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Insulin inhibits the expression of ICAM-1 by human aortic endothelial cells through stimulation of nitric oxide
Track 4. Basic Research
one, and two weeks. Result of TRAP assays indicated no change in telomerase activity in cells grown for three days in high glucose medium (105fl2Z of control). Cells grown in high glucose medium for one week and two weeks showed increased telomerase activity (145f37% of control, 128+20% of control, respectively). The corresponding cell counts at 3 day, 1 week, and 2 week were 109fll% of control, 80flO% of control, and 75f12% of control, respectively. This is the first report on the effect of high glucose on telomerase activity. The results from this study indicate that reduced proliferation of endothelial cells observed under high glucose condition may be in part due to altered telomerase activity.
P1451
Involvement of Iron in the High Glucose Toxicity to Cultured Endothelial Cells KAYOKO SASAKI I**,Kanae Hashida3, Shiro Bannai ‘, Nobuo Makino3. ’ Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; 2 Research Fellowship division, Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo, Japan; 3 Center for Humanity and Sciences, Ibaraki Prefectural University of Health Sciences, Ami, Ibaraki, Japan
Oxidative stress is an important factor in the diabetic angiopathy caused by hyperglycemia, and transition metals such as iron and copper mediate the injury. In ex viva studies, high glucose (HG) is generally toxic to cells, but it is known that subcultured human umbilical vein cells (HUVEC) are rather insensitive to HG. We hypothesized that the susceptibility to hyperglycemia depends on the cellular iron and have carried out the iron quantitation and loading experiments on cultured HUVEC. Iron contents of the cells at population doubling levels (PDL) of 0 to 15 were determined, and HWEC were found to lose iron rapidly during culture. Iron loading to the cells using Fe(III)/8-hydroxyquinoline(8HQ) increased the cellular iron content linearly to Fe/8HQ concentration in the loading medium. When 0.1 @M Fe/8HQ was used for loading, the iron content of the cells at PDL 15 increased about 10%. Under the conditions, the cytotoxicity of Fe itself was negligible, whereas the susceptibility to HaOr showed a significant increase. Although growth of the cells at PDL 15 was only slightly decreased by HG (22% by 44 mM glucose medium in 48 h), the growth of the Fe-loaded cells in HG medium (Fe/HG cells) showed a considerable decrease (by 75%). Furthermore, lipid peroxides content of the Fe/HG cells increased significantly, and growth inhibition on the Fe/I-IG cells was recovered by ascorbic acid, catalase, aldose reductase inhibitor or pyruvate. It was concluded that iron (most possibly free or loosely bound iron) plays an important role in the cellular hyperglycemia susceptibility, probably through the enhancement of oxidative stress. It is expected that the finding will offer a new idea and useful tool for the study of diabetic complications.
P1452
Insulin Inhibits the Expression of ICAM- by Human Aortic Endothelial Cells through Sthnulation of Nitric Oxide AHMAD ALIADA, Rana Saadeh, Ezzat Assian. Diabetes-Endocrinology Center of Western New York, Millard Fillmore Hospital, State University of New York, Buffalo, NZ United States of America
Intercellular adhesion molecule-l @CAM-l) is expressed by various cell types in human atherosclerotic lesions. It serves as a ligand for LFA- 1 on leucocytes and is partially responsible for the adhesion of lymphocytes, granulocytes and monocytes to cytokine stimulated endothelial cells and the subsequent transendothelial migration. The data from this research demonstrate clearly that insulin decreases the expression of ICAM- by human aortic endothelial cells (HABC) in a dose-dependent manner after incubation for 2 days. This decrease is associated with a concomitant increase in endothelial nitric oxide synthase (e-NOS) expression also
S369
induced by insulin. Furthermore, this effect appears to be mediated through nitric oxide (NO) release. N O-Nitro-L-Arginine (L-NNA), a NOS inhibitor, does indeed inhibit insulin-induced decrease in ICAMexpression in HAEC on the mRNA and protein levels. The inhibitory effect of insulin on ICAM-I expression, allied with its potent vasodilatory effect and anti-platelet effect are all potentially anti-atherogenic. These observations further add to the concept that insulin is probably not the mediator of atherosclerosis in insulin resistant states.
