Conditioned medium from contracting skeletal muscle cells reverses insulin resistance and dysfunction of endothelial cells

ObjectiveObese adipose tissue has been characterized with chronic inflammation associated with elevated secretion of inflammatory cytokines and declined secretion of anti-inflammatory cytokines which can impair endothelial function in an endocrine manner. Adipose tissue hypoxia plays a role in the changes of cytokines. Physical exercise/muscle contraction may help preventing cardiovascular disease through improving insulin resistance and endothelium function. However the mechanism is unclear. Skeletal muscle is an endocrine tissue. Contracting muscles secrete myokines which may play roles in the beneficial effect of exercise. In this study, the conditioned medium from electrical pulse stimulation (EPS) regulated skeletal muscle cells was used to explore the mechanism of contraction on endothelial dysfunction and insulin resistance induced by conditioned medium from hypoxic adipocytes.Methods3T3-L1 adipocytes were incubated under normoxia or hypoxia condition, respectively. The supernatant was collected as adipocyte conditioned medium (CM-N and CM-H). C2C12 mouse skeletal muscle cells were stimulated with EPS for 12 h. The supernatant was collected as muscle cells conditioned medium (CM-EPS). Human umbilical vein endothelial cells (HUVECs) were incubated with adipocyte CM and muscle cells CM together. Macrophages migration to HUVECs was detected with transwell system. The mRNA expressions of E-selectin, ICAM-1, MCP-1 and IL-6 were measured by real-time PCR. The phosphorylation of IKKα/β, NF-κB, Akt, AMPK, eNOS and SOCS3 protein levels were detected by Western blot. Concentration of NO was measured by ELISA kit. HUVECs apoptosis was detected by flow cytometry.ResultsCM-EPS reduced the increase of mRNA expressions of E-selectin, ICAM-1, MCP-1 and IL-6 in HUVECs induced by CN-H. The phosphorylations of IKKα/β and NF-κB, SOCS3 protein level and endothelial cells apoptosis, which were raised by CM-H, were significantly reduced by CM-EPS. CM-EPS reversed the effects of CM-H on Akt and eNOS phosphorylations and NO production in HUVECs. CM-EPS directly stimulated the phosphorylation of AMPK, which caused the following phosphorylation of eNOS in HUVECs.ConclusionIn summary, CM-EPS reversed endothelial cells inflammation, apoptosis, insulin resistance and dysfunction induced by CM-H.     

Insulin-mediated stimulation of protein kinase Akt: A potent survival signaling cascade for endothelial cells

Insulin exerts potent antiapoptotic effects in neuronal cells and has been suggested to promote angiogenesis. Therefore, we investigated whether insulin inhibits tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Because insulin has been shown to stimulate the protein kinase Akt, we investigated whether activation of Akt contributes to the apoptosis-suppressive effect of insulin and characterized the downstream signaling pathway. Incubation with insulin dose-dependently prevented apoptosis induced by TNF-alpha (50 ng/mL). The extent of apoptosis suppression by insulin was similar to the effect of vascular endothelial growth factor. Pharmacological inhibition of Akt activation or overexpression of a dominant-negative Akt mutant prevented the antiapoptotic effect of insulin. Furthermore, we investigated the effect of TNF-alpha on Akt phosphorylation by Western blot analysis with the use of a phosphospecific Akt antibody. Incubation of HUVECs with TNF-alpha induced a marked dephosphorylation of Akt. Insulin counteracted this TNF-alpha-induced dephosphorylation of Akt. Furthermore, we investigated the downstream signaling events. Akt has been shown to mediate its apoptosis-suppressive effects via phosphorylation of Bad or caspase-9. However, incubation with insulin did not lead to enhanced phosphorylation of Bad at Ser 136 or Ser 112. In contrast, insulin inhibited caspase-9 activity and prevented caspase-9-induced apoptosis. Mutation of the Akt site within caspase-9 significantly reduced the apoptosis-suppressive effect of insulin. The present study demonstrates an important role for insulin-mediated Akt activation in the prevention of endothelial cell apoptosis, which may importantly contribute to cell homeostasis and the integrity of the endothelium. In endothelial cells, Akt seems to mediate its antiapoptotic effect, at least in part, via phosphorylation of caspase-9 rather than Bad.     

