To study whether the diacylglycerol (Dia) signaling pathway is stimulated by advanced glycosylation end products (AGEP) and to test the effect of vitamin E and aminoguanidine (AG) on the elevation of Dia induced by AG...To study whether the diacylglycerol (Dia) signaling pathway is stimulated by advanced glycosylation end products (AGEP) and to test the effect of vitamin E and aminoguanidine (AG) on the elevation of Dia induced by AGEP in cultured human umbilical vein endothelial cells (HUVECs) Methods The effects of AGEP on Dia levels in cultured HUVEC were studied with radio enzymatic assay Quantitative measurements of 32 P phosphatidic acid were achieved by thin layer chromatography and autoradiography Results The Dia levels in HUVECs were increased by AGEP modified bovine serum albumin (AGEP BSA) in a dose dependent, biphasic manner The early phase was rapid and transient, peaking at 15?s; the late phase reached the maximal level at 10?min and then decayed slowly Dia levels in HUVEC exposed to different concentrations (50, 100 and 200?mg/L) of AGEP BSA (341±14, 678±16, and 873±18?pmol/L, respectively vs control 225±10?pmol/L) and AGEP BSA samples with various glycosylation times (4, 8 and 12 weeks) were significantly increased (270±12, 394±16, and 556±19?pmol/L) as compared with the controls 50 and 100?mmol/L of vitamin E can reduce AGEP BSA induced Dia levels from 873±18?pmol/L to 764±29 and 441±21?pmol/L in HUVEC, respectively In AG treated (100?mmol/L) groups, the same concentration (100 and 200?mg/L) of AGEP BSA induced elevation of Dia was decreased to 312±8 and 351±13?pmol/L, respectively Glycosylated low density lipoprotein (LDL) did not affect Dia levels Conclusion AGEP causes a robust stimulation of the Dia/protein kinase C pathway in HUVEC Vitamin E can attenuate the AGEP BSA induced elevation of Dia levels AG can suppress the ability of AGEP BSA to increase Dia levels in HUVEC展开更多
Objective To determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in...Objective To determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the presence or absence of inflammatory mediators.Methods Cultured human umbilical vein endothelial cells (HUVECs) were exposed to AGEs-BSA for 6, 12, 24, and 36 hours, and exposed to AGEs-BSA glycosylated with different concentrations of glucose, tumor necrosis factord-α (TNF-α), interferon (IFN-γ), TNF-α + IFN-y and AGEs-BSA + TNF-α for 24 hours, respectively. Expression of PECAM-1 mRNA was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with β-actin as an internal standard, and sequencing of RT-PCR products was performed to confirm the specificity of amplification for PECAM-1 gene. The endothelial cell surface expression of PECAM-1 was determined by flow cytometry (FCM).Results There were no significant changes in the expression of PECAM-1 mRNA and protein when the cells were exposed to AGEs-BSA with different concentrations or periods ( P>0. 05). However, PECAM-1 expression was reduced in the cells treated with TNF-α, IFN-y, TNF-α + IFN-γ and AGEs-BSA + TNF-α. The level of PECAM-1 treated with AGEs-BSA + TNF-α was lower than that of TNF-α treated alone (P<0. 01).Conclusions AGEs-BSA had no effect on the expression of PECAM-1 mRNA and protein in cultured HUVEC. With the presence of inflammatory mediator TNF-α, AGEs-BSA decreased the level of PECAM-1, which might reduce the adhesion interaction between adjacent endothelial cells, enhance the permeability of endothelial cells, and might be implicated in the endothelial dysfunction and pathogenesis of atherosclerosis in patients with diabetes mellitus. The significance of this phenomenon in intracellular signal transduction remains to be determined.展开更多
