Effects of pyrrolidine dithiocarbamate on the function of mTOR complex 1 and 2

第一项研究的目的是检查小硫醇化合物吡咯烷二硫代氨基甲酸酯（PDTC）对2型糖尿病大鼠肝糖原合成和肝再生的影响，并探讨这些影响的机制。本实验观察了正常饮食（NC）、高脂饮食（HFD）诱导的2型糖尿病大鼠（DM）和DM+ PDTC干预1周的胰岛素抵抗大鼠（DM+PDTC）的空腹血糖和糖原沉积以及肝脏中与糖尿病发生相关的两个关键基因的表达。通过Western印迹法评估肝提取物中Akt、GSK 3和FoxO 1的磷酸化，同时进行间接免疫荧光染色以确定FoxO 1的细胞分布。DM组大鼠空腹血糖明显升高，肝糖原含量明显降低。Akt/GSK 3通路的激活和随后FoxO 1的失活在DM大鼠肝脏中大大减少。相比之下，PDTC治疗通过增加Akt/GSK 3信号传导和随后的FoxO 1失活和核输出保护DM大鼠免受高空腹血糖和糖原沉积的损失。重要的是，磷酸烯醇式丙酮酸羧激酶和葡萄糖-6-磷酸酶mRNA的表达在DM+PDTC组中与DM大鼠相比显著降低。这项研究表明，PDTC通过Akt依赖性下调FoxO 1活性，增强糖原合成，同时减少糖尿病大鼠的糖原生成。 营养依赖性激酶mTOR存在于两种复合物中：mTOR复合物1（mTORC 1），其对雷帕霉素敏感并磷酸化直接靶点，如核糖体蛋白S6激酶（S6 K1），和mTORC 2，其对雷帕霉素不敏感并磷酸化Akt。mTOR的生理作用包括参与代谢、蛋白质合成、凋亡途径、转录因子调节和细胞周期。先前的研究表明，mTORC 1/S6 K1通路的过度激活导致胰岛素受体底物1和2的抑制性丝氨酸磷酸化，随后损害胰岛素作用。相反，S6 K1基因敲除小鼠在高脂饮食下仍然对胰岛素敏感，这可以追溯到从S6 K1到胰岛素受体底物1的负反馈回路的丢失。 根据我们的结果，我们假设PDTC通过抑制mTORC 1/S6 K1负反馈回路改善了2型糖尿病大鼠的肝脏胰岛素反应（糖原合成增加和血管生成减少）。目前正在进行研究，使用基于免疫沉淀的激酶测定和蛋白质印迹，以确定PDTC是否能够在各种胰岛素抵抗模型和衰老中恢复胰岛素受体-胰岛素受体底物1-PI-3激酶途径。 第二项研究的主要目的是在用PDTC或IL-6处理长达8小时后，对人肝源性HepG 2细胞进行整体基因表达谱分析。我们之前报道过PDTC通过损伤STAT 3的激活和下游信号传导来拮抗细胞对IL-6的反应性。在此，通过无偏途径分析方法，基因阵列分析显示响应于PDTC与IL-6的表达变化的显著和时间差异。与代谢途径、炎症、翻译和线粒体功能相关的大量基因发生了变化，核糖体蛋白基因和DDIT 4主要在PDTC下上调，但在IL-6下下调。定量PCR和Western印迹分析验证了微阵列数据，并显示了mTOR负调节因子DDIT 4响应于PDTC与IL-6的相互表达模式。PDTC的施用导致Akt的快速和持续活化，随后通过DDIT 4表达的上调阻断IL-6介导的mTOR复合物1（mTORC 1）功能的增加。相反，在DDIT 4敲低细胞中，PDTC导致的IL-6依赖性mTOR激活的下降减慢。细胞的整体蛋白质生物合成能力被IL-6严重削弱，但在雷帕霉素非依赖性途径中被PDTC增加。这些结果证明了PDTC对mTORC 1功能的关键作用，并提供了PDTC可以通过诱导DDIT 4逆转IL-6相关信号传导的证据。 IL-6循环水平升高是慢性炎性疾病病理生理学中涉及的许多因素之一。有趣的是，IL-6被认为是一种蛋白水解诱导因子，因为它调节肌肉蛋白水解系统，并且在老年肌肉减少大鼠中观察到的肌肉萎缩与蛋白酶体途径活性增强和肌肉蛋白合成减少有关。最近的临床前和临床研究表明，PDTC及其衍生物靶向泛素-蛋白酶体系统，这可能解释了PDTC在恶病质小鼠模型中对肌肉和脂肪组织损失的保护作用。最近的研究结果表明，泛素-蛋白酶体系统的抑制诱导mTORC 1信号转导的抑制。我们假设PDTC抑制mTORC 1的能力源于DDIT 4的蛋白酶体稳定，DDIT 4是mTORC 1的短寿命负阻遏物。这些发现表明，细胞对蛋白酶体抑制和PDTC激活mTORC 2/Akt通路的敏感性可能代表了一种增强骨骼肌卫星细胞增殖和分化的新策略。

The aims of the first study were to examine the effects of the small thiol compound pyrrolidine dithiocarbamate (PDTC) on hepatic glycogen synthesis and gluconeogenesis in type 2 diabetic rats and explore the mechanism underlying these effects. Fasting blood glucose and glycogen deposition, together with expressions of two key genes related to gluconeogenesis was studied in the liver of rats fed a normal diet (NC), high fat diet (HFD)-induced insulin resistant rats made type 2 diabetic by a single ip injection of streptozotocin (DM), and an DM with intervention of PDTC (DM+PDTC) for 1 week. The phosphorylation of Akt, GSK3β and FoxO1 was assessed in liver extracts by Western blot while indirect immunofluorescence staining was performed to determine the cellular distribution of FoxO1. The DM rats exhibited obvious increase in fasting blood glucose, but a decrease in hepatic glycogen content as compared with the NC group. Activation of the Akt/GSK3β pathway and subsequent inactivation of FoxO1 were greatly reduced in DM rat livers. By contrast, PDTC treatment protected DM rats against high fasting blood glucose and the loss in glycogen deposition through an increase in Akt/GSK3β signaling and subsequent inactivation and nuclear export of FoxO1. Importantly, expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs was significantly reduced in the DM+PDTC group as compared with the DM rats. This study suggests that PDTC enhances glycogen synthesis while reducing gluconeogenesis in DM rats through Akt-dependent down-modulation of FoxO1 activity. The nutrient-dependent kinase mTOR exists in two complexes: mTOR Complex1 (mTORC1), which is rapamycin-sensitive and phosphorylates direct targets such as ribosomal protein S6 kinase (S6K1), and mTORC2, which is rapamycin-insensitive and phosphorylates Akt. The physiological roles of mTOR include