The role of radix hedysari polysaccharide on the human umbilical vein endothelial cells (HUVECs) induced by high glucose

BackgroundDiabetes mellitus can cause a wide variety of vascular complications and it is one of the major risk factors for cardiovascular diseases (CVD). High glucose can induce vascular endothelial cell apoptosis. In this study, we investigated the effect of radix hedysari polysaccharide (HPS) on the depression of apoptosis of human umbilical vein endothelial cells (HUVECs) induced by high glucose.MethodsHUVECs were treated with media containing 30 mM glucose in the presence or absence of vitamin C or HPS. The level of intracellular reactive oxygen species (ROS) and apoptosis of HUVECs was measured with flow cytometry. Expression of c-Jun NH₂-terminal kinase (JNK) and caspase-3 were testified by real-time quantitative RT-PCR and immunofluorescence.ResultsHigh glucose was capable of eliciting the overexpression of JNK during the treatment procedure. Moreover, we found that the caspase-3 became overexpressed in apoptosis induced by high glucose; HPS could inhibit apoptosis under high glucose and suppress the generation of ROS and the overexpression of JNK and caspase-3. The effect of HPS on ROS quenching, inhibition of JNK and caspase-3 overexpression at the concentration of 100 μg/ml was similar to that of vitamin C at the concentration of 100 μM.ConclusionThe findings of the present study may suggest that HPS play a protection role on HUVECs against apoptosis induced by high glucose.     

FGF21 protects human umbilical vein endothelial cells against high glucose-induced apoptosis via PI3K/Akt/Fox3a signaling pathway

Aims

Diabetic macroangiopathy is the main cause of morbidity and mortality in patients with diabetes. Endothelial cell injury is a pathological precondition for diabetic macroangiopathy. Fibroblast growth factor 21 (FGF21) is a key metabolic regulator which has recently been suggested to protect cardiac myocytes and vascular cells against oxidative stress-induced injury in vitro and vivo. In this study, we aimed to investigate the protective capacity of FGF21 in human umbilical vein endothelial cells (HUVECs) against high glucose (HG)-induced apoptosis via phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt)/FoxO3a pathway.

Akt mediates insulin rescue from apoptosis in brown adipocytes: effect of ceramide.

We have recently shown that insulin can rescue serum deprived adipocytes from apoptosis in a PI 3 kinase and MAP kinase dependent manner. This study investigated the contribution of Akt and p70S6-kinase in insulin rescue from two different apoptotic triggers, serum deprivation and ceramide treatment. Insulin rescued serum-deprived immortalized brown adipocytes from apoptosis through phosphatidylinositol (PI) 3-kinase and Akt pathways, but independently of p70S6-kinase, as demonstrated by the use of inhibitors such as LY294002 or Rapamycin, and transfection experiments with dominant-negative constructs of Akt or p85 subunit of PI 3-kinase. A constitutively active Akt construct mimicked the insulin survival effect, decreasing the percentage of hypodiploid cells, the percentage of apoptopic cells and precluding the formation of apoptotic nuclei. We propose that the insulin survival effect on immortalized brown adipocytes is mediated through activation of Akt. However, insulin and EGF failed to rescue brown adipocytes from ceramide-induced apoptosis, as determined by DNA laddering, hypodiploid cells and apoptotic nuclei. Ceramide treatment blunted Akt activity but not PI 3-kinase activity, and insulin and EGF were unable to activate Akt. Ceramide also caused apoptosis in cells transfected with a constitutively active Akt construct, since phosphorylation of Akt was impaired under these experimental conditions. This study suggests that activation of Akt may be an absolute requirement for the survival of brown adipocytes.     