To investigate the effect of advanced glycosylation end products (AGEs) on the activity of protein kinase C (PKC) in human peripheral blood mononuclear cells (PBMC) and to observe whether aminoguanidine (AG) can influ...To investigate the effect of advanced glycosylation end products (AGEs) on the activity of protein kinase C (PKC) in human peripheral blood mononuclear cells (PBMC) and to observe whether aminoguanidine (AG) can influence the effect of AGEs Methods After PBMC were isolated from human peripheral blood and incubated with different concentrations of AGEs BSA for various periods, total PKC activity in PBMC was determined by measuring the incorporation of 32 P from [γ 32 P] ATP into a special substrate using Promega PKC assay kit Results AGEs BSA increased the total PKC activity in PBMC from 83 43±6 57?pmol/min/mg protein to 116 8±13 82?pmol/min/mg protein with a peak at 15?min AGEs BSA also increased the total PKC activity in a concentration dependent manner from 83 1±6 4?pmol/min/mg protein (control) to 119 1±13 3?pmol/min/mg protein (control vs AGEs BSA 400?mg/L, P <0 01) Furthermore, AGEs BSA induced an elevation of PKC activity in a glycosylating time related manner, from 80 9±8 2 (control) to 118 3±11 5?pmol/min/mg protein (glycosylation for 12 wk, P <0 01) The total PKC activity stimulated by AGEs BSA pretreated with AG (100, 200?mg/L) was markedly lower than that of AGEs BSA group not pretreated with AG ( P <0 05, P <0 01) Conclusions AGEs BSA increased the total PKC activity in PBMC in a concentration and incubation time dependent manner The ability of AGEs BSA to stimulate PKC activity was markedly decreased by pretreatment of AGEs BSA with AG展开更多
Background Galectin-3 is the most recently identified advanced glycosylation end products （AGEs） binding protein. This study aimed to investigate the effects of AGEs and rosiglitazone on the expression and secretion...Background Galectin-3 is the most recently identified advanced glycosylation end products （AGEs） binding protein. This study aimed to investigate the effects of AGEs and rosiglitazone on the expression and secretion of galectin-3 in cultured human renal mesangial cells （HRMCs）. Methods HRMCs were incubated with different concentrations of AGE-bovine serum albumin （BSA） （0, 50, 100, 200, and 400 mg/L） for different time （0, 24, 36, 48, and 72 hours）, and exposed to AGE-BSA in the presence of different concentrations of rosiglitazone （1, 10, and 100 μmol/L）. The mRNA and protein expression of galectin-3 in HRMCs were analyzed by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting. The culture medium of HRMCs was collected and concentrated, and the content of galectin-3 in the medium was detected by Western blotting. Results Both RT-PCR and Western blotting revealed that AGE-BSA up-regulated the expression of galectin-3 in HRMCs in a concentration- （P 〈0.05） and time-dependent （P 〈0.05） manner compared with the control. Compared with the control, AGE-BSA elevated the content of galectin-3 in the culture medium of HRMCs time- and concentrationdependently （P 〈0.05, respectively）. Both protein and mRNA expression of galectin-3, and its content in the medium of HRMCs exposed to different concentrations of rosiglitazone in the presence of AGE-BSA were increased compared with those of cells exposed to AGE-BSA alone （P 〈0.05）. Rosiglitazone increased the expression and secretion of galectin-3 in a dose-dependent manner （P 〈0.05）. Conclusions AGEs up-regulates the expression and secretion of galectin-3 in HRMCs. Rosiglitazone further enhances the upregulation of galectin-3 in HRMCs induced by AGEs, which suggests that rosiglitazone may play a role of reno-protection via up-regulation of galectin-3.展开更多
To study the relationship between advanced glycosylation end products (AGE) and protein kinase C (PKC), and their effects on renal alteration in diabetic rats Methods Insulin or aminoguanidine was administered to di...To study the relationship between advanced glycosylation end products (AGE) and protein kinase C (PKC), and their effects on renal alteration in diabetic rats Methods Insulin or aminoguanidine was administered to diabetic rats Blood glucose, hemoglobin A 1C (HbA 1C ), glomerular tissue extracts AGE (GTE AGE), PKC, glomerular basement membrane thickness (GBMT) and urine protein/creatinine (Pr/Cr) ratio in diabetic rats were measured and analysed Results Levels of blood glucose, HbA 1C and AGE, PKC activity, the Pr/Cr ratio and GBMT were all significantly increased ( P values all less than 0 01) in diabetic rats Insulin could decrease the formation of HbA 1C and AGE, and improve PKC activity Aminoguanidine had no influence on PKC activity ( P >0 05) although it decreased the formation of AGE Both drugs could delay the increase of urine Pr/Cr ratio and GBMT ( P <0 05 or P <0 01) Conclusions Chronic hyperglycemia may lead to an increase of PKC activity HbA 1C and AGE may not directly contribute to alterations of PKC activity, but the increase of PKC activity could promote the action of AGE on GBM thickening It is important to inhibit the formation of AGE and reduce the PKC activity so as to prevent or delay the development of diabetic nephropathy展开更多
