Abstract：
OBJECTIVE: Alterations in circadian rhythms increase the likelihood of developing type 2 diabetes and CVD. Circadian rhythms are controlled by several core clock genes, which are expressed in nearly every cell, including immune cells. Immune cells are key players in the pathophysiology of type 2 diabetes, and participate in the atherosclerotic process that underlies cardiovascular risk in these patients. The role of the core clock in the leukocytes of people with type 2 diabetes and the inflammatory process associated with it are unknown. We aimed to evaluate whether the molecular clock system is impaired in the leukocytes of type 2 diabetes patients and to explore the mechanism by which this alteration leads to an increased cardiovascular risk in this population.
METHODS: This is an observational cross-sectional study performed in 25 participants with type 2 diabetes and 28 healthy control participants. Clinical and biochemical parameters were obtained. Peripheral blood leukocytes were isolated using magnetic bead technology. RNA and protein lysates were obtained to assess clock-related gene transcript and protein levels using real-time PCR and western blot, respectively. Luminex XMAP technology was used to assess levels of inflammatory markers. Leukocyte-endothelial interaction assays were performed by perfusing participants\' leukocytes or THP-1 cells (with/without CLK8) over a HUVEC monolayer in a parallel flow chamber using a dynamic adhesion system.
RESULTS: Participants with type 2 diabetes showed increased BMAL1 and NR1D1 mRNA levels and decreased protein levels of circadian locomotor output cycles kaput (CLOCK), cryptochrome 1 (CRY1), phosphorylated basic helix-loop-helix ARNT like 1 (p-BMAL1) and period circadian protein homologue 2 (PER2). Correlation studies revealed that these alterations in clock proteins were negatively associated with glucose, HbA1c, insulin and HOMA-IR levels and leukocyte cell counts. The leukocyte rolling velocity was reduced and rolling flux and adhesion were enhanced in individuals with type 2 diabetes compared with healthy participants. Interestingly, inhibition of CLOCK/BMAL1 activity in leukocytes using the CLOCK inhibitor CLK8 mimicked the effects of type 2 diabetes on leukocyte-endothelial interactions.
CONCLUSIONS: Our study demonstrates alterations in the molecular clock system in leukocytes of individuals with type 2 diabetes, manifested in increased mRNA levels and decreased protein levels of the core clock machinery. These alterations correlated with the impaired metabolic and proinflammatory profile of the participants with type 2 diabetes. Our findings support a causal role for decreased CLOCK/BMAL1 activity in the increased level of leukocyte-endothelial interactions. Overall, our data suggest that alterations in core clock proteins accelerate the inflammatory process, which may ultimately precipitate the onset of CVD in patients with type 2 diabetes.
METHODS: This is an observational cross-sectional study performed in 25 participants with type 2 diabetes and 28 healthy control participants. Clinical and biochemical parameters were obtained. Peripheral blood leukocytes were isolated using magnetic bead technology. RNA and protein lysates were obtained to assess clock-related gene transcript and protein levels using real-time PCR and western blot, respectively. Luminex XMAP technology was used to assess levels of inflammatory markers. Leukocyte-endothelial interaction assays were performed by perfusing participants\' leukocytes or THP-1 cells (with/without CLK8) over a HUVEC monolayer in a parallel flow chamber using a dynamic adhesion system.
RESULTS: Participants with type 2 diabetes showed increased BMAL1 and NR1D1 mRNA levels and decreased protein levels of circadian locomotor output cycles kaput (CLOCK), cryptochrome 1 (CRY1), phosphorylated basic helix-loop-helix ARNT like 1 (p-BMAL1) and period circadian protein homologue 2 (PER2). Correlation studies revealed that these alterations in clock proteins were negatively associated with glucose, HbA1c, insulin and HOMA-IR levels and leukocyte cell counts. The leukocyte rolling velocity was reduced and rolling flux and adhesion were enhanced in individuals with type 2 diabetes compared with healthy participants. Interestingly, inhibition of CLOCK/BMAL1 activity in leukocytes using the CLOCK inhibitor CLK8 mimicked the effects of type 2 diabetes on leukocyte-endothelial interactions.
CONCLUSIONS: Our study demonstrates alterations in the molecular clock system in leukocytes of individuals with type 2 diabetes, manifested in increased mRNA levels and decreased protein levels of the core clock machinery. These alterations correlated with the impaired metabolic and proinflammatory profile of the participants with type 2 diabetes. Our findings support a causal role for decreased CLOCK/BMAL1 activity in the increased level of leukocyte-endothelial interactions. Overall, our data suggest that alterations in core clock proteins accelerate the inflammatory process, which may ultimately precipitate the onset of CVD in patients with type 2 diabetes.
