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.
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。