HUVECs from newborns with a strong family history of diabetes show increased apoptosis by flow cytometry with annexin V

A family history of type 2 diabetes mellitus (DM) increases the probability to develop DM and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) isolated from healthy newborns with familiar background of diverse diseases show early alterations such as less resistance to shear stress. The aim of this study was to evaluate the apoptosis by flow cytometry in HUVECs obtained from healthy newborns with (experimental) and without (control) a strong family history of DM, exposed to different glucose concentrations.MethodsHUVECs were incubated in M-199 culture media (containing a 5 mmol/L physiological glucose concentration) or supraphysiological glucose concentrations (15 or 30 mmol/L), for 48 h. Apoptosis was quantified by flow cytometry with annexin V and a polycaspase assay kit (FLICA, Immunochemistry Technologies LLC), and cell death was measurement by propidium iodide (PI, St Cruz Biotechnology Inc) positive stain.ResultsExperimental HUVECs showed higher levels of apoptosis in the presence of increasing glucose concentration (p < 0.01), whereas control HUVECs showed low levels of apoptosis.ConclusionsOur results suggest that HUVECs, isolated from healthy newborns with a strong family history of DM, have an abnormal predisposition to apoptosis.     

The preventive effect of uncarboxylated osteocalcin against free fatty acid-induced endothelial apoptosis through the activation of phosphatidylinositol 3-kinase/Akt signaling pathway

Objective

Increasing evidence suggests that osteocalcin (OC), one of the osteoblast-specific proteins, has been associated with atherosclerosis, but results are conflicting. The aim of this study was to elucidate the independent effect of uncarboxylated osteocalcin (ucOC), an active form of osteocalcin which has been suggested to have an insulin sensitizing effect, on vascular endothelial cells.

Materials and Methods

We used human aortic endothelial cells and treated them with ucOC. Linoleic acid (LA) was used as a representative free fatty acid. Apoptosis was evaluated using various methods including a terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling analysis kit and Western blotting for cleaved caspase 3, cleaved poly (ADP-ribose) polymerase and Bcl-xL. The phosphorylations of Akt and endothelial nitric oxide synthase (eNOS) as well as the level of NO were measured to confirm the effect of ucOC on insulin signaling pathway.

Results

Pretreatment of ucOC (30 ng/ml) prevented LA-induced apoptosis in insulin-stimulated endothelial cells; effects were abolished by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin. Treatment of ucOC (ranged from 0.3 to 30 ng/ml) significantly increased the phosphorylation of Akt and eNOS and nitric oxide secretion from endothelial cells in a PI3-kinase dependent manner.

Conclusions

Our study is the first to demonstrate the independent effect of ucOC on vascular endothelial cells. Our results further suggest that ucOC could have beneficial effects on atherosclerosis.     

Sphingosine-1-Phosphate Protects Intestinal Epithelial Cells from Apoptosis Through the Akt Signaling Pathway

Objective The regulation of apoptosis of intestinal mucosal cells is important in maintenance of normal intestinal physiology. Summary Sphingosine-1-phosphate (S1P) has been shown to play a critical role in cellular protection to otherwise lethal stimuli in several nonintestinal tissues. Methods The current study determines whether S1P protected normal intestinal epithelial cells (IECs) from apoptosis and whether Akt activation was the central pathway for this effect. Results S1P demonstrated significantly reduced levels of apoptosis induced by tumor necrosis factor-alpha (TNF-α)/cycloheximide (CHX). S1P induced increased levels of phosphorylated Akt and increased Akt activity, but did not affect total amounts of Akt. This activation of Akt was associated with decreased levels of both caspase-3 protein levels and of caspase-3 activity. Inactivation of Akt by treatment with the PI3K chemical inhibitor LY294002 or by overexpression of the dominant negative mutant of Akt (DNMAkt) prevented the protective effect of S1P on apoptosis. Additionally, silencing of the S1P-1 receptor by specific siRNA demonstrated a lesser decrease in apoptosis to S1P exposure. Conclusion These results indicate that S1P protects intestinal epithelial cells from apoptosis via an Akt-dependent pathway.     