P1453
Nitric Oxide Synthase Inhibition Disappears the Training Effect on Insulin Action Y. OSHIDA, Y.Q. Han, N. Fuku, K. Kitakoshi, Y. Sato. Nagoya University, Nagoya, Japan
Recent studies have demonstrated that nitric oxide (NO) involves the regulation of hemodynamics and contributes to exercise- and insulin-mediated glucose uptake in skeletal muscle. The purpose of this study was to investigate whether NO synthase inhibitioninfluences the improved insulin action by voluntary running. NO synthase inhibitor, No-monomethel-L-arginine (LNMMA, lmg/kg/min), or saline was infused during the sequential euglycemic clamp procedure in sedentary control and voluntary running rata. Plasma insulin levels during the 3- and 30-mIJ/kg/min insulin infusion were 30-40 and 300-400~ U/ml, respectively and blood glucose was clamped at a fasting level by adjustment of i.v. glucose infusion. Glucose disposal rate (GDR) was regarded as an index of whole body insulin action. At the 3-mU/kg/min insulin infusion, voluntary running showed a significant increase in GDR compared with the sedentary controls given saline (23.9f0.6 vs 18.5fl.lmg/kg/min, p
P1454
Plasma Homocysteine Concentrations in CAD in Asian Indians C. SNEHALATHA, A. Ramachandran, K. Satyavani, S. Sivasankari, Vijay Viswanathan. Diabetes Research Centre, Madras, India Aim: To study the association of fasting plasma homocysteine with coronary artery disease (CAD) in Asian Indians. Subjects and Methods: Fasting concentrations of plasma total homocysteine (Hey) was measured in 137 men, aged ~25 years who underwent coronary angiography while investigating for chest pain. Among them 71 had no significant CAD (mean age 49.2 f 9.5 years and mean BMI 24.8f3.5 kg/m*) and 66 had CAD (mean age 57.8f8.1 years, mean BMI 24.8f3.3 kg/m’) (Pt0.001). Hey was measured by Elisa kit (Axis Biochemicals - Oslo, Norway). Among the total group, there were 32 non diabetic, non CAD, 31 non diabetic CAD, 39 diabetic non CAD and 35 diabetic CAD. Results: The geometric mean values of Hey in non CAD and CAD subjects were 12.4f2.4 (SD) and 14.3f1.8 (SD) u mohl, respectively; the difference was statistically non significant. The mean values for Hey in non diabetic non CAD, non diabetic CAD, diabetic non CAD and diabetic CAD were 15.2, 15.2, 10.5 and 13.5 u mol/l respectively. There was no significant intra group difference. Conclusion: The data showed no association between plasma Hey and CAD either in the diabetic or the non diabetic study subjects. The presence of a high Hey concentration in the non diabetic, non CAD control group
one, and two weeks. Result of TRAP assays indicated no change in telomerase activity in cells grown for three days in high glucose medium (105fl2Z of control). Cells grown in high glucose medium for one week and two weeks showed increased telomerase activity (145f37% of control, 128+20% of control, respectively). The corresponding cell counts at 3 day, 1 week, and 2 week were 109fll% of control, 80flO% of control, and 75f12% of control, respectively. This is the first report on the effect of high glucose on telomerase activity. The results from this study indicate that reduced proliferation of endothelial cells observed under high glucose condition may be in part due to altered telomerase activity.
P1451
Involvement of Iron in the High Glucose Toxicity to Cultured Endothelial Cells KAYOKO SASAKI I**,Kanae Hashida3, Shiro Bannai ‘, Nobuo Makino3. ’ Department of Biochemistry, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; 2 Research Fellowship division, Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo, Japan; 3 Center for Humanity and Sciences, Ibaraki Prefectural University of Health Sciences, Ami, Ibaraki, Japan
Oxidative stress is an important factor in the diabetic angiopathy caused by hyperglycemia, and transition metals such as iron and copper mediate the injury. In ex viva studies, high glucose (HG) is generally toxic to cells, but it is known that subcultured human umbilical vein cells (HUVEC) are rather insensitive to HG. We hypothesized that the susceptibility to hyperglycemia depends on the cellular iron and have carried out the iron quantitation and loading experiments on cultured HUVEC. Iron contents of the cells at population doubling levels (PDL) of 0 to 15 were determined, and HWEC were found to lose iron rapidly during culture. Iron loading to the cells using Fe(III)/8-hydroxyquinoline(8HQ) increased the cellular iron content linearly to Fe/8HQ concentration in the loading medium. When 0.1 @M Fe/8HQ was used for loading, the iron content of the cells at PDL 15 increased about 10%. Under the conditions, the cytotoxicity of Fe itself was negligible, whereas the susceptibility to HaOr showed a significant increase. Although growth of the cells at PDL 15 was only slightly decreased by HG (22% by 44 mM glucose medium in 48 h), the growth of the Fe-loaded cells in HG medium (Fe/HG cells) showed a considerable decrease (by 75%). Furthermore, lipid peroxides content of the Fe/HG cells increased significantly, and growth inhibition on the Fe/I-IG cells was recovered by ascorbic acid, catalase, aldose reductase inhibitor or pyruvate. It was concluded that iron (most possibly free or loosely bound iron) plays an important role in the cellular hyperglycemia susceptibility, probably through the enhancement of oxidative stress. It is expected that the finding will offer a new idea and useful tool for the study of diabetic complications.
