Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.     

Protective effect of procyanidin single active ingredient B2 on human endothelial progenitor cells under high glucose stimulation

AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.     

Effect of insulin on the SGK1 expression and extracellular matrix synthesis in human mesangial cells cultured in high glucose

AIM:To study the effect of insulin on the serum and glucocorticoid-inducible kinase 1 (SGK1) expression and extracellular matrix synthesis in human glomerular mesangial cells (HMC) cultured in high glucose. METHODS:The HMCs were cultured in the presence of 5.5 or 25 mmol/L glucose with or without 100 nmol/L insulin (i.e. NG, HG, NI and HI groups). 4 h latter, expressions of SGK1, insulin receptor substrate-1 (IRS1) and IRS2 in corresponding groups were detected by immunofluorescence or examined by Western blotting. The phosphorylation of IRS1 and IRS2 was measured by immunoprecipitation. 24 h latter, connective tissue growth factor(CTGF) and fibronectin (FN) were also examined by RT-PCR and ELISA, respectively. RESULTS:Compared with NG, the SGK1 protein expression in HG, NI and HI groups was significantly higher (P<0.01). High glucose mainly caused IRS2 protein and its phosphorylation level increase (P<0.01). When treated with 100 nmol/L insulin, IRS1 protein and its phosphorylation in HI group apparently elevated while slight inhibition of IRS2 protein expression and its phosphorylation were observed (HI vs HG, P<0.05). High glucose enhanced the expression of CTGF and FN, and insulin strengthened this effect.CONCLUSION:Insulin and high glucose up-regulate the expression of SGK1 in mesangial cells through different target molecular pathways and ultimately enhance ECM synthesis. The effect of insulin is highly associated with IRS1 signaling cascades.     

Inhibitory effect of 5-fluorouracil on ultra-microstructure and insulin secretion of pancreatic β cells

AIM: To investigate the molecular mechanisms of β cell dysfunction induced by 5-fluorouracil (5-FU) in islet β cell line (NIT-1 cells). METHODS: The NIT-1 cells were treated with different concentrations of 5-FU. The content of insulin in the culture medium was determined by radioimmunoassay. Cell apoptosis was observed by flow cytometry with annexin V/PI staining. The ultra-microstructural changes of NIT-1 cells were observed under transmission electron microscope. The expression of pancreatic and duodenal homeobox protein 1(PDX-1) at mRNA and protein levels in NIT-1 cells was examined by RT-PCR and Western blotting, respectively. RESULTS: Exposed to the low glucose concentration (5.6 mmol/L), insulin secretion in NIT-1 cells was not significantly decreased following a 24 h treatment with 5.0 to 40.0 mg/L 5-FU (P>0.05). On the contrary, the high glucose (16.7 mmol/L)-stimulated insulin secretion in NIT-1 cells was inhibited by 5.0 to 40.0 mg/L of 5-FU in a dose-dependent manner after 24 h of incubation (P<0.01). The apoptosis rate of NIT-1 cells was significantly increased as compared to those in the control levels(P<0.05). The structural changes of mitochondria were the main apoptotic changes under transmission electron microscope. Significant down-regulation of PDX-1 expression at mRNA and protein levels was observed in NIT-1 cells treated with 5-FU at the concentration of 10.0 mg/L to 40.0 mg/L(P<0.05).CONCLUSION: 5-FU inhibits the insulin secretion in islet β cell induced by high glucose. A relative deficiency in insulin secretion following 5-FU treatment is related to the changes of β cell ultra-microstructure and the reduction of β cell numbers, by which an increase in apoptosis of pancreatic β cells is induced. Down-regulation of PDX-1 expression may play a pivotal role in increasing the apoptosis of pancreatic β cells induced by 5-FU in high-glucose condition.     