文摘To study whether the diacylglycerol (Dia) signaling pathway is stimulated by advanced glycosylation end products (AGEP) and to test the effect of vitamin E and aminoguanidine (AG) on the elevation of Dia induced by AGEP in cultured human umbilical vein endothelial cells (HUVECs) Methods The effects of AGEP on Dia levels in cultured HUVEC were studied with radio enzymatic assay Quantitative measurements of 32 P phosphatidic acid were achieved by thin layer chromatography and autoradiography Results The Dia levels in HUVECs were increased by AGEP modified bovine serum albumin (AGEP BSA) in a dose dependent, biphasic manner The early phase was rapid and transient, peaking at 15?s; the late phase reached the maximal level at 10?min and then decayed slowly Dia levels in HUVEC exposed to different concentrations (50, 100 and 200?mg/L) of AGEP BSA (341±14, 678±16, and 873±18?pmol/L, respectively vs control 225±10?pmol/L) and AGEP BSA samples with various glycosylation times (4, 8 and 12 weeks) were significantly increased (270±12, 394±16, and 556±19?pmol/L) as compared with the controls 50 and 100?mmol/L of vitamin E can reduce AGEP BSA induced Dia levels from 873±18?pmol/L to 764±29 and 441±21?pmol/L in HUVEC, respectively In AG treated (100?mmol/L) groups, the same concentration (100 and 200?mg/L) of AGEP BSA induced elevation of Dia was decreased to 312±8 and 351±13?pmol/L, respectively Glycosylated low density lipoprotein (LDL) did not affect Dia levels Conclusion AGEP causes a robust stimulation of the Dia/protein kinase C pathway in HUVEC Vitamin E can attenuate the AGEP BSA induced elevation of Dia levels AG can suppress the ability of AGEP BSA to increase Dia levels in HUVEC
基金This study was supported by the grants from the Jiangsu Technologic Foundation (No. BJ98324).
文摘Objective To determine whether advanced glycosylation end products modified bovine serum albumin (AGEs-BSA) affects endothelial cell lateral junction protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the presence or absence of inflammatory mediators.Methods Cultured human umbilical vein endothelial cells (HUVECs) were exposed to AGEs-BSA for 6, 12, 24, and 36 hours, and exposed to AGEs-BSA glycosylated with different concentrations of glucose, tumor necrosis factord-α (TNF-α), interferon (IFN-γ), TNF-α + IFN-y and AGEs-BSA + TNF-α for 24 hours, respectively. Expression of PECAM-1 mRNA was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) with β-actin as an internal standard, and sequencing of RT-PCR products was performed to confirm the specificity of amplification for PECAM-1 gene. The endothelial cell surface expression of PECAM-1 was determined by flow cytometry (FCM).Results There were no significant changes in the expression of PECAM-1 mRNA and protein when the cells were exposed to AGEs-BSA with different concentrations or periods ( P>0. 05). However, PECAM-1 expression was reduced in the cells treated with TNF-α, IFN-y, TNF-α + IFN-γ and AGEs-BSA + TNF-α. The level of PECAM-1 treated with AGEs-BSA + TNF-α was lower than that of TNF-α treated alone (P<0. 01).Conclusions AGEs-BSA had no effect on the expression of PECAM-1 mRNA and protein in cultured HUVEC. With the presence of inflammatory mediator TNF-α, AGEs-BSA decreased the level of PECAM-1, which might reduce the adhesion interaction between adjacent endothelial cells, enhance the permeability of endothelial cells, and might be implicated in the endothelial dysfunction and pathogenesis of atherosclerosis in patients with diabetes mellitus. The significance of this phenomenon in intracellular signal transduction remains to be determined.