P1452
Insulin Inhibits the Expression of ICAM- by Human Aortic Endothelial Cells through Sthnulation of Nitric Oxide AHMAD ALIADA, Rana Saadeh, Ezzat Assian. Diabetes-Endocrinology Center of Western New York, Millard Fillmore Hospital, State University of New York, Buffalo, NZ United States of America
Intercellular adhesion molecule-l @CAM-l) is expressed by various cell types in human atherosclerotic lesions. It serves as a ligand for LFA- 1 on leucocytes and is partially responsible for the adhesion of lymphocytes, granulocytes and monocytes to cytokine stimulated endothelial cells and the subsequent transendothelial migration. The data from this research demonstrate clearly that insulin decreases the expression of ICAM- by human aortic endothelial cells (HABC) in a dose-dependent manner after incubation for 2 days. This decrease is associated with a concomitant increase in endothelial nitric oxide synthase (e-NOS) expression also
S369
induced by insulin. Furthermore, this effect appears to be mediated through nitric oxide (NO) release. N O-Nitro-L-Arginine (L-NNA), a NOS inhibitor, does indeed inhibit insulin-induced decrease in ICAMexpression in HAEC on the mRNA and protein levels. The inhibitory effect of insulin on ICAM-I expression, allied with its potent vasodilatory effect and anti-platelet effect are all potentially anti-atherogenic. These observations further add to the concept that insulin is probably not the mediator of atherosclerosis in insulin resistant states.
P1453
Nitric Oxide Synthase Inhibition Disappears the Training Effect on Insulin Action Y. OSHIDA, Y.Q. Han, N. Fuku, K. Kitakoshi, Y. Sato. Nagoya University, Nagoya, Japan
Recent studies have demonstrated that nitric oxide (NO) involves the regulation of hemodynamics and contributes to exercise- and insulin-mediated glucose uptake in skeletal muscle. The purpose of this study was to investigate whether NO synthase inhibitioninfluences the improved insulin action by voluntary running. NO synthase inhibitor, No-monomethel-L-arginine (LNMMA, lmg/kg/min), or saline was infused during the sequential euglycemic clamp procedure in sedentary control and voluntary running rata. Plasma insulin levels during the 3- and 30-mIJ/kg/min insulin infusion were 30-40 and 300-400~ U/ml, respectively and blood glucose was clamped at a fasting level by adjustment of i.v. glucose infusion. Glucose disposal rate (GDR) was regarded as an index of whole body insulin action. At the 3-mU/kg/min insulin infusion, voluntary running showed a significant increase in GDR compared with the sedentary controls given saline (23.9f0.6 vs 18.5fl.lmg/kg/min, p
P1454
Plasma Homocysteine Concentrations in CAD in Asian Indians C. SNEHALATHA, A. Ramachandran, K. Satyavani, S. Sivasankari, Vijay Viswanathan. Diabetes Research Centre, Madras, India Aim: To study the association of fasting plasma homocysteine with coronary artery disease (CAD) in Asian Indians. Subjects and Methods: Fasting concentrations of plasma total homocysteine (Hey) was measured in 137 men, aged ~25 years who underwent coronary angiography while investigating for chest pain. Among them 71 had no significant CAD (mean age 49.2 f 9.5 years and mean BMI 24.8f3.5 kg/m*) and 66 had CAD (mean age 57.8f8.1 years, mean BMI 24.8f3.3 kg/m’) (Pt0.001). Hey was measured by Elisa kit (Axis Biochemicals - Oslo, Norway). Among the total group, there were 32 non diabetic, non CAD, 31 non diabetic CAD, 39 diabetic non CAD and 35 diabetic CAD. Results: The geometric mean values of Hey in non CAD and CAD subjects were 12.4f2.4 (SD) and 14.3f1.8 (SD) u mohl, respectively; the difference was statistically non significant. The mean values for Hey in non diabetic non CAD, non diabetic CAD, diabetic non CAD and diabetic CAD were 15.2, 15.2, 10.5 and 13.5 u mol/l respectively. There was no significant intra group difference. Conclusion: The data showed no association between plasma Hey and CAD either in the diabetic or the non diabetic study subjects. The presence of a high Hey concentration in the non diabetic, non CAD control group