摘要：
目的：昼夜节律的改变增加了2型糖尿病和CVD的可能性。昼夜节律由几个核心时钟基因控制，几乎在每个细胞中都有表达，包括免疫细胞。免疫细胞是2型糖尿病病理生理学的关键参与者，并参与这些患者心血管风险的动脉粥样硬化过程。核心时钟在2型糖尿病患者白细胞中的作用以及与其相关的炎症过程尚不清楚。我们旨在评估2型糖尿病患者白细胞中的分子钟系统是否受损，并探索这种改变导致该人群心血管风险增加的机制。
方法：这是一项观察性横断面研究，在25名2型糖尿病参与者和28名健康对照参与者中进行。获得临床和生化参数。使用磁珠技术分离外周血白细胞。使用实时PCR和蛋白质印迹获得RNA和蛋白质裂解物以评估时钟相关基因转录物和蛋白质水平，分别。LuminexXMAP技术用于评估炎症标志物的水平。通过使用动态粘附系统在平行流动室中在HUVEC单层上灌注参与者的白细胞或THP-1细胞（有/没有CLK8）进行白细胞-内皮细胞相互作用测定。
结果：患有2型糖尿病的参与者显示BMAL1和NR1D1mRNA水平升高，昼夜节律运动输出周期kaput（CLOCK）的蛋白质水平降低，cryprochrome1(CRY1)，磷酸化的碱性螺旋-环-螺旋ARNT样1（p-BMAL1）和周期昼夜节律蛋白同源物2（PER2）。相关研究表明，时钟蛋白的这些变化与葡萄糖呈负相关，HbA1c,胰岛素和HOMA-IR水平和白细胞计数。与健康参与者相比，2型糖尿病患者的白细胞滚动速度降低，滚动通量和粘附增强。有趣的是,使用CLOCK抑制剂CLK8抑制白细胞中CLOCK/BMAL1活性,模拟了2型糖尿病对白细胞-内皮相互作用的影响.
结论：我们的研究表明，2型糖尿病患者的白细胞分子钟系统发生了改变，表现为核心时钟机制的mRNA水平升高和蛋白质水平降低。这些改变与2型糖尿病参与者的代谢和促炎特征受损相关。我们的发现支持CLOCK/BMAL1活性降低在白细胞-内皮细胞相互作用水平升高中的因果作用。总的来说,我们的数据表明核心时钟蛋白的改变加速了炎症过程，这可能最终促使2型糖尿病患者发生CVD。
方法：这是一项观察性横断面研究，在25名2型糖尿病参与者和28名健康对照参与者中进行。获得临床和生化参数。使用磁珠技术分离外周血白细胞。使用实时PCR和蛋白质印迹获得RNA和蛋白质裂解物以评估时钟相关基因转录物和蛋白质水平，分别。LuminexXMAP技术用于评估炎症标志物的水平。通过使用动态粘附系统在平行流动室中在HUVEC单层上灌注参与者的白细胞或THP-1细胞（有/没有CLK8）进行白细胞-内皮细胞相互作用测定。
结果：患有2型糖尿病的参与者显示BMAL1和NR1D1mRNA水平升高，昼夜节律运动输出周期kaput（CLOCK）的蛋白质水平降低，cryprochrome1(CRY1)，磷酸化的碱性螺旋-环-螺旋ARNT样1（p-BMAL1）和周期昼夜节律蛋白同源物2（PER2）。相关研究表明，时钟蛋白的这些变化与葡萄糖呈负相关，HbA1c,胰岛素和HOMA-IR水平和白细胞计数。与健康参与者相比，2型糖尿病患者的白细胞滚动速度降低，滚动通量和粘附增强。有趣的是,使用CLOCK抑制剂CLK8抑制白细胞中CLOCK/BMAL1活性,模拟了2型糖尿病对白细胞-内皮相互作用的影响.
结论：我们的研究表明，2型糖尿病患者的白细胞分子钟系统发生了改变，表现为核心时钟机制的mRNA水平升高和蛋白质水平降低。这些改变与2型糖尿病参与者的代谢和促炎特征受损相关。我们的发现支持CLOCK/BMAL1活性降低在白细胞-内皮细胞相互作用水平升高中的因果作用。总的来说,我们的数据表明核心时钟蛋白的改变加速了炎症过程，这可能最终促使2型糖尿病患者发生CVD。
