对体外炎性肌细胞萎缩的影响。进一步，我们将研究特异性的ptp1b抑制是否也能在体内预防炎症性胰岛素抵抗和肌肉萎缩。为此，胰岛素敏感性将通过体内高胰岛素/正血糖钳夹以及肌细胞和肌肉中肌细胞特异性PTP1B和TC-PTP敲除小鼠的葡萄糖摄取测定来量化。ptp对细胞和纤维特异性胰岛素受体磷酸化的影响将通过近端连接试验进行研究。特定PTP缺失或PTP抑制对炎性肌无力的影响将通过肌肉力量测量来评估。我们的目标是建立PTP1B和TC-PTP作为ICUAW的治疗靶点，并为一种新的因果导向的方法铺平道路，以克服胰岛素抵抗和预防危重患者的炎症性肌肉无力。

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.