Establishment and verification of insulin resistance model in mouse skeletal muscle cells

AIM:To establish a stable and repeatable insulin resistance model of skeletal muscle cells in vitro, so as to promote the exploration of the pathological mechanism of insulin resistance and the development and screening of related drugs. METHODS:C2C12 mouse myoblasts were used to induce differentiation in normal differentiation medium and differentiation medium containing glucose at 40 and 60 mmol/L, respectively. The effects of glucose at different concentrations on cell convergence, fusion and formation of multinucleated myotubes were observed under phase contrast microscope every day. After 1, 3, 5 and 7 d of differentiation, 2-NBDG assay was used to detect the effects of different interventions on C2C12 basal glucose uptake and insulin-stimulated glucose uptake. The effects of different interventions on the protein expression of glucose transporter 4 (GLUT4) after 5 d and 7 d of differentiation were determined by Western blot. The effects of different interventions on the distribution of GLUT4 protein after 5 d of differentiation were detected by immunofluorescence staining. RESULTS:After treated with glucose at 60 mmol/L, the morphological observation showed that high glucose treatment significantly inhibited the growth and differentiation of C2C12 cells after 3 d. High glucose treatment significantly inhibited basal glucose uptake and insulin-stimulated glucose uptake of the C2C12 cells after 5 d and 7 d (P<0.01). No difference between insulin-stimulated GLUT4 expression and basal GLUT4 expression after 5 d and 7 d of high glucose treatment was observed (P>0.05), but there was significant difference between control group and 60 mmol/L group (P<0.05) determined by Western blot. Immunofluorescence staining observation showed that the distribution of GLUT4 protein in the C2C12 cell membrane was significantly decreased after 5 d of high glucose treatment (P<0.01). Glucose treatment (40 mmol/L) also played a role to some extent, but the effect was not as obvious and stable as 60 mmol/L glucose. CONCLUSION:A stable insulin resistance model of mouse skeletal muscle cells in vitro was successfully established by high glucose stimulation. The treatment of glucose at 60 mmol/L for 5 d was the best. Morphological observation and detection of basic and insulin-stimulated glucose uptake and GLUT4 protein expression and distribution evaluates the insulin resistance level of skeletal muscle cells in vitro.     

Effects of L-carnosine on NIT-1 islet cells

AIM: To investigate the protective effect of L-carnosine on insulin secretion, proliferation and apoptosis of β-cells impaired by high glucose. METHODS: NIT-1 cells were pre-treated with glucose at concentrations of 11.1 mmol/L (control level) and 33.3 mmol/L (high level) for 72 h, and then the cells were stimulated with various concentrations of glucose (0, 5 and 25 mmol/L) and/or L-carnosine (0, 1 and 20 mmol/L). The level of insulin in the medium was measured by radioimmunoassay. To detect the effect of L-carnosine on proliferation and apoptosis, NIT-1 cells were divided into 4 groups according to different culture conditions for 72 h: group C (with 11.1 mmol/L glucose), group H (with 33.3 mmol/L glucose), group H+A (with 33.3 mmol/L glucose+ 1 mmol/L L-carnosine) and group H+B (with 33.3 mmol/L glucose +20 mmol/L L-carnosine). Proliferous or apoptotic cells were identified by BrdU labeling and flow cytometry (labeling with annexin V-FITC/PI),respectively. Total RNA was extracted and the mRNA expression of caspase-3 and bcl-2 was measured by RT-PCR. The caspase-3 activity was also checked by fluorometric assay kit. RESULTS: The insulin in high-level glucose group was lower than that in control-level glucose group. L-carnosine at concentration of 20 mmol/L notably increased the insulin secretion of the cells pre-treated with glucose at control level or high level. The proliferation and apoptosis were both increased in group H compared with group C, but the total cell counts declined because the apoptotic rate was higher than the proliferation rate. L-carnosine at concentration of 1 mmol/L significantly increased the proliferation rate and decreased the apoptotic rate. The mRNA level of caspase-3 was decreased and the mRNA level of bcl-2 was increased after the cells were treated with L-carnosine at concentration of 1 mmol/L. L-carnosine at concentrations of both 1 mmol/L and 20 mmol/L significantly decreased the caspase-3 activity. CONCLUSION: L-carnosine at high level directly stimulates insulin secretion in NIT-1 cells, and L-carnosine at normal level promotes the cell proliferation and inhibits apoptosis induced by high concentration of glucose. Caspsase-3 and Bcl-2 may be partly involved in this process.     

