Irisin Improves Endothelial Dysfunction And Ameliorates Atherosclerosis In Apolipoprotein E-Null Diabetic Mice

BackgroundDiabetes mellitus (DM) is a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action or both. DM represents a major health burden, affecting about 371 million people worldwide and half of these patients died of DM less than 60-year-old according to the International Diabetes Federation. The epidemiology of diabetes reveals that 75% of patients died of diabetic angiopathy, and the major cause of death is macro-angiopathy. Atherosclerosis is the most important pathologic change of diabetic macro-angiopathy. Vascular endothelial injury is not only the principal pathogenesis of atherosclerosis, but also the early pathophysiological changes. High glucose induces oxidative stress, DNA damage, disturbance of mitotic cycle, delayed replication and apoptosis in endothelial cells. Therefore, it has important significance to protect vascular endothelium and improve atherosclerosis.It has been proven that physical activity improves atherosclerosis, the mechanisms-involved are associated with alleviating insulin resistance, losing weight, improving vascular endothelial dysfunction and ameliorating inflammation of arteries. But these mechanisms are unable to explain the improvement of atherosclerosis. Scholars have been suspicious of that skeletal muscle is able to produce some "myokines" to improve atherosclerosis. Irisin may be this kind of myokines. Irisin mainly secreted by skeletal muscle and physical activity increased the production of irisin. The circulating irisin converts white fat into the more thermogenic beige fat, a process known as white fat "browning". Animal experiments reveals that irisin reduced insulin resistance and weight in fat C57 mice. Moreno-Navarrete JM et.al showed that irisin concentrations decreased with the weight gain. So as the comparison of normal subjects to the type 2 diabetic patients. Our previous study indicates that the irisin concentrations in newly diagnosed type 2 diabetic patients were positively associated with vascular endothelial function. But the association of irisin and atherosclerosis has not been investigated. Those studies mentioned above revealed that irisin was associated with the improvement of insulin resistance and weight gain, and the circulating irisin concentrations were positively associated vascular endothelial function. In summary, irisin may be the myokine that improve atherosclerosis. Therefore, we hypothesized that irisin can improve endothelial dysfunction and ameliorate atherosclerosis induced by diabetes and high glucose-induced endothelial cells apoptosis. This study was aim to investigated the effects of irisin on diabetes-induced endothelial dysfunction, atherosclerosis and high glucose-induced endothelial cells apoptosis and the mechanisms involved.AimsTherefore, the aims of the present study were:(1) to determine whether irisin attenuate endothelial dysfunction in ApoE-/- diabetic mice; (2) to investigate whether irisin ameliorate atherosclerosis in ApoE-/- diabetic mice; (3) and the underlying mechanisms; (4) to explore the effect of irisin on high glucose-induced endothelial cells apoptosis; (5) and the mechanisim-involved.MethodsPart I. Animal experiment1. A total of thirty male 4-week-old ApoE-/- mice (The Jackson Laboratory, USA) were housed in a specific-pathogen-free environment with unrestricted access to water and high-fat diet (45% kcal fat,35% kcal carbohydrates, and 20% kcal protein). A total of twenty mice were rendered diabetic by five daily intraperitoneal injections of streptozotocin (Sigma, USA) at a dose of 50mg/kg. Non-diabetic mice received the vehicle citrate buffer alone (n=10). The intraperitoneal glucose tolerance test (IPGTT) was performed on day 2 after the last injection of streptozotocin or citrate buffer. Diabetic animals were further divided into subgroups matching serum glucose and body weight, then treated with irisin (Phoenix Pharmaceuticals, USA) or normal saline (NS). For diabetic+irisin group, each mouse received an intravenous injection of irisin (2μg per mouse in a total of 100μL NS) twice a week, non-diabetic (n=10) or diabetic +NS group (n=10) received an equivalent volume of NS.2. Body weight and blood glucose:The body weight serum glucose and were followed weekly. After irisin or normal saline treatment for 4 or 12 weeks, IPGTT was performed to detect the glucose tolerance.3. Vascular endothelium-dependent vasodilation:endothelium-dependent vasodilation was perform after irisin or normal saline treatment for 4 weeks, three cases in each group was chosen. The phosphorylation of Akt and eNOS in aortas was detected by Western blot.4. Serum metabolic and inflammatory characteristics:the serum levels of insulin, glycosylated hemoglobin (HbAlc), tumor necrosis factor-a (TNF-α), C-reactive protein (CRP), interleukin-6 (IL-6), oxidized-low density lipoprotein (ox-LDL) and irisin were measured.5. Atherosclerotic area of aortas:after treatment for 12 weeks, whole aortas were stained with oil red O, the extent of atherosclerosis was expressed as the percent of the lesion area extending from the ascending aorta to the iliac bifurcation. To quantify luminal cross-sectional area involved by atherosclerotic plaque, sections obtained from the abdominal aorta were stained with hematoxylin and eosin.6. The inflammatory levels of aortas:Immunohistochemistry was performed to detect the macrophages and T-lymphocytes infiltrating in aortas. The expression levels of inflammatory cytokines (IL-6, IL-10, TNF-α, ICAM-1, VCAM-1 and MCP-1) were measured by RT-PCR.Part II. Cell experiment1. HUVECs culture:Endothelial cells were seeded at a density of 5×103/cm2 flask in M199 (Gibco, USA), supplemented with 20% fetal bovine serum (FBS, Gibco, USA), 10μg/ml heparin,1% penicillin/streptomycin, and 50μg/ml endothelial cell growth factor (ECGF, Sigma, USA). The cultures were maintained at 37℃ with a 5% CO2 atmosphere and the media were refreshed every third day. Endothelial cells of the third to fifth passages were used for experiments. Cells were starved in M199 containing 1% FBS for 20h to synchronize before cytokines treatment.2. Groups and measurement:Experiment one, HUVECs were divided into six groups and pretreated with inhibitors against PI3K (LY294002, 10μmol/L, LY group) or eNOS (L-NAME,500μmol/L, L-NAME group) for 30min before irisin (100ng/ml) was added. The cells were then cultured for 48h with media containing 5.5mmol/L glucose (NG group) or 30mmol/L glucose (HG group). Endothelial cells apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining. Intracellular ROS generation was detected by using the peroxide-sensitive fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA). The glutathione peroxidase type 1 (GPX-1), catalase (CAT) and superoxide dismutase (SOD) in HUVECs were measured by quantitative RT-PCR. The P-Akt, Akt, P-eNOS and eNOS in HUVECs were detected by Western blot. Experiment two, HUVECs were transfected with Scr-siRNA or eNOS-siRNA for 24h, followed by cultured in normal (5.5mmol/L) or high (20mmol/L) glucose for 48h in the presence or absence of irisin (100ng/ml). After treatment, the cells were double-stained with Annexin V-FITC/PI (BD, USA) and analyzed by flow cytometry.Statistical AnalysisAll analyses were performed with SPSS 19.0. Data are expressed as mean±SD. Differences between 2 groups were tested with unpaired Student’s t test. Data of multiple groups were compared using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) t-test. Statistical significance was defined as P<0.05.ResultsPart I. Irisin Improves Endothelial Dysfunction and Ameliorates Atherosclerosis in Apolipoprotein E-Null Diabetic Mice1. All twenty ApoE-/- mice were rendered diabetes. None of the mice were died during the experiments.2. The Effects of Irisin on Metabolic and inflammatory Characteristics of ApoE-/- MiceAfter treatment for 4 weeks or 12 weeks, IPGTT indicated that irisin caused a significant improvement in glucose tolerance, but no difference of fasting insulin concentrations between irisin group and diabetes group has been observed. Compared with control group, the body weight, insulin concentration in diabetes group were significantly decreased. While, the blood glucose and HbAlc were significantly increased. Irisin had no notable effects on body