Effects of Antidiabetic Agents on Coronary Cells
Effects of Antidiabetic Agents on Coronary Cells
Agents that were used in this study were the following: insulin lispro (100 pM, 1 nM), C-peptide (1 nM), pioglitazone (2.5 μM), metformin (500 μM), glimepiride (1 μM), rosuvastatin (10 nM), candesartan (100 nM) and BLX-1002 (1 μM). Of the compounds tested, we were unable to detect any effect of C-peptide on either proliferation or apoptosis of the cells. All agents have been tested in all conditions, but only compounds that were able to exert significant effects are displayed in the graphs.
As shown in Figure 1 among the agents tested, BLX-1002 and insulin (the latter at both 100 pM and 1 nM) increased cell viability. Exposure of the cells to metformin also resulted in a slight, but statistically significant, increase in cell viability. Under the same conditions, cell viability was also augmented by rosuvastatin, pioglitazone, candesartan and glimepiride.
(Enlarge Image)
Figure 1.
Insulin, metformin, BLX-1002, rosuvastatin, pioglitazone, candesartan and glimepiride increase HCAEC viability. Cells were incubated for 48 h in medium supplemented with 0.5% FBS in the presence or absence of insulin lispro (100 pM and 1 nM), metformin (500 μM), BLX-1002 (1 μM), rosuvastatin (10 nM), pioglitazone (2.5 μM), candesartan (100 nM) and glimepiride (1 μM). Viability was assessed with Trypan blue exclusion. * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001 for chance differences compared to controls by Student's t-test.
Since the increased viability noted above (Figure 1) could be explained by increased proliferation, decreased apoptosis, or a combination thereof, we investigated if the drugs exert any effect on HCAECs' DNA synthesis. Rates of [H]thymidine incorporation were analyzed after a 24 hour exposure to the agents at 5 mM glucose. The mitogenic effect of the compounds was also verified by measuring protein concentrations of the samples to reflect an increase in cell number. As shown in Figure 2 insulin, BLX-1002, metformin and rosuvastatin increased both DNA synthesis and protein concentration significantly.
(Enlarge Image)
Figure 2.
Insulin, BLX-1002, metformin and rosuvastatin stimulate HCAEC proliferation at normal glucose concentration. In order to study the effects of agents on proliferation of HCAECs, cells were incubated at 5 mM of glucose in medium supplemented with 0.5% FBS for 24 h in the presence or absence of insulin lispro (100 pM), metformin (500 μM), BLX-1002 (1 μM), rosuvastatin (10 nM). Eight hours prior to the end of the incubation, cells were pulsed with [H]thymidine. Cells were then harvested and [H]thymidine incorporation into DNA was measured using a microplate scintillation & luminescence counter, and the protein concentration of the samples was measured using DC™ Protein Assay. * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001 for chance differences vs. controls by Student's t-test.
Since insulin, BLX-1002, metformin and rosuvastatin were able to increase the proliferation of HCAECs in 5 mM glucose (Figure 2), we further investigated whether they had any effect on cell proliferation at high glucose. HCAECs were therefore exposed to 11 mM of glucose, to simulate a diabetic setting, in the presence or absence of the agents. Similar to the effect observed at 5 mM glucose, insulin and BLX-1002 were both able to increase DNA synthesis and protein concentration (Figure 3). In contrast to their effects at 5 mM glucose, metformin and rosuvastatin did not exert any effect on cell proliferation under these conditions (data not shown).
(Enlarge Image)
Figure 3.
Insulin and BLX-1002 stimulate HCAEC proliferation in high glucose. HCAECs were incubated at 11 mM of glucose in medium supplemented with 0.5% FBS for 24 h in the presence or absence of insulin lispro (100 pM) or BLX-1002 (1 μM). Eight hours prior to the end of the incubation, cells were pulsed with [H]thymidine. Cells were then harvested and [H]thymidine incorporation into DNA was measured using a microplate scintillation & luminescence counter and the protein concentration of the samples was measured using DC™ Protein Assay. * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001 for a chance difference vs. control by Student's t-test.
Plasma FFA levels are increased in type-2 diabetic patients and the FFA palmitate is known to induce apoptosis of endothelial cells. We therefore evaluated how the agents affected the activity of cleaved caspase-3, a crucial mediator of apoptosis, in HCAECs after 24 hour exposure to 0.125 mM palmitate. As shown in Figure 4 palmitate significantly increased caspase-3 activity, thus confirming its pro-apoptotic effect, in HCAECs. Insulin, BLX-1002, metformin, pioglitazone and candesartan significantly decreased the palmitate-induced caspase-3 activation, suggesting a protective effect of the drugs against lipotoxicity in HCAECs. Glimepiride significantly decreased basal caspase-3 activity with ~20% (data not shown), which might explain why an increased viability of the cells in the presence of glimepiride is noted (Figure 1).
