The earliest site at which the vascular complications develop in diabetic patients is the endothelium. The damage to endothelial cells plays an important role in endothelial dysfunction.1,2 As a result, endothelial dysfunction plays an important role in the pathogenesis of diabetic vascular disease. There are a number of in vitro studies providing evidence that hyperglycemia can induce endothelial cell apoptosis.3,4 It has been reported that variability in glycemic control can be more deleterious to endothelial cells than a constant high concentration of glucose.5 This condition is similar to that observed in poorly controlled diabetes. Adiponectin is present in serum as trimers, hexamers, and high molecular weight complexes. A proteolytic cleavage product of adiponectin, known as globular adiponectin (gAD), also circulates in human plasma.6 Hypoadiponectinemia is correlated with increased atherosclerotic disease, such as coronary artery disease.7-9 In cultured endothelial cells, adiponectin has been shown to exhibit anti-apoptotic effects.10-12 Additionally, adiponectin promotes endothelial cell survival through its ability to stimulate the adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway.10 Downregulation of adiponectin receptors (adipoRs) may blunt adiponectin induced signaling.13 Regulation of adipoR1 has recently been shown to be a possible element involved in glucose and lipid metabolism in diabetic states.14 Treatment with adiponectin or ectopic expression of its receptors has been shown to increase AMPK phosphorylation and fatty acid oxidation in muscles, although this effect does not occur when using dominant-negative AMPK.13,15 Conversely, targeted disruption of adipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, resulting in increased endogenous glucose production and insulin resistance.
Unknown is whether human umbilical vein endothelial cell (HUVEC) apoptosis is associated with adipoRs and AMPK. To artificially create poorly controlled diabetic conditions, HUVECs were incubated with an intermittent high-glucose concentration, and cells were pretreated with gAD. We evaluated the effects of gAD on intermittent high-glucose-induced apoptosis, adipoR1, adipoR2 expression, and AMPK activation in HUVECs.
Cell culture conditions
HUVECs (ECV-304, GDC023, China) were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco). Cells were grown in a monolayer at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Throughout the entire experiment, cells were used at passages 4-6. Cells received the following fresh media every 8 hours: continuous normal glucose medium (5.5 mmol/L), continuous high-glucose medium (25 mmol/L), and alternating normal and high-glucose media. Osmotic control was assured by incubating cells both in continuous 25 mmol/L mannitol and in alternating concentrations of 5.5 and 25 mmol/L mannitol. HUVECs were exposed to the experimental conditions for 1, 3, and 5 days. HUVECs were cultured to detect viability using the D-(+)-glucose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma, USA) assay for 1, 3, and 5 days and to detect apoptosis by flow cytometry only at 5 days.
HUVECs were cultured with 0, 0.5, 1.0, and 3.0 μg/ml gAD (BioVendor, Czech Republic) in alternating normal and high-glucose media every 8 hours for 5 days. We detected HUVEC apoptosis by flow cytometry.
HUVECs were first transfected with the adipoR1-specific small-interfering RNA (siRNA) for 24 hours and then stimulated with 3 μg/ml gAD in alternating normal and high-glucose media every 8 hours for 5 days. HUVECs apoptosis was detected by flow cytometry.
HUVECs were cultured in both gAD and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) (Sigma) in alternating normal and high-glucose media every 8 hours for 5 days. Western blotting analysis was used to detect the expression of AMPKα and phosphorylated AMPKα.
HUVECs were subjected to treatment with the AMPK inhibitor adenine-9-β-D-arabino-furanoside (araA) (Sigma), gAD, and AICAR in alternating normal and high-glucose media every 8 hours for 5 days.
MTT assay to detect HUVEC viability
HUVECs were plated onto 96-well microtiter plates at a density of 1×104 cells/well for 24 hours. The cells were then incubated with FBS-free medium for 24 hours. Ten microliters of a sterile, filtered MTT solution (5 mg/ml) in phosphate-buffered saline (PBS, pH 7.4) was added to each well to attain a final concentration of 0.5 mg MTT/ml. After 4 hours, the non-reacted dye was removed, and 150 μl of dimethyl sulfoxide was added to dissolve the insoluble formazan crystals. After 20 minutes of incubation, a microplate reader was used to determine the OD value (3550-UV, BioRad). The following formula was used to calculate the inhibition rate of HUVECs proliferation: (1-(experimental group OD value-negative control OD value) / (positive control OD value-negative control OD value)) ×100%.
Flow cytometry to detect HUVECs apoptosis
HUVECs were collected, washed with PBS, and modulated at a density of 1×106/ml. Both 5 μl Annexin V-FITC and 10 μl propidium iodide (PI, 10 μg/ml, Sigma) were added into 100 μl of suspended cells and then incubated for 15 minutes in a dark room at room temperature. Ten thousand cells were collected and analyzed using CellQuest software, version 3.0 (Becton Dickinson Immunocytometry Systems).