应用RNA干扰技术研究游离脂肪酸对人脐静脉内皮细胞凋亡的影响

目的 应用RNA干扰(RNAi)技术探讨游离脂肪酸(FFA)对人脐静脉内皮细胞(HUVEC)凋亡的影响.方法 利用体外同源重组技术构建糖原合成酶激酶-3β(GSK-3β)特异的siRNA腺病毒表达载体,在人胚胎肾293A细胞中包装并扩增病毒,应用空斑实验法测定病毒滴度.选用GsK-3β特异的RNAi腺病毒感染HUVEC,采用Western blot方法检测其对GSK-3β和β-catenin 蛋白水平的影响.FFA贮存液按油酸盐:棕榈酸盐2:1配制,RNAi腺病毒和FFA共同干预HUVEC,采用流式细胞术、Hoechst 33258染色检测细胞凋亡.实验分为0.75 mmol/L FFA组、GSK-3β特异性RNAi腺病毒(Ad-640)+FFA组、未重组RNAi腺病毒(Ad-DEST)+FFA组、正常细胞组.采用方差分析进行数据比较.结果 构建的RNAi腺病毒感染HUVEC可以显著抑制GSK-3β蛋白的表达,上调细胞内β-catenin蛋白水平.0.75 mmol/L FFA具有促进细胞凋亡率的作用,荧光染色可见细胞出现典型的凋亡形态学改变;Ad-640+0.75 mmol/L FFA组与0.75 mmol/L FFA组(F=26.42,P<0.01)及Ad-DEST+0.75 mmol/L FFA组(F=23.34,P<0.01)相比细胞凋亡率显著减少,凋亡形态学改变亦明显减少,而Ad-DEST+FFA组与FFA组细胞凋亡率无显著差异(F=5.56,P>0.05),凋亡形态学改变相似.结论 构建的CSK-3β特异性RNAi腺病毒对FFA作用下的HUVEC具有部分保护作用.     

Intermittent hypoxia-induced autophagy via AMPK/mTOR signaling pathway attenuates endothelial apoptosis and dysfunction in vitro

Purpose

The aim of this study was to examine whether or not intermittent hypoxia (IH) upregulated autophagy and the contributions of autophagy to endothelial apoptosis and dysfunction in human umbilical vein endothelial cells (HUVECs).

Method

HUVECs were incubated under normoxia and IH conditions. After 3-, 6-, 12-, and 24-h exposure, the autophagic vacuoles and autophagosomes were observed by transmission electron microscopy and monodansylcadaverine staining. The protein levels of autophagy-related biomarkers and AMPK/mTOR pathway were measured by Western blot. The apoptosis-related proteins and the percentage of apoptotic cells were evaluated by Western blot and flow cytometry, respectively, while the levels of endothelial function biomarkers were assessed by ELISA.

Results

IH induced autophagy, as determined by the increased numbers of the autophagic vacuoles, autophagosomes, and by the elevated levels of Beclin-1 protein, the LC3II/LC3I ratio, and p62 degradation. IH-induced autophagic flux peaked at 12-h duration and weakened at 24 h. IH increased the ratio of p-AMPK/AMPK and decreased the ratio of p-mTOR/mTOR, while compound C restored the alteration. A significant decrease in the Bcl-2 level and the Bcl-2/Bax ratio and a significant increase in the protein expression levels of Bax and cleaved caspase 3 and in the percentage of apoptosis were observed under IH exposure. Moreover, the NO level was reduced, while the ET-1 and VEGF levels were raised under IH condition. These alterations were suppressed by the pretreatment of 3-methyladenine.

Conclusions

IH upregulates autophagy through AMPK/mTOR pathway in HUVECs in vitro, which might be protective against endothelial apoptosis and dysfunction caused by IH.

     

Extracellular Vesicles Derived from Intermittent Hypoxia–Treated Red Blood Cells Impair Endothelial Function Through Regulating eNOS Phosphorylation and ET-1 Expression

Purpose

Intermittent hypoxia (IH), a main characteristic of obstructive sleep apnea (OSA) syndrome, has been known as a dominant cause of OSA-related endothelial dysfunction and hypertension. However, the underlying mechanism still remains unclear. Extracellular vesicles (EVs), small vesicles secreted by various cells, can be absorbed by endothelial cells and then influence vascular function. The aim of this research is to clarify whether and how EVs shedding from red blood cells (RBCs) are involved in IH-induced endothelial dysfunction.

Methods

EVs were extracted by ultracentrifugation. After the identification of property and purity, EVs from IH-exposed RBCs (IH REVs) and normoxia-exposed RBCs (NOR REVs) or from OSA and non-OSA patient RBCs were utilized to treat C57BL/6 mouse aortas or human umbilical vein endothelial cells (HUVECs) for mechanistic exploration.