Mechanisms of PGF2α on the glucose-induced insulin secretion in NIT-1β cells

AIM:To investigate the cellular mechanisms by which PGF₂α promotes glucose-stimulated insulin secretion in NIT-1 beta cells. METHODS:Using the radioimmunoassay (RIA), the amount of the PGF₂α augmentation of glucose stimulated insulin secession was determined in different conditions, and the confocal laser scanning methods by Fluo-3AM as a fluorescent probe were used to analyze the changes of intracellular calcium in NIT-1β cells. RESULTS:At the lower glucose (0, 5.5 mmol/L), PGF₂α (5 μmol/L) failed to potentiate insulin secretion (P>0.05). However, in the presence of 16.5 mmol/L glucose, PGF₂α increased significantly in insulin secretion (P<0.05). Neither the AC inhibitor ddA nor the GC inhibitor Ly-83583 altered PGF₂α-potentiated insulin secretion in the presence of 16.5 mmol/L (P<0.05 or P<0.01). Otherwise, the PLC inhibitor U-73122 and the PKC blocker calphostin C both counteracted the insulinotropic of PGF₂α (P<0.01 or P<0.05). Moreover, exposure of the NIT-1β cells to 5 μmol/L PGF₂α induced a rapid increase of intracellular calcium (P<0.01). The inhibitor, ddA or Ly-83583 had no impact on PGF₂α-induced elevation of the intracellular calcium (P<0.01). Pretreatment of the cells with U-73122 completely prevented the calcium response induced by PGF₂α (P<0.01). CONCLUSION:Efects of PGF₂α was independent of cAMP or cGMP, potentiated glucose (16.5 mmol/L)-induced insulin secretion in NIT-1β cells through stimulation of phospholipase C, which subsequently mediated the elevation of intracellular calcium and activation of protein kinase C.     

Reverse effect of protein kinase C inhibitor Ro-31-8220 on the hypertrophy of cardiomyocytes of neonatal rats induced by high glucose levels

AIM: To study the effect of protein kinase C (PKC) inhibitor Ro-31-8220 on the hypertrophy of cardiomyocytes of neonatal rats induced by high glucose levels, and to investigate the role of PKC and its downstream signal transduction pathway. METHODS: Using cultured neonatal cardiac myocytes as a model, the cells were divided into: (1) control group (glucose 5.5 mmol/L); (2) different high glucose level (10 mmol/L,15 mmol/L, 20 mmol/L, 25.5 mmol/L); (3) high glucose level (25.5 mmol/L) + PKC inhibitor Ro-31-8220 (50 nmol/L); (4) high glucose level (25.5mmol/L) + NF-κB inhibitor (BAY11-7082, 5 mmol/L). The cellular diameters and protein level were measured and the expression of PKC-α, PKC-β2, p-PKC-α, p-PKC-β2, NF-κB and c-Fos were determined by Western blotting. RESULTS: Neonatal cardiomyocytes cultured in high glucose concentration showed increased cellular diameters, protein level and higher expressions of PKC-α, PKC-β2, p-PKC-α, p-PKC-β2, NF-κB and c-Fos, which was consistent with the increased glucose levels and had statistical significance compared to control group (P<0.01). PKC inhibitor Ro-31-8220 reversed these changes induced by high glucose concentration as showed by decreased cellular diameters, protein level and expression of PKC-α, PKC-β2, p-PKC-α, p-PKC-β2, NF-κB and c-Fos, which had statistical significance compared to high glucose groups (P<0.01). CONCLUSION: High glucose levels induce hypertrophy of cardiomyocytes. PKC inhibitor Ro-31-8220 reverses the effect of high glucose on the cardiac myocytes, which may be via PKC/NF-κB/c-Fos pathway.     