involvement in metabolism, protein synthesis, apoptosis pathways, transcription factor regulation and the cell cycle. Previous studies have shown that overactivation of the mTORC1/S6K1 pathway results in inhibitory serine phosphorylation of insulin receptor substrate 1 and 2, with subsequent impairment in insulin action. Conversely, S6K1 knockout mice remain exquisitely insulin sensitive on a high-fat diet, which has been traced to the loss of a negative-feedback loop from S6K1 to the insulin receptor substrate 1. From our results, we hypothesize that PDTC improved hepatic insulin responses (increase in glycogen synthesis and reduction in gluconeogenesis) in rats made type 2 diabetic through inhibition of the mTORC1/S6K1 negative feedback loop. Studies are currently underway, using immunoprecipitation-based kinase assays and Western blots, to determine if PDTC enables recovery of the insulin receptor-insulin receptor substrate 1-PI-3 kinase pathway in various models of insulin resistance and in aging. The principal aim of the second study was to perform a global gene expression profiling of human liver-derived HepG2 cells after treatment with PDTC or IL-6 for up to 8 h. We have reported earlier that PDTC antagonizes the cellular responsiveness to IL-6 through impairment in STAT3 activation and downstream signaling. Here, through an unbiased pathway analysis method, gene array analysis showed dramatic and temporal differences in expression changes in response to PDTC vs. IL-6. A significant number of genes associated with metabolic pathways, inflammation, translation and mitochondrial function were changed, with ribosomal protein genes and DDIT4 primarily up-regulated with PDTC but down-regulated with IL-6. Quantitative PCR and Western blot analyses validated the microarray data and showed the reciprocal expression pattern of the mTOR negative regulator, DDIT4, in response to PDTC vs. IL-6. Administration of PDTC resulted in a rapid and sustained activation of Akt, and subsequently blocked IL-6-mediated increase in mTOR complex 1 (mTORC1) function through upregulation in DDIT4 expression. Conversely, the decline in IL-6-dependent mTOR activation by PDTC was slowed down in DDIT4-knockdown cells. The overall protein biosynthetic capacity of the cells was severely blunted by IL-6 but increased in a rapamycin-independent pathway by PDTC. These results demonstrate a critical effect of PDTC on mTORC1 function and provide evidence that PDTC can reverse IL-6-related signaling via induction of DDIT4. Elevated circulating levels of IL-6 is one of many factors involves in the pathophysiology of chronic inflammatory diseases. It is interesting that IL-6 is considered to be a proteolysis-inducing factor due to its modulation of muscle proteolytic systems and that the muscle wasting observed in aged, sarcopenic rats is associated with enhanced activity of the proteasome pathway and reduced synthesis of muscle proteins. Both preclinical and clinical studies have recently shown that PDTC and derivatives target the ubiquitin-proteasome system, which might explain the protection offered by PDTC against muscle and adipose tissue loss in a mouse model of cachexia. Recent findings indicate that suppression of the ubiquitin-proteasome system induces inhibition of mTORC1 signaling. We hypothesize that the ability of PDTC to inhibit mTORC1 stems from proteasomal stabilization of DDIT4, the short-lived negative repressor of mTORC1. These findings suggest that cellular sensitivity to proteasome inhibition and mTORC2/Akt pathway activation by PDTC may represent a novel strategy to enhance skeletal muscle satellite cell proliferation and differentiation.