Results

Functional results demonstrated that REVs from OSA patients dramatically impaired endothelium-dependent relaxations (EDRs). Similarly, in vivo and ex vivo studies showed that IH REVs caused significant endothelial dysfunction compared to control group. Further results presented that IH REVs blocked endothelial nitric oxide synthase (eNOS) phosphorylation through inhibiting PI3K/Akt pathway and enhanced endothelin-1 (ET-1) expression through activating Erk1/2 pathway in endothelial cells. Meanwhile, endothelial dysfunction caused by IH REVs was reversed by Akt activator SC79 as well as Erk kinase inhibitor PD98059, suggesting that PI3K/Akt/eNOS and Erk1/2/ET-1 pathways were implicated in IH REV-induced impaired EDRs.

Conclusions

This study reveals a novel role of REVs in endothelial dysfunction under IH and dissects the relevant mechanism involved in this process, which will help to establish a comprehensive understanding of OSA or IH-related endothelial dysfunction from a new scope.

     

Effects of antioxidants on homocysteine thiolactone-induced apoptosis in human umbilical vein endothelial cells

Background and objectives Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine thiolactone (HcyT), one of the homocysteine metabolites in vivo, is toxic both in vivo and in vitro. The aim of this study was to investigate the effect of HcyT on apoptotic damage in human umbilical vein endothelial cells (HUVECs) and the role of antioxidants in the reduction of HcyT-induced apoptosis. Methods HUVECs were cultured in DMEM supplemented with 20% heat inactivated fetal bovine serum cell cultures were maintained in a humidified 5% CO2 atmosphere at 37℃. Cytotoxicity was determined by MTT assay, which consists of hypodiploid cells with propidium iodide labeling and intracellular reactive oxygen species levels using 2',7'-dichlorofluorescein diacetate as the probe by flow cytometry. Results HcyT (250-2000μM) induced HUVECs apoptosis in a time- and concentration-dependent manner. Reactive oxygen species levels rose in response to increasing HcyT concentrations at 24-h incubation. The reduction of cell apoptosis by N-acetylcysteine, vitamin E, or pyrrolidine dithiocarbamate, occurred simultaneously with a significant decrease in intracellular reactive oxygen species levels. Conclusion HcyT exerts its cytotoxic effects on endothelial cells through an apoptotic mechanism involving cellular reactive oxygen species production. The capacity of N-acetylcysteine, vitamin E, and pyrrolidine dithiocarbamate to scavenge HcyT-induced cellular reactive oxygen species correlates well with their efficiency to protect against HcyT-promoted apoptotic damage. The protective effect of pyrrolidine dithiocarbamate on cell apoptosis indicates HcyT-generated hydrogen peroxide may provoke cell apoptosis via activating nuclear factor-kappa binding protein.     

Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis

AIMS: Visfatin is a novel adipokine whose plasma concentrations are altered in obesity and obesity-related disorders; these states are associated with an increased incidence of cardiovascular disease. We therefore investigated the effect of visfatin on vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-2, MMP-9) production and the potential signalling cascades. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), visfatin significantly and dose-dependently up-regulated gene expression and protein production of VEGF and MMPs and down-regulated expression of tissue inhibitors of MMPs (TIMP-1 and TIMP-2). The gelatinolytic activity of MMPs (analysed by zymography) correlated with mRNA and western blot findings. Interestingly, visfatin significantly up-regulated VEGF receptor 2 expression. Inhibition of VEGFR2 and VEGF [by soluble FMS-like tyrosine kinase-1 (sFlt1)] down-regulated visfatin-induced MMP induction. Visfatin induced dose- and time-dependent proliferation and capillary-like tube formation. Importantly, visfatin was noted to have anti-apoptotic effects. In HUVECs, visfatin dose-dependently activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt) and ERK(1/2) (extracellular signal-regulated kinase) pathways. The functional effects and MMP/VEGF induction were shown to be dependent on the MAPK/PI3K-Akt/VEGF signalling pathways. Inhibition of PI3K/Akt and ERK(1/2) pathways led to significant decrease of visfatin-induced MMP and VEGF production and activation, along with significant reduction in endothelial proliferation and capillary tube formation. CONCLUSION: Our data provide the first evidence of visfatin-induced endothelial VEGF and MMP production and activity. Further, we show for the first time the involvement of the MAPK and PI3K/Akt signalling pathways in mediating these actions, as well as endothelial cell proliferation. Collectively, our findings provide novel insights into visfatin-induced endothelial angiogenesis.     