Hydrogen sulfide attenuates human umbilical vein endothelial cell senescence via modulation of Sirt1/eNOS pathway

AIM: To explore the role of Sirt1/eNOS signalling pathway in the protective effect of hydrogen sulphide (H₂S) against endothelial cell senescence induced by high glucose.METHODS: High glucose (33 mmol/L) was applied to induce senescence in primary human umbilical vein endothelial cells (HUVECs). The cell viability, the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells and the plasminogen activator inhibitor 1 (PAI-1) expression were detected to assess the senescence model. Mean while, Sirt1 siRNA was used to examine the effect of Sirt1 on eNOS expression and the senescence-related parameters.RESULTS: Treatment of HUVECs with high glucose decreased the cell viability slowly with a larger proportion of the cells stained with SA-β-Gal, and the protein expression of PAI-1 was dramatically increased. The increased cell viability, reduced SA-β-Gal positive cells and decreased protein expression of PAI-1 were detected after sodium hydrosulfide (NaHS, 100 μmol/L) treatment. Furthermore, NaHS treatment upregulated the protein expression of Sirt1 and eNOS, and eventually increased the production of nitric oxide (NO).CONCLUSION: Exogenous H₂S modulates Sirt1/eNOS/NO pathway to prevent HUVECs against high glucose-induced senescence.     

Effects of PPARβ and NO on high glucose and insulin-induced cardiomyocyte hypertrophy

AIM: To investigate the role of peroxisome proliferator-activated receptor β(PPARβ)-nitric oxide(NO) signal pathway in cardiomyocyte hypertrophy induced by high glucose(25.5 mmol/L) and insulin(0.1 μmol/L)(HGI). METHODS: The cardiomyocyte hypertrophy was characterized in rat primary cardiomyocytes by measuring the cell surface area, protein content, and the mRNA expression of atrial natriuretic factor(ANF). The mRNA and protein expression were measured by real-time PCR and Western blotting, respectively. The activity of NO synthase(NOS) and NO content were measured by a reagent kit through ultraviolet spectroscopy. RESULTS: HGI induced profound change of hypertrophic morphology, and significantly increased the cell surface area, protein content and mRNA expression of ANF(P<0.01), but decreased the expression of PPARβ at mRNA and protein levels(P<0.05). At the same time, the expression of inducible NOS(iNOS) was obviously elevated(P<0.01), which occurred in parallel with the rising NOS activity and NO concentration(P<0.01). GW0742(1 μmol/L), a selective PPARβ agonist, inhibited the cardiomyocyte hypertrophy induced by HGI(P<0.01), and up-regulated the expression of PPARβ at both mRNA and protein levels. Meanwhile, GW0742 also inhibited the increases in iNOS expression, NOS activity, and NO content induced by HGI, which were abolished by GSK0660(1 μmol/L), a selective PPARβ antagonist(P<0.01). CONCLUSION: PPARβ down-regulation and the following iNOS-NO activation are involved in the cardiomyocyte hypertrophy induced by HGI.     