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Figure 4.
Insulin, metformin, BLX-1002, pioglitazone and candesartan protect HCAECs against palmitate-induced caspase-3 activation. Palmitate-induced caspase-3 activity, a measure of apoptosis, was evaluated using the EnzChek® Caspase-3 Assay Kit. HCAECs were incubated in medium containing 5 mM glucose, supplemented with 0.5% FBS, with or without insulin lispro (1 nM), BLX-1002 (1 μM), metformin (500 μM), pioglitazone (2.5 μM), candesartan (100 nM) in the presence of 0.125 mM palmitate or vehicle (ethanol and BSA) for 24 h. * denotes P < 0.05 for a chance difference vs. controls by ANOVA. # denotes P < 0.05 for a chance difference vs. palmitate by ANOVA.
To corroborate the anti-apoptotic effect of the agents above, we investigated their influence on palmitate-induced DNA fragmentation by analyzing cytoplasmic DNA-histone nucleosome complexes generated during apoptosis. Palmitate alone gave rise to a markedly increased apoptosis of the HCAECs (Figure 5). Co-incubation with insulin, BLX-1002, metformin, pioglitazone or candesartan significantly countered the palmitate-induced apoptosis, thus confirming the protective effect of these agents against lipoapoptosis in HCAECs.
(Enlarge Image)
Figure 5.
Insulin, metformin, BLX-1002, pioglitazone and candesartan protect HCAECs against palmitate-induced DNA fragmentation. To further corroborate the protective effects of the agents against lipotoxicity-induced apoptosis, DNA fragmentation was evaluated using the Cell Death Detection kit ELISA. Cells were exposed for 24 h to 0.125 mM palmitate or vehicle at 5 mM glucose with or without insulin lispro (1 nM), BLX-1002 (1 μM), metformin (500 μM), pioglitazone (2.5 μM), candesartan (100 nM). * denotes P < 0.05 for a chance difference vs controls by ANOVA, # denotes P < 0.05 for a chance difference vs palmitate by ANOVA.
Results
Agents that were used in this study were the following: insulin lispro (100 pM, 1 nM), C-peptide (1 nM), pioglitazone (2.5 μM), metformin (500 μM), glimepiride (1 μM), rosuvastatin (10 nM), candesartan (100 nM) and BLX-1002 (1 μM). Of the compounds tested, we were unable to detect any effect of C-peptide on either proliferation or apoptosis of the cells. All agents have been tested in all conditions, but only compounds that were able to exert significant effects are displayed in the graphs.
Cell Viability of HCAECs is Increased by the Agents
As shown in Figure 1 among the agents tested, BLX-1002 and insulin (the latter at both 100 pM and 1 nM) increased cell viability. Exposure of the cells to metformin also resulted in a slight, but statistically significant, increase in cell viability. Under the same conditions, cell viability was also augmented by rosuvastatin, pioglitazone, candesartan and glimepiride.
(Enlarge Image)
Figure 1.
Insulin, metformin, BLX-1002, rosuvastatin, pioglitazone, candesartan and glimepiride increase HCAEC viability. Cells were incubated for 48 h in medium supplemented with 0.5% FBS in the presence or absence of insulin lispro (100 pM and 1 nM), metformin (500 μM), BLX-1002 (1 μM), rosuvastatin (10 nM), pioglitazone (2.5 μM), candesartan (100 nM) and glimepiride (1 μM). Viability was assessed with Trypan blue exclusion. * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001 for chance differences compared to controls by Student's t-test.
Insulin, BLX-1002, Metformin and Rosuvastatin Stimulate Proliferation of HCAECs at Normal Glucose Concentration
Since the increased viability noted above (Figure 1) could be explained by increased proliferation, decreased apoptosis, or a combination thereof, we investigated if the drugs exert any effect on HCAECs' DNA synthesis. Rates of [H]thymidine incorporation were analyzed after a 24 hour exposure to the agents at 5 mM glucose. The mitogenic effect of the compounds was also verified by measuring protein concentrations of the samples to reflect an increase in cell number. As shown in Figure 2 insulin, BLX-1002, metformin and rosuvastatin increased both DNA synthesis and protein concentration significantly.
(Enlarge Image)
Figure 2.