Serum vascular endothelial growth factor in cyanotic congenital heart disease functionally contributes to endothelial cell kinetics in vitro

BackgroundRemarkable amounts of neovascularization develop in patients with cyanotic congenital heart disease who have low pulmonary blood flow and systemic cyanosis, but the factors functionally responsible for angiogenesis in cyanotic congenital heart disease have not been determined.Methods and resultsTo investigate the functional angiogenic molecules in serum from these patients, serum angiogenic activity was studied in 21 patients (systemic oxygen saturation: 82 ± 1.9%) and in 17 healthy controls. Patient serum was more active in stimulating the tube formation of human umbilical vein endothelial cells (HUVECs) into capillary-like structures than control serum (150% vs 104% of internal control; p < 0.001). This increased serum angiogenic activity normalized after total cardiac repair (p < 0.001). The migration activity of HUVECs was also accelerated in patient serum (p = 0.007). To identify the molecules in patient serum affecting tube formation of HUVECs, we examined the effects of an inhibitor or a neutralizing antibody against various angiogenic molecules on in vitro angiogenesis. Both the soluble vascular endothelial growth factor (VEGF) receptor 1 and the VEGF receptor 2 tyrosine kinase inhibitor SU5416 reduced the basal serum angiogenic activity of patients and this was reversed by a supplement of recombinant human VEGF.ConclusionOur results indicate that serum VEGF functionally contributes to vascular endothelial cell kinetics in patients with cyanotic congenital heart disease.     

FFAs: Do they play a role in vascular disease in the insulin resistance syndrome?

The insulin resistance syndrome, otherwise known as the metabolic syndrome, describes a cluster of cardiovascular and metabolic abnormalities, which are strongly associated with overweight and obesity. The importance of the syndrome is due to its increased rates of cardiovascular morbidity and mortality. Insulin resistance is also characterized by elevated free fatty acid (FFA) levels. In otherwise healthy human subjects, elevation of FFA impairs endothelial function. This appears to be largely the result of blunting of nitric oxide-dependent tone, most likely at the level of the endothelial isoform of nitric oxide synthase (_eNOS). Some of the potential mediatory mechanisms include oxidative stress, proinflammatory cytokines, C-reactive protein, or endogenous inhibitors of _eNOS. Regardless of the mechanism(s) that mediates the effects of increased FFA on the vasculature, impaired vascular function is likely to account, at least in part, for the increase in cardiovascular mortality in subjects with the insulin resistance syndrome.     

The glucagon-like peptide 1 analog liraglutide reduces TNF-α-induced oxidative stress and inflammation in endothelial cells

ObjectiveGlucagon-like peptide 1 (GLP-1), one of the incretin hormones, has been reported to increase positive inotropic activity in cardiac myocytes and protect against myocardial injury. However, the effects upon endothelial cells and the mechanisms involved are not fully understood. We assessed the hypothesis that GLP-1 has protective effects against inflammation and oxidative stress on human endothelial cells.Methods and resultsThe effects of the GLP-1 analog liraglutide upon TNF-α-induced injury of the human umbilical vein endothelial cells (HUVECs) were evaluated. First, ROS induced by TNF-α was measured by staining with CM-H₂DCFDA. Intracellular ROS production of HUVECs was significantly decreased in a dose-dependent manner until 30 nM while liraglutide inhibited the induction of gp91^phox and p22^phox, subunit of NADPH oxidase, by TNF-α? In addition, protein levels of SOD-2, catalase and GPx were significantly increased by liraglutide. Second, rapid translocation of PKC-α into the membrane following TNF-α was evident. Liraglutide significantly inhibited this very rapid TNF-α-induced translocation of PKC-α into membrane at 2.5 min. Third, liraglutide significantly inhibited NF-κB activation and upregulated I-κB family while phosphorylation of IKK-α/β, which is upstream of NF-κB signaling, was also downregulated after 15 min of TNF-α treatment. Finally, liraglutide inhibited apoptosis of HUVEC and expression of Pentraxin-3 induced by TNF-α.ConclusionLiraglutide exerts marked anti-oxidative and anti-inflammatory effects on endothelial cells with inhibition of PKC-α, NADPH oxidase, NF-κB signaling and upregulation of protective anti-oxidative enzymes.     