Icariin attenuates high glucose-induced insulin resistance in C2C12 myotubes

AIM: To observe the potential effects of icariin on high glucose-induced insulin resistance in C2C12 myotubes and to investigate its underlying mechanisms. METHODS: The insulin resistance model was induced by high glucose (25 mmol/L) in the C2C12 myotubes. The effects of icariin on Akt phosphorylation at T308, glucose transporter 4 (GLUT4) membrane translocation, and glucose uptake were investigated in high glucose-treated C2C12 myotubes. The protein levels of phosphorylated proteins were determined by Western blot. The glucose uptake was measured by colorimetric method. The small interfering RNA (siRNA) was used to knockdown the expression of p38 MAPK. RESULTS: Icariin significantly increased insulin-stimulated Akt T308 phosphorylation in C2C12 myotubes treated with high glucose. Treatment with icariin at 25, 50 and 75 μmol/L for 24 h increased Akt T308 phosphorylation in a dose-dependent manner (P<0.05 or P<0.01). Treatment with icariin at 50 μmol/L for 12, 24 and 36 h increased Akt T308 phosphorylation in a time-dependent manner (P<0.05 or P<0.01). In addition, treatment with icariin at 50 μmol/L for 24 h significantly enhanced the expression of GLUT4 on plasma membrane (P<0.01) and 2-deoxyglucose (2-DG) uptake (P<0.01). Treatment with icariin recovered high glucose-reduced p38 MAPK phosphorylation (P<0.01). Pharmacological or genetic inhibition of p38 MAPK abolished the protective impacts of icariin on insulin-stimulated Akt T308 phosphorylation (P<0.01), GLUT4 plasma membrane translocation (P<0.01), and 2-DG uptake under high glucose condition (P<0.05). CONCLUSION: Icariin attenuates high glucose-induced insulin resistance in C2C12 myotubes by activating p38 MAPK.     

Mechanism of acylation stimulating protein resistance induced by fatty acid in 3T3-L1 adipocytes and preadipocytes

AIM: To evaluate the potential acylation stimulating protein (ASP) resistance in both adipocytes and preadipocytes under the conditions by which insulin resistance is produced by the stimulation of free fatty acids (FFA), and to explore the mechanism of ASP resistance on post-receptor level. METHODS: 3T3-L1 preadipocytes were induced to differentiate. Then the cells were treated with oleate or palmitate at concentration of 0 mmol/L (FFA-free DMEM/F12), 0.125 mmol/L, 0.5 mmol/L or 1.0 mmol/L overnight. Glucose transport was assessed by ［3H］ 2-deoxyglucose uptake to evaluate insulin resistance and ASP resistance. Both non-FFA treated and FFA treated 3T3-L1 cells were cultured with ASP at concentration of 5.0 μmol/L for 4 h, then the cell proteins were extracted, and the expressions of guanine nucleotide binding protein beta (Gβ), guanine nucleotide-binding protein alpha-q/11(Gαq/11), phosphorylated-protein kinase Cα (p-PKCα) and phosphorylated-protein kinase Cζ (p-PKCζ) were measured by Western blotting. RESULTS: Both adipocytes and preadipocytes were responsive to ASP. ASP stimulation increased glucose transport by 198% in adipocytes and by 287% in preadipocytes (P<0.01 vs PBS). FFA at concentration of 0.125 mmol/L did not change ASP-stimulated glucose transport significantly, but high dose of oleate or palmitate effectively reduced the ASP response with a significant reduction by 47% (P<0.05 for oleate) and 34% (P<0.05 for palmitate) at 1 mmol/L FFA in adipocytes. Similarly in preadipocytes, glucose uptake rates were decreased by 43% (P<0.05 for oleate) and 62% (P<0.01 for palmitate) at 1 mmol/L FFA. Effects were comparable to those obtained with insulin. After overnight incubation with oleate or palmitate in adipocytes and preadipocytes, Gβ, Gαq/11, p-PKCα and p-PKCζ were downregulated both in the absence of ASP treatment and in the presence of ASP treatment in adipocytes. At concentration of 1.0 mmol/L, oleate inhibited the expressions of ASP-induced Gβ, Gαq/11, p-PKCα and p-PKCζ in adipocytes by 47％, 44%, 39% (P<0.05, P<0.01) and 20% (P>0.05), respectively. Palmitate also effectively blocked the expressions of ASP (at concentration of 1.0 mmol/L)-induced Gβ, Gαq/11, p-PKCα and p-PKCζ by 50%, 43%, 44% and 43% (P<0.05, P<0.01) in adipocytes. In preadipocytes, oleate only inhibited ASP-induced p-PKCα and p-PKCζ significantly by 39% and 19%, respectively (P<0.05). However, overnight exposure of 3T3-L1 preadipocytes to 1 mmol/L palmitate leaded to 45%, 50%, 52% and 21% (P<0.05, P<0.01) inhibition of ASP-induced expressions of Gβ, Gαq/11, p-PKCα and p-PKCζ, respectively. CONCLUSION: Oleate and palmitate inhibit ASP-mediated stimulation of glucose transport both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance under the condition of insulin resistance induced by FFA in a cellular model. The mechanism of action involves both changes in expression of C5L2 as well as signaling parameters. Fatty acid-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and obesity phenotype.     