Insulin, BLX-1002, metformin and rosuvastatin stimulate HCAEC proliferation at normal glucose concentration. In order to study the effects of agents on proliferation of HCAECs, cells were incubated at 5 mM of glucose in medium supplemented with 0.5% FBS for 24 h in the presence or absence of insulin lispro (100 pM), metformin (500 μM), BLX-1002 (1 μM), rosuvastatin (10 nM). Eight hours prior to the end of the incubation, cells were pulsed with [H]thymidine. Cells were then harvested and [H]thymidine incorporation into DNA was measured using a microplate scintillation & luminescence counter, and the protein concentration of the samples was measured using DC™ Protein Assay. * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001 for chance differences vs. controls by Student's t-test.
Insulin and BLX-1002 Stimulate Proliferation of HCAECs at High Glucose
Since insulin, BLX-1002, metformin and rosuvastatin were able to increase the proliferation of HCAECs in 5 mM glucose (Figure 2), we further investigated whether they had any effect on cell proliferation at high glucose. HCAECs were therefore exposed to 11 mM of glucose, to simulate a diabetic setting, in the presence or absence of the agents. Similar to the effect observed at 5 mM glucose, insulin and BLX-1002 were both able to increase DNA synthesis and protein concentration (Figure 3). In contrast to their effects at 5 mM glucose, metformin and rosuvastatin did not exert any effect on cell proliferation under these conditions (data not shown).
(Enlarge Image)
Figure 3.
Insulin and BLX-1002 stimulate HCAEC proliferation in high glucose. HCAECs were incubated at 11 mM of glucose in medium supplemented with 0.5% FBS for 24 h in the presence or absence of insulin lispro (100 pM) or BLX-1002 (1 μM). Eight hours prior to the end of the incubation, cells were pulsed with [H]thymidine. Cells were then harvested and [H]thymidine incorporation into DNA was measured using a microplate scintillation & luminescence counter and the protein concentration of the samples was measured using DC™ Protein Assay. * denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001 for a chance difference vs. control by Student's t-test.
Insulin, BLX-1002, Metformin, Pioglitazone and Candesartan Suppress Lipoapoptosis in HCAECs
Plasma FFA levels are increased in type-2 diabetic patients and the FFA palmitate is known to induce apoptosis of endothelial cells. We therefore evaluated how the agents affected the activity of cleaved caspase-3, a crucial mediator of apoptosis, in HCAECs after 24 hour exposure to 0.125 mM palmitate. As shown in Figure 4 palmitate significantly increased caspase-3 activity, thus confirming its pro-apoptotic effect, in HCAECs. Insulin, BLX-1002, metformin, pioglitazone and candesartan significantly decreased the palmitate-induced caspase-3 activation, suggesting a protective effect of the drugs against lipotoxicity in HCAECs. Glimepiride significantly decreased basal caspase-3 activity with ~20% (data not shown), which might explain why an increased viability of the cells in the presence of glimepiride is noted (Figure 1).
(Enlarge Image)
Figure 4.
Insulin, metformin, BLX-1002, pioglitazone and candesartan protect HCAECs against palmitate-induced caspase-3 activation. Palmitate-induced caspase-3 activity, a measure of apoptosis, was evaluated using the EnzChek® Caspase-3 Assay Kit. HCAECs were incubated in medium containing 5 mM glucose, supplemented with 0.5% FBS, with or without insulin lispro (1 nM), BLX-1002 (1 μM), metformin (500 μM), pioglitazone (2.5 μM), candesartan (100 nM) in the presence of 0.125 mM palmitate or vehicle (ethanol and BSA) for 24 h. * denotes P < 0.05 for a chance difference vs. controls by ANOVA. # denotes P < 0.05 for a chance difference vs. palmitate by ANOVA.
To corroborate the anti-apoptotic effect of the agents above, we investigated their influence on palmitate-induced DNA fragmentation by analyzing cytoplasmic DNA-histone nucleosome complexes generated during apoptosis. Palmitate alone gave rise to a markedly increased apoptosis of the HCAECs (Figure 5). Co-incubation with insulin, BLX-1002, metformin, pioglitazone or candesartan significantly countered the palmitate-induced apoptosis, thus confirming the protective effect of these agents against lipoapoptosis in HCAECs.
(Enlarge Image)
Figure 5.
Insulin, metformin, BLX-1002, pioglitazone and candesartan protect HCAECs against palmitate-induced DNA fragmentation. To further corroborate the protective effects of the agents against lipotoxicity-induced apoptosis, DNA fragmentation was evaluated using the Cell Death Detection kit ELISA. Cells were exposed for 24 h to 0.125 mM palmitate or vehicle at 5 mM glucose with or without insulin lispro (1 nM), BLX-1002 (1 μM), metformin (500 μM), pioglitazone (2.5 μM), candesartan (100 nM). * denotes P < 0.05 for a chance difference vs controls by ANOVA, # denotes P < 0.05 for a chance difference vs palmitate by ANOVA.
