| BackgroundPatients with diabetes have a high incidence of cardiovascular morbidity and an increased prevalence of cardiovascular risk factors, such as dyslipidemia, hypertension, obesity, insulin resistance and prothrombotic factors. A synergistic cross-talk among these risk factors contributes to the derangement of endothelial function, which results in the impaired ability of endothelium to regulate vascular tone by the deranged release of several vasoactive substances, such as prostacyclin, which exerts profound cardiovascular effects, including inflammation, thrombogenesis, cell growth and peripheral circulation. An inactivation of cycloxygenase and a depressed formation of prostacyclin have been described in diabetes. Especially, beneficial effects of prostacyclin analogues therapies for diabetic complications have been confirmed. However, as a potential cardiovascular regulating substance, why the circulating levels of prostacyclin is decreased in diabetes remains unclear.Coupling factor 6 (CF6) is an essential component of the energy-transducing stalk of mitochondrial adenosine triphosphate (ATP) synthase and acts as an essential component for proton ducting and ATP synthesis. Recent studies showed that CF6 was also localized on the surface of endothelial cell and released into blood by shear stress or tumor necrosis factor. By inhibiting the synthesis of prostacyclin, CF6 functions as an endogenous vasoconstrictor in the fashion of systemic hormone. In addition, circulatory CF6 promoted the expression of asymmetric dimethylarginine in endothelial cells by enhancing its synthesis and suppressing its degradation. As a novel vasoactive peptide, plasma level of CF6 was markedly increased in patients with cardiovascular disease, such as hypertension, acute myocardial infarction (AMI) and end-stage renal disease. These studies suggest that CF6 may play an important pathogenic role in cardiovascular disease. Considering the decreased prostacyclin level and high incidence of cardiovascular morbidity and mortality, the level and implication of CF6, as a novel inhibitor of prostacyclin, in diabetes is unknown; also, the relationship between CF6 and traditional cardiovascular risk factors and its secretary regulation in diabetes is not yet investigated.Objectives1. To investigate the alternations of CF6 in patients with type 2 diabetes and its complications;2. To analyzed interrelation of plasma CF6 level to plasma prostacyclin and clinical traits in type2 diabetes;3. To illuminate the mechanism responsible for the changes of circulating CF6 level under diabetic condition and the role of PKC or MAPKs signal pathway relevant to diabetes.MethodsAccording to the WHO diagnostic criteria for diabetes mellitus, a total of 70 Chinese patients with type 2 diabetes (37 men and 33 women), who were finally pass the including and excluding criteria exam, were consecutively recruited Apr. through Oct. 2005. Patients with ketoacidosis, acute infection, severe liver or renal disease, congestive heart failure, malignant tumor and administration of aspirin or thiazolidinediones within the recent 4 weeks were excluded from the study. All the patients received routine diabetes medications (Insulin, oral or combined hypoglycemic agents). Fifty-six age- and sex-matched healthy subjects were enrolled through our hospital health check clinic and had no known illnesses. The study was reviewed and approved by the Ethics Committee of the Shandong University School of Medicine, and informed consent was obtained from each subject before initiation of the study.Then, a detailed clinical database was established to collect patient demographic information including age, gender, smoking habit, family history of diabetes, coronary artery disease, use of medications, and past medical history. Physical exam was also carried out to document height, weight, body mass index, blood pressure.Based on the previous finding that plasma CF6 was significantly related to glucose level in diabetic patients, we further investigated the potential effect of high glucose on CF6 secretion. Human umbilical vein endothelial cells (HUVECs) were isolated from the human umbilical cords from normal pregnancies and successfully cultured and identified. Before treatment, HUVECs within passages 2 and 4 were deprived of growth factors and cultured commonly. Once confluent, the cells were exposed to medium containing final concentrations of 5.6mmol/L D-glucose (control group), 10mmol/L, 20mmol/L and 30mmol/L. D-glucose (high glucose group), or 5.6mmol/L D-glucose plus 24.4mmol/L mannitol as an osmotic control. Inhibitory reagents were added into the medium 2 h before the 30 mmol/L D-glucose in signal pathway experiments except time- and dose-dependent ones. Cell viability in all experiment groups was assessed by trypan blue staining.Biochemical analyses were followed after samples collecting. Circulating CF6 and prostacyclin levels were measured by radioimmunoassay; CF6 gene expression analyzed by real-time RT-PCR and Western blot analysis for total and phosphorylated p38, JNK and ERK1/2 MAPK protein level. The data were analyzed with the statistical software package SPSS11.5 and differences were considered statistically significant with P< 0.05.Results1. Demographic Data of Patients: No significant differences were found in age, gender, smoking and BMI between patients with type 2 diabetes and healthy controls. As expected, the fasting plasma levels of FBG, triglyceride and LDL in patients were significantly higher than those in healthy controls.2. The plasma CF6 level was markedly higher 23.9% in patients with type2 diabetes (262.1±49.9pg/ml) than that in controls (P<0.01). Meanwhile, the level of 6-keto-PGF1a was significantly lower 16.5% in patients than that in controls. Significantly, plasma CF6 level was inversely correlated to plasma 6-keto-PGF1a, level (r=0.52 , P <0.01) in type 2 patients.3. Plasma CF6 level in patients with current smoker was obviously higher than that in ex-smokers; Plasma CF6 was significantly correlated with cholesterol, apoB or glucose. By a multiple stepwise regression analysis, CF6 level was independently correlated with 6-keto-PGF1a (R2=0.272, r=-0.521, P<0.01).4. In vitro, CF6 gene expression and protein release enhanced in a time- and dose-dependent manner by incubation of HUVECs with increasing glucose concentrations (10, 20 and 30mmol/L).5. The p38 MAPK was significantly phosphorylated by exposure to high glucose and the enhanced p38 MAPK phosphorylation was significantly suppressed by the PKC inhibitor and by the p38 MAPK inhibitor. Meanwhile, compared with 30 mmol/L glucose alone, high glucose plus PKC inhibitor or p38 inhibitor significantly suppressed the stimulatory effect of high glucose on CF6 production, the inhibition rate being -44.5% (P<0.01) and -22.8% (P<0.01), respectively.6. Importantly, HUVECs co-incubated with 100nmol/L insulin showed significantly decreased CF6 mRNA expression and protein release induced by high glucose.Conclusions1. As a novel endogenous prostacyclin inhibitor, CF6 is significantly increased in diabetes mellitus, and it is correlated to plasma 6-keto-PGF1a level and maybe an independent risk factor for the reduced prostacyclin level in diabetes.2. Plasma CF6 level is influenced by cardiovascular risk factors clustered in diabetes, which suggests that CF6 release is related to endothelial injury. High glucose may acts as a newly identified stimulus for CF6 release, especially in diabetes mellitus.3. The PKC and p38 MAPK signal pathway maybe involved in high glucose induced CF6 production, which infers a potential target for CF6 regulating.4. Insulin may play an important role in the CF6 modulation in diabetes. |
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