Effects of high-glucose on proliferation and apoptosis in endothelial progenitor cells of type 2 diabetes mellitus

AIM: This study aimed to observe the effects of high-glucose on proliferation and apoptosis of endothelial progenitor cells (EPCs) in type 2 diabetes mellitus patients,and tried to elucidate their possible role.METHODS: Various concentrations of glucose were added to the culture system of EPCs from 25 cases of type 2 diabetes mellitus patients (DM group) and 25 cases of healthy volunteers (control group).MTT assays were used to detect the proliferative rates.Annexin-V/PI stains were used to detect the apoptotic rates,and RT-PCR to detect the expression level of bcl-2 and bax.RESULTS: Proliferative activity of EPCs in both control group and DM group were attenuated when concentration of glucose was 33 mmol/L,while apoptotic rates increased.No significant change of proliferative rate and apoptotic rate of EPCs in DM group and control group in the presence of 5 mmol/L glucose was observed.The expression level of bax of EPCs in both DM group and control group increased while expression level of bcl-2 did not change much in the presence of 33 mmol/L glucose.CONCLUSION: High-glucose attenuates proliferative activity of EPCs and increases the apoptotic rate.Upregulation of bax may be its possible role.     

Effect of ubiquitin-proteasome pathway on expression of Smad7 in cultured glomerular mesangial cells induced by high concentration of glucose

AIM:To observe the expression of Smad7 and Smad ubiquition regulatory factor-Smurf2 in rat glomerular mesangial cells (GMC) stimulated by the high concentration of glucose, and to investigate the effect of the ubiquition on Smad signaling by adding MG132 as a proteasome differential inhibitor.METHODS:Cultured rat GMC were divided into normal group (the concentration of glucose:5.6 mmol/L), high glucose group (20 mmol/L, 30 mmol/L, respectively), therapy group (30 mmol/L glucose with MG132).The expressions of Smurf2 and Smad7 in each group were measured by indirect immunofluorescence and laser scanning confocal microscope.RESULTS:(1) The expression of Smurf2 in GMC in normal group was weak (25.93±3.35) whereas the expression of Smad7 was strong (64.09±7.43).(2) The expression of Smurf2 in high glucose group was stronger than that in normal group (P<0.05), in a concentration-dependent manner, 20 mmol/L high glucose (56.99±7.00), 30 mmol/L high glucose (96.36±9.19), respectively.The expression of Smad7 in high glucose group was weakened (P<0.05), 20 mmol/L high glucose (45.33±6.67), 30 mmol/L high glucose (30.20±4.41), respectively.(3) In therapy group, the expression of Smurf2 was found weakened and expression of Smad7 was enhanced.CONCLUSION:(1) High glucose increases the expression of Smurf2 and decreases the expression of Smad7 in glomerular mesangial cells.(2) Ubiquition-proteasome pathway (UPP) is related with the regulation of Smad signal transduction pathways in diabetic nephropathy.     