Arterial insulin resistance in Yucatan micropigs with diet-induced obesity and metabolic syndrome

AimMetabolic syndrome affects a large proportion of the population and increases cardiovascular disease risk. Because metabolic syndrome often co-exists clinically with atherosclerosis, it is difficult to distinguish the respective contributions of the components to vascular abnormalities. Accordingly, we utilized a porcine dietary model of metabolic syndrome without atherosclerosis to investigate early abnormalities of vascular function and signaling.MethodsThirty-two Yucatan micropigs were fed either a high-fat, high-simple-sugar, high-calorie (HFHS) or standard chow diet (STD) for 6 months. Neither diet contained added cholesterol. Blood pressure and flow-mediated vasodilatation were assessed at baseline and 6 months. Aortas were harvested at 6 months to assess histology, insulin signaling, and endothelial nitric oxide (eNOS) phosphorylation.ResultsHFHS pigs developed characteristics of metabolic syndrome including obesity, dyslipidemia, and insulin resistance, but without histologic evidence of atherosclerosis. Although arterial intima-media thickness did not differ between groups, vascular dysfunction in HFHS was manifest by increased blood pressure and impaired flow-mediated vasodilation of the femoral artery. Compared with STD, aortas from HFHS exhibited increased p85α expression and Ser307 IRS-1 phosphorylation, and blunted insulin-stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase activity. In the absence of insulin stimulation, aortic Akt Ser473-phosphorylation was greater in HFHS than in STD. With insulin stimulation, Akt phosphorylation increased in STD, but not HFHS. Insulin-induced Ser1177-phosphorylation of eNOS was decreased in HFHS, compared with STD.ConclusionsPigs with metabolic syndrome develop early vascular dysfunction and aortic insulin signaling abnormalities, and could be a useful model for early human vascular abnormalities in this condition.     

1,25-Dihydroxyvitamin D improved the free fatty-acid-induced insulin resistance in cultured C2C12 cells

BACKGROUND: Epidemiological evidence has indicated that vitamin D deficiency increased the risk of insulin resistance in metabolic syndrome. The present study was conducted to test the hypothesis that 1,25-dihydroxyvitamin D may improve the free fatty-acid (FFA)-induced insulin resistance in muscle cells. METHOD: The insulin resistance of muscle cell model was established by treatment of FFA in differentiated C2C12 cells. Glucose uptake of C2C12 myotubes was analysed by the 3H-labelled 2-deoxyglucose uptake assay. The diameter of myotubes was measured under the condition of glutaraldehyde-induced autofluorescense. Tyrosine phosphorylated insulin receptor substrate 1 (IRS-1) was measured by immunoprecipitation. Serine phosphorylated IRS-1 and protein kinase B (Akt), extracellular signal-related kinase (ERK), c-Jun amino-terminal kinases (JNK) as well as their phosphorylated form were analysed by Western blots. RESULTS: Compared with a vehicle-treated group, FFA treatment in myotubes was associated with 70.6% reduction in insulin-mediated uptake of glucose, a five-fold increase in serine phosphorylation of IRS-1, 76.9% decrease in tyrosine phosphorylation of IRS-1 and 81.8% decrease in phosphorylation of Akt. Supplement of 1,25-dihydroxyvitamin D improved the FFA-induced inhibition of glucose uptake in a dose- dependent (p < 0.001) and time-dependent manner (p < 0.01). This was accompanied by increase in tyrosine phosphorylation of IRS-1 and phosphorylated Akt and decrease in serine phosphorylation of IRS-1 (p < 0.001). 1,25-Dihydroxyvitamin D also inhibited the FFA-induced reduction in myotube diameter by 35.3% (p < 0.001). JNK phosphorylation was reduced by 126.7% with treatment of 1,25-dihydroxyvitamin D (p < 0.001). 1,25-Dihydroxyvitamin D had no effect on FFA-induced ERK phosphorylation (p = 0.84). CONCLUSION: 1,25-Dihydroxyvitamin D improved the FFA-induced insulin resistance in muscle cells. Copyright (c) 2008 John Wiley & Sons, Ltd.