Reverse transcription polymerase chain reaction (RT-PCR) to detect adipoR mRNA
HUVECs were cultured in normal glucose medium (5.5 mmol/L), high-glucose medium (25 mmol/L), and alternating normal and high-glucose media for 5 days. One group of cells was cultured in alternating normal and high-glucose media with 3 μg/ml gAD. HUVECs were washed with ice-cold PBS and then treated with TRIzol (Takara, Japan). RNA was extracted according to a TRIzol isolation protocol. A total of 2 μg RNA was reverse-transcribed using an RNA PCR kit into cDNA in a final reaction solution of 20 μl containing 1.0 mmol/L dNTP, 2 μl 10×RT buffer, 5 mmol/L MgCl2, 20 U RNase inhibitor, 2.5 μmol oligo(dT) primer, 5 U avian myeloblastosis virus (AMV) reverse transcriptase, and 2 μg RNA and RNase-free dH2O (Takara). The mixtures were heated according to the following conditions: 15 minutes at 37°C, repeated three times, and 5 seconds at 85°C. The extracted cDNA was used for RT-PCR or stored at -80°C.
RT-PCR was performed according to the manufacturer's instructions. All quantifications were performed using human GAPDH as the internal standard. Triplicate RT-PCR analyses were performed for each sample. Values were averaged for each sample, and the relative amounts of adipoR1 and adipoR2 mRNA were normalized against GAPDH. Data analysis was performed using Light Cycler software, version 3.5. The following sequences were used: GAPDH (forward primer, 5′-GCACCGTCAAGGCTGAAC-3′; reverse primer, 5′-TGGTG-AAGACGCCAGTGGA-3′), adipoR1 (forward primer, 5′-TCCTAAGCACCGGCAGACAAG-3′; reverse primer, 5′-CTTGACAAAGCCCTCAGCGATAGTA-3′), adipoR2 (forward primer, 5′-GCACTATGTCATCTCGGAGGG-3′; reverse primer 5′-GCCATCAGCATCAACCAGC-3′).
The reaction conditions were the following: 1 cycle for 10 seconds at 95°C, 40 cycles for 5 seconds at 95°C, 34 seconds at 60°C.
siRNA affects HUVEC apoptosis
HUVECs were seeded in six-well plates. Cells were cultured in RPMI-1640 without FBS and antibiotic. After 24 hours, the subculture was transfected according to the manufacturer's instructions with 15 pmol adipoR1 siRNA using Lipofectamine (Invitrogen, USA).
The adipoR1 sense siRNA sequence was 5′-GGACAACGACUAUCUGCUATT-3′. The siRNA was obtained from Shanghai GenePharma Co., Ltd. Following transfection, HUVECs were cultured in normal glucose medium (5.5 mmol/L) and cultured in alternating normal and high-glucose media every 8 hours for 5 days. Finally, one group of cells was cultured in alternating normal and high-glucose media with 3 μg/ml gAD. We detected HUVECs apoptosis using flow cytometry.
Western blotting analysis to detect phosphorylated AMPKα expression of HUVECs
HUVECs were cultured with 3 μg/ml gAD and 1 mmol/L AICAR in alternating normal and high-glucose media every 8 hours for 5 days. After implementing identical conditions, other groups received an additional 0.5 mmol/L araA.
HUVECs were washed twice with ice-cold PBS and lysed in a buffer containing 20 mmol/L Tris-HCl, 0.1 mmol/L Na3VO4, 25 mmol/L NaF, 25 mmol/L β-glycerophosphate, 2 mmol/L EDTA, 2 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 2 μg/ml aprotinin, and 2 μg/ml leupeptin, pH 7.5. The lysed cells were first gently mixed for 10 minutes on ice and then centrifuged at 10 000 × g for 15 minutes at 4°C. Using bovine serum albumin (BSA) as the standard, the extracts' protein concentrations were determined according to the bicinchoninic acid (BCA) protein assay method. We subjected 20 μg of the protein extracts to 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electro-blotted the material onto nitrocellulose membranes. After blocking and washing, membranes were incubated overnight with rabbit anti-human AMPKα (1:400) and phosphorylated AMPKα (pAMPK, 1:200) (Cell Signaling, USA) antibody at 4°C. They were then further incubated for 2 hours with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:1000, Sigma). The immune complexes were visualized by enhanced chemiluminescence methods, and the band intensity was measured and quantified. The AMPKα and pAMPK band intensities were relative to the β-actin band intensity. The resulting images were analyzed by Scion Image software.