Role of asiatic acid in treatment of insulin resistance in mouse adipocytes

AIM：To investigate the effects of asiatic acid, one of triterpenoids from Psidium guajava leaves, on the proliferation and differentiation of 3T3-L1 preadipocytes, and glucose and lipid metabolism of insulin-resistant adipocytes. METHODS：The proliferation of 3T3-L1 preadipocytes was tested by MTT assay, and the accumulation of lipid droplets in differentiated preadipocytes was measured by oil red O staining. The insulin-resistant cell model was established by exposure of the cells to dexamethasone. The cellular glucose uptake was determined by glucose oxidase-peroxidase assay. The free fat acid (FFA) concentration was detected by colorimetric method. Secreted adiponectin were measured by ELISA. The protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and protein tyrosine phosphatase 1B (PTP1B) in insulin-resistant adipocytes were analyzed by Western blotting. RESULTS：Compared with medium group, asiatic acid increased the proliferation of 3T3-L1 preadipocytes and inhibited their differentiation at a concentration range of 10~100 μmol/L (P<0.05 or P<0.01). At concentrations of 30 μmol/L and 100 μmol/L, asiatic acid enhanced cellular glucose uptake in the insulin-resistant adipocytes both in basic and insulin-stimulation states. Asiatic acid decreased FFA production (P<0.05), and down-regulated the protein expression of PTP1B (P<0.05, or P<0.01). However, no effect on the secretion of adiponectin and the protein expression of PPARγ was observed (P>0.05). CONCLUSION：Asiatic acid enhances glucose uptake and inhibits FFA production in insulin-resistant adipocytes via down-regulating the protein expression of PTP1B, all of which play the roles of increasing insulin signaling sensitivity to improve insulin resistance.     

CD137 signaling molecules promote proliferation of pulmonary artery endothelial cells by aerobic glycolysis

AIM:To investigate whether CD137 signaling molecules promote the proliferation of pulmonary artery endothelial cells (PAECs) by aerobic glycolysis. METHODS:The experiments of mouse PAECs were performed as follows. (1) Stimulating factors TNF-α (10 μg/L), ET-1 (10 mmol/L) and 5-HT (1 μmol/L) were used to stimulate the cells for 24 h. (2) After stimulation with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group (treatment with 5 mg/L CD137L recombinant protein to activate CD137-CD137L signaling), c-Myc inhibitor group (pretreatment with 10 μmol/L c-Myc inhibitor 10074-G5, dissolved in DMSO, for 30 min, followed by treatment with 5 mg/L CD137L recombinant protein) and DMSO group (pretreated with DMSO at the same volume to c-Myc inhibitor group for 30 min followed by CD137L recombinant protein treatment). (3) After stimulated with TNF-α for 24 h, the cells were divided into control group, CD137 agonist group and 2-deoxyglucose (2-DG) group (pretreatment with 10 mmol/L glycolysis inhibitor 2-DG for 30 min followed by CD137L recombinant protein treatment).The expression of membrane protein and total protein of CD137 in the PAECs was detected by flow cytometry and Western blot, respectively. The protein levels of glycolytic enzymes such as hexokinase (HK2), 6-phosphofructo-2-kinase/fructose-2,6-diphosphatase 3 (PFKFB3) and c-Myc were measured by Western blot. The enzyme activity of HK2 and PFKFB3 was detected by HK2 kit and PFK kit, respectively. Glucose oxidase method was used to measure the glucose uptake rate, and lactate colorimetric assay was conducted for analyzing lactic acid production. CCK-8 assay and EdU staining were used to detect proliferation of the PAECs. RESULTS:Compared with control group, TNF-α, ET-1 and 5-HT significantly increased the expression of CD137 membrane protein and total protein in the PAECs (P<0.05). The protein levels and enzyme activity of HK2 and PFKFB3 protein in CD137 agonist group were significantly higher than those in control group (P<0.05). Compared with control group, the lactic acid production and glucose consumption in CD137 agonist group were significantly increased. The protein level of c-Myc was significantly higher than that in control group after stimulation with CD137L recombinant protein, while c-Myc inhibitor 10074-G5 significantly inhibited the promoting effect of CD137L recombinant protein on glycolysis (P<0.05). The results of CCK-8 assay and EdU staining showed that the cell proliferation in CD137 agonist group was significantly increased compared with control group, while glycolysis inhibitor 2-DG significantly inhibited the proliferation-enhancing effect of CD137 signaling activation on the cells (P<0.05). CONCLUSION:CD137 signaling molecules may modulate the aerobic glycolysis by up-regulating c-Myc, thus promoting the proliferation of mouse PAECs.     