基金ThisprojectwassupportedbytheNationalNaturalScience FoundationofChina (No .39470 314 )
文摘To investigate the effect of advanced glycosylation end products (AGEs) on the activity of protein kinase C (PKC) in human peripheral blood mononuclear cells (PBMC) and to observe whether aminoguanidine (AG) can influence the effect of AGEs Methods After PBMC were isolated from human peripheral blood and incubated with different concentrations of AGEs BSA for various periods, total PKC activity in PBMC was determined by measuring the incorporation of 32 P from [γ 32 P] ATP into a special substrate using Promega PKC assay kit Results AGEs BSA increased the total PKC activity in PBMC from 83 43±6 57?pmol/min/mg protein to 116 8±13 82?pmol/min/mg protein with a peak at 15?min AGEs BSA also increased the total PKC activity in a concentration dependent manner from 83 1±6 4?pmol/min/mg protein (control) to 119 1±13 3?pmol/min/mg protein (control vs AGEs BSA 400?mg/L, P <0 01) Furthermore, AGEs BSA induced an elevation of PKC activity in a glycosylating time related manner, from 80 9±8 2 (control) to 118 3±11 5?pmol/min/mg protein (glycosylation for 12 wk, P <0 01) The total PKC activity stimulated by AGEs BSA pretreated with AG (100, 200?mg/L) was markedly lower than that of AGEs BSA group not pretreated with AG ( P <0 05, P <0 01) Conclusions AGEs BSA increased the total PKC activity in PBMC in a concentration and incubation time dependent manner The ability of AGEs BSA to stimulate PKC activity was markedly decreased by pretreatment of AGEs BSA with AG
文摘Background Galectin-3 is the most recently identified advanced glycosylation end products （AGEs） binding protein. This study aimed to investigate the effects of AGEs and rosiglitazone on the expression and secretion of galectin-3 in cultured human renal mesangial cells （HRMCs）. Methods HRMCs were incubated with different concentrations of AGE-bovine serum albumin （BSA） （0, 50, 100, 200, and 400 mg/L） for different time （0, 24, 36, 48, and 72 hours）, and exposed to AGE-BSA in the presence of different concentrations of rosiglitazone （1, 10, and 100 μmol/L）. The mRNA and protein expression of galectin-3 in HRMCs were analyzed by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting. The culture medium of HRMCs was collected and concentrated, and the content of galectin-3 in the medium was detected by Western blotting. Results Both RT-PCR and Western blotting revealed that AGE-BSA up-regulated the expression of galectin-3 in HRMCs in a concentration- （P 〈0.05） and time-dependent （P 〈0.05） manner compared with the control. Compared with the control, AGE-BSA elevated the content of galectin-3 in the culture medium of HRMCs time- and concentrationdependently （P 〈0.05, respectively）. Both protein and mRNA expression of galectin-3, and its content in the medium of HRMCs exposed to different concentrations of rosiglitazone in the presence of AGE-BSA were increased compared with those of cells exposed to AGE-BSA alone （P 〈0.05）. Rosiglitazone increased the expression and secretion of galectin-3 in a dose-dependent manner （P 〈0.05）. Conclusions AGEs up-regulates the expression and secretion of galectin-3 in HRMCs. Rosiglitazone further enhances the upregulation of galectin-3 in HRMCs induced by AGEs, which suggests that rosiglitazone may play a role of reno-protection via up-regulation of galectin-3.
文摘To study the relationship between advanced glycosylation end products (AGE) and protein kinase C (PKC), and their effects on renal alteration in diabetic rats Methods Insulin or aminoguanidine was administered to diabetic rats Blood glucose, hemoglobin A 1C (HbA 1C ), glomerular tissue extracts AGE (GTE AGE), PKC, glomerular basement membrane thickness (GBMT) and urine protein/creatinine (Pr/Cr) ratio in diabetic rats were measured and analysed Results Levels of blood glucose, HbA 1C and AGE, PKC activity, the Pr/Cr ratio and GBMT were all significantly increased ( P values all less than 0 01) in diabetic rats Insulin could decrease the formation of HbA 1C and AGE, and improve PKC activity Aminoguanidine had no influence on PKC activity ( P >0 05) although it decreased the formation of AGE Both drugs could delay the increase of urine Pr/Cr ratio and GBMT ( P <0 05 or P <0 01) Conclusions Chronic hyperglycemia may lead to an increase of PKC activity HbA 1C and AGE may not directly contribute to alterations of PKC activity, but the increase of PKC activity could promote the action of AGE on GBM thickening It is important to inhibit the formation of AGE and reduce the PKC activity so as to prevent or delay the development of diabetic nephropathy