Each experiment was repeated three times. SPSS version 11.5 (SPSS Inc., USA) was used for statistical analysis, and data were expressed as mean ± standard deviation (SD). We used single-factor analysis of variance to compare the data. Values of P <0.05 were considered statistically significant.
We measured endothelial cell viability using the MTT assay. In contrast to the control group (5.5 mmol/L), alternating normal and high-glucose media inhibited endothelial cell viability in a time-dependent manner (P <0.01). When endothelial cells were placed in continuous high-glucose media (25 mmol/L), their viability was inhibited on days 3 and 5, compared with the control group (P <0.01). In comparison to the cells placed in continuous high-glucose media, endothelial cell viability decreased in alternating normal and high-glucose media on days 3 and 5 (P <0.01, Figure 1).
gAD protects HUVECs from apoptosis
We measured endothelial cell apoptosis using Annexin V-FITC/PI double staining. Endothelial cell apoptosis increased when alternating normal and high-glucose media and in continuous high-glucose media for 5 days compared with the control group (P <0.05). Endothelial cell apoptosis also increased when alternating normal and high-glucose media for 5 days compared with the continuous high-glucose group (P <0.05, Figure 2).
Endothelial cells were incubated in different concentrations of gAD (0, 0.5, 1.0, and 3 μg/ml). Only 3 μg/ml gAD inhibited endothelial cell apoptosis when cultured with alternating normal and high-glucose media for 5 days (Figure 3).
Relationship between gAD and adipoRs
The expression of adipoR1 and adipoR2 in HUVECs was detected using RT-PCR. The expression of adipoR1 decreased sharply when culturing cells in both alternating normal and high-glucose media and continuous high-glucose media for 5 days compared with the control group (P <0.01). The expression of adipoR1 decreased sharply when alternating normal and high-glucose media for 5 days compared with continuous high-glucose media (P <0.01). The expression of adipoR1 increased in cells when alternating normal and high-glucose media in the presence of 3 μg/ml gAD compared with cells without gAD (P <0.01). No differences in adipoR2 expression were observed in any of the experimental conditions (Figure 4).
Protective effect of gAD was decreased in HUVECs transfected by siRNA
HUVECs were first transfected with adipoR1-specific siRNA and then stimulated with gAD. Compared with cells stimulated with only gAD and not transfected, these HUVECs exhibited a significant increase in apoptosis (P <0.01, Figure 5). However, HUVECs also exhibited significantly decreased apoptosis compared with HUVECs without transfection.
gAD activates pAMPKα (Thr-172) in HUVECs
The protein expression levels of AMPKα and pAMPKα were detected using Western blotting. The AMPK activator AICAR (1 mmol/L) significantly increased pAMPKα (Thr-172) in HUVECs that had been cultured in alternating normal and high-glucose media for 5 days compared with the control group. gAD (3 μg/ml) alone or combined with AICAR induced the same results. araA inhibited, whereas AICAR and gAD stimulated, AMPKα activity under the same conditions (Figure 6).
The present study demonstrates that gAD protects human umbilical vein endothelial cells against apoptosis through the up-regulation of adipoR1 and AMPK activity. Besides these observations, we have also found that intermittent cultured in alternating normal and high-glucose media high glucose levels, in contrast to permanent high-glucose levels, induce a greater decrease in expression of adipoR1.
Apoptosis is genetically programmed cell death that occurs during pathological conditions, such as atherosclerosis. Vascular endothelia are under continuous exposure to many factors that may activate death or survival pathways in vascular cells. Apoptosis of vascular endothelial cells is linked to endothelial dysfunction. Endothelial cell damage caused by increased glucose leads to their dysfunction and plays a key role in the pathogenesis of diabetic vascular disease.1,2 Recently, it has been revealed that cell apoptosis is more pronounced under intermittent high-glucose than stable high-glucose conditions.16 Glucose fluctuations may also be involved in the development of vascular injury in diabetics. We found that intermittent high-glucose concentrations, similar to those observed in poorly controlled diabetic patients, play a key role in HUVECs apoptosis. In the present study, we found that fluctuating glucose levels may be more harmful than a permanent high-glucose concentration for endothelial cells. Similar result has been reported.5 However, the precise mechanism remains to be determined. We sought to elucidate the possible role and mechanism of gAD in preventing endothelial cell apoptosis induced by intermittent and stable high glucose.