Protective effect of anti-aging Klotho protein on human umbilical vein endothelial cells treated with high glucose

AIM: To study the protective effect of anti-aging Klotho protein on human umbilical vein endothelial cells (HUVECs) treated with high glucose (HG).METHODS: HUVECs were cultured in vitro, and divided into PBS control group, 5.5 mmol/L glucose group, 33.3 mmol/L glucose group, 0.1 μmol/L Klotho+33.3 mmol/L glucose group, 1 μmol/L Klotho+33.3 mmol/L glucose group, and 10 μmol/L Klotho+33.3 mmol/L glucose group. The viability of the HUVECs was measured by MTT assay. The content of malondialdehyde (MDA), and the activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione (GSH) in cell culture supernatants were observed. The production of reactive oxygen species (ROS) in HUVECs was analyzed by flow cytometry. The levels of nitric oxide (NO), endothelin (ET-1), intercellular adhesion molecule-1 (ICAM-1) in HUVEC culture medium were detected by ELISA. The protein expression of nuclear factor-kappa B (NF-κB) in the HUVECs was determined by Western blot. RESULTS: Compared with PBS control group, 33.3 mmol/L glucose significantly decreased the HUVEC viability, increased ROS, LDH and MDA levels, reduced the activities of SOD and GSH, decreased the NO secretion, and induced the ET-1 and ICAM-1 secretion and the protein expression of NF-κB in HUVECs. When HUVECs were treated with Klotho protein at different concentrations combined with 33.3 mmol/L glucose, the cell viability was increased significantly, the ROS, LDH and MDA levels were decreased significantly, the antioxidant SOD and GSH activities were significantly increased, the secretion of NO was increased, but ET-1 and ICAM-1 releases and protein expression of NF-κB were significantly reduced.CONCLUSION: Anti-aging Klotho protein promotes the viability of HUVECs treated with HG, reduces the oxidative damage and ROS production, and restores the normal secretory function of HUVECs, thus playing a protective role in vascular endothelial cells through reducing the protein expression of NF-κB.     

Ubiquitin-dependent degradation of SnoN/Ski protein in glomerular mesangial cells induced by high glucose

AIM: To observe the protein expression of SnoN/Ski and ubiqutin ligase Arkadia in rat glomerular mesangial cells (GMCs) exposed to the high glucose. METHODS: Cultured rat glomerular mesangial HBZY-1 cells were divided into control group, 20 mmol/L glucose group, 30 mmol/L glucose group, 30 mmol/L glucose+MG132 group (culture medium containing 30 mmol/L glucose and 0.5 μmol/L specific proteasome inhibitor MG132), and mannitol group. The expression levels of SnoN, Ski and Arkadia were measured by Western blotting analysis, immunofluorescence and laser scanning confocal microscopy. RESULTS: In control GMCs, the expression of SnoN/Ski was rich and Arkadia was weak. After stimulated with high glucose, the expression of SnoN/Ski was decreased and Arkadia was gradually increased (P<0.05). Compared with high glucose group, the levels of SnoN/Ski and Arkadia were mostly reverted by adding the proteasome inhibitor MG132 at concentration of 0.5 μmol/L (P<0.01). The expression levels of SnoN/Ski and Arkadia were not significantly changed in mannitol group in comparison with control group (P>0.05). CONCLUSION: High glucose decreases the expression of SnoN/Ski through ubiquitin-dependent degradation of SnoN/Ski. The degradation of SnoN/Ski mediated by Arkadia may play an important role in the pathogenesis of diabetic nephropathy.