weight, plasma glucose, HbAlc and insulin. The levels of inflammatory cytokines (TNF-a, CRP, IL-6 and ox-LDL) were significantly increased in diabetic mice compared with non-diabetic, treatment with irisin markedly reduced these cytokines in diabetic mice.3. Irisin improved aortic endothelium-dependent vasodilation in ApoE-/-diabetic miceDiabetes decreased the endothelium-dependent vasodilation in response to Ach, while irisin treatment markedly improved the vasodilation (82.43±4.79% vs.61.54± 4.62% vasodilation at 10-4 mmol/L ACh, P<0.05 vs. diabetic+NS group). In contrast, the endothelium-independent vasodilation in response to SNP did not differ among the 3 groups (P>0.05). Diabetes decreased Akt and eNOS phosphorylation in aortas of mice, irisin treatment significantly increased Akt and eNOS phosphorylation.4. Irisin alleviated atherosclerotic plaque area in ApoE-/- diabetic miceAtherosclerotic lesion increased in the aortas of ApoE-/- mice after induction of diabetes. By Oil Red O staining, we found that atherosclerotic plaque area of en face sections were significantly decreased in irisin-treated mice compared with the diabetic +NS (22.57±2.17% vs.35.09±2.38%, P< 0.05). Similarly, as compared with NS treatment, irisin decreased plaque area of cross sections (19.36±1.85% vs.25.53± 7.87%, P< 0.05 vs. diabetic+NS group).5. Irisin reduced inflammatory infiltration in atherosclerotic plaque and down-regulated mRNA expression levels of inflammatory cytokines in aortasQuantification of CD68-and CD90-positive area revealed that diabetes increased the infiltration of macrophages and T lymphocytes in atherosclerotic plaque. Irisin treatment significantly reduced inflammatory infiltration within plaque compared with diabetic+NS (CD68:30.5±2.79% vs.41.34±9.13%, P<0.05; CD90:28.11±4.24% vs.35.74±9.1%, P<0.05). In addition, the inflammatory cytokines in aortas were assessed by quantitative RT-PCR. Irisin treatment dramatically down-regulated the mRNA expression levels of IL-6, TNF-a, ICAM-1, VCAM-1 and MCP-1, but not that of IL-10 (P>0.05).Part II Irisin Prevented High Glucose-Induced Endothelial Cells Apoptosis and Oxidative Stress via PI3K-Akt-eNOS Signaling Pathway1. Irisin Prevented High Glucose-Induced Endothelial Cells Apoptosis via PI3K-Akt-eNOS Signaling PathwayExposure of endothelial cells to high glucose (30mmol/L) for 48h resulted in a significant increase in apoptosis (25.96±3.46% vs.6.23±1.61%, P<0.05 vs. NG group). Irisin reduced high glucose-induced endothelial cells apoptosis (20.6±2.79%, P<0.05 vs. HG group). However, the protective role of irisin in endothelial apoptosis was blocked by the addition of LY294002 or L-NAME. Suppression of eNOS expression by siRNA significantly restrained the effect of irisin on endothelial apoptosis compared with Scr-siRNA transfection (25.87±4.1% vs.15.35±2.9%, P<0.05).2. Irisin Inhibited High Glucose-Induced Oxidative Stress via PI3K-Akt-eNOS Signaling PathwayHigh glucose-induced ROS generation in endothelial cells was dramatically increased compared with NG group (P<0.05), whereas irisin treatment markedly inhibited it (P<0.05 vs. HG group). However, the inhibitory effect of irisin on ROS generation was abolished by LY294002 or L-NAME. On the other hand, the mRNA expression levels of GPX-1, CAT and SOD were reduced in high glucose-treated endothelial cells. Irisin treatment significantly up-regulated antioxidant enzymes expression (P<0.05), and the effect could also be blocked by LY294002 or L-NAME.ConclusionsSystemic administration of irisin decreased serum inflammatory cytokines, improved endothelial dysfunction, activated Akt-eNOS signaling pathway, reduced atherosclerotic en face and cross section plaque area, decreased the macrophages and T lymphocytes infiltrated in plaque and down-regulated mRNA expression levels of inflammatory cytokines in aortas, protected high glucose-induced endothelial cells apoptosis and oxidative stress through PI3K-Akt-eNOS signaling pathway. Irisin improves endothelial dysfunction and ameliorates atherosclerosis induced by diabetes and reduced high glucose-induced endothelial cells apoptosis. Irisin could be therapeutic for atherosclerotic vascular diseases.