Previous articles have shown that adiponectin exerts an anti-apoptosis effect,10-12 anti-inflammatory effect,17-19 anti-atherosclerotic effect,20,21 anti-insulin resistance,22 and reduces oxidative stress.23,24 Hypoadiponectinemia and endothelial dysfunction are linked to cardiovascular disease and atherosclerosis.25 In the present experiment, macrovascular endothelial cell apoptosis was more overt under intermittent high-glucose conditions than under permanent high-glucose conditions. gAD at 3 μg/ml promoted the survival of HUVECs subjected to intermittent high-glucose concentrations. Thus, we conclude that gAD exerts an anti-apoptosis effect and is a beneficial factor for protecting endothelial cells in intermittent and stable high glucose.
gAd activates adiponectin receptors (mainly adipoR1), whereas adipoR2 engages mainly full length adiponectin. gAd has a higher binding affinity for adipoR1 and full length adiponectin for adipoR2.26 Overexpression of adipoRs in HUVECs significantly enhanced some of the protective effects of adiponectin.27 The present experiment demonstrated the expression of adipoR1 in HUVECs. The quantity of adipoR1 mRNA increased 2.09-fold when HUVECs were incubated with 3 μg/ml gAD in cells placed in intermittent high-glucose media. The expression of adipoR1 mRNA decreased more sharply in the presence of intermittent high-glucose media compared with permanent high-glucose media. However, no differences in adipoR2 mRNA expression were observed between these groups. Recent reports indicate that in L6 myoblasts, hyperglycemia caused a significant reduction in adipoR1 mRNA expression, whereas hyperglycemia had no effect on the expression level of adipoR2 mRNA.28-30 gAD is rapid and potent stimulator of adipoR1. We found that gAD decreased HUVEC apoptosis induced by fluctuating hyperglycemia. This phenomenon was partially counteracted when cells were transfected with adipoR1-specific siRNA. Using siRNA to efficiently knockdown adipoR1 expression may attenuate the protective effects of adiponectin on reactive oxygen species production and caspase 3 activity.31 This indicates that gAD inhibited HUVEC apoptosis induced by fluctuating hyperglycemia by partially connecting with adipoR1. We provide direct evidence that adipoR1, not adipoR2, is involved in mediating high glucose-evoked apoptosis in HUVECs. We also provide evidence that gAD significantly inhibits endothelial cell apoptosis and increases adipoR1 expression when incubated with intermittent high-glucose media. Thus, we conclude that the regulation of adipoR1 expression, not adipoR2 expression, may be an important mechanism for determining gAD function in endothelial cells.
Previous studies found that the effects of adiponectin are associated with AMPK.32-35 AMPK is a multi-subunit protein kinase that functions as a sensor of the intracellular energy state.36 AMPK phosphorylation is hypothesized to be the “fuel gauge” for cells. Increasing evidence suggests that AMPK, a central regulator of cellular energy metabolism, plays a key role in modulating vascular reactivity. The present study directly showed that gAD rapidly activated AMPK in HUVECs. gAD is a dominating activator of AMPK and has high affinity for adipoRl, which activates AMPK. AMPK is a primary metabolic and anti-apoptotic coordinator.12,35 Blocking AMPK activation by utilizing a dominant-negative mutant inhibited the effect of acetyl coenzyme-A carboxylase phosphorylation, fatty-acid combustion, glucose uptake, and lactate production in myocytes, indicating that the stimulation of glucose utilization and fatty-acid combustion by adiponectin occurs via AMPK activation.15 The present study confirmed that gAD activates AMPK in HUVECs. In endothelial cells, adiponectin enhances production of nitric oxide, suppresses production of reactive oxygen species, and protects cells from inflammation that results from exposure to high glucose levels or tumor necrosis factor, through activation of AMP-activated protein kinase and cyclic AMP-dependent protein kinase (also known as protein kinase A) signaling cascades.37 Previous investigations have shown that AMPK is involved in the signaling pathway for the metabolic effects of adiponectin.36,38 The present study directly observed AMPK (Thr-172) phosphorylation in endothelial cells after gAD treatment. The present results further indicate that gAD exerts an anti-apoptotic effect in endothelial cells partially via the AMPK pathway. The AMPK pathway is critically involved in anti-apoptotic properties of adiponectin.39
In conclusion, adipoR1, not adipoR2, has been shown to be involved in mediating intermittent high-glucose-evoked apoptosis in HUVECs. gAD can reverse the decreased expression of adipoR1 in HUVECs. Additionally, gAD induced AMPK phosphorylation, suggesting that gAD inhibits HUVEC apoptosis induced by an intermittent high-glucose concentration through an adipoR1/AMPK-dependent pathway. Glucose fluctuations, compared with constant high-glucose levels, may be more harmful for endothelial cells. Adiponectin receptor agonists and gAD sensitizers may be relevant in the development of novel therapeutic strategies for treating endothelial cell dysfunction in high glucose.
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