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Atherogenic, fibrotic and glucose utilising actions of glucokinase activators on vascular endothelium and smooth muscle.

Al-aryahi S, Kamato D, Getachew R, Zheng W, Potocnik SJ, Cohen N, Guidone D, Osman N, Little PJ - Cardiovasc Diabetol (2014)

Bottom Line: Some anti-hyperglycaemic drugs have been found to have adverse cardiovascular effects in their own right, limiting their therapeutic role.GKA RO28-1675 did not increase glucose consumption in endothelial cells indicating the absence of glucokinase in those cells.No direct deleterious actions, in terms of atherogenic changes or excessive vasoactive effects were seen on cells or vessels of the cardiovascular system in response to GKAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Discipline of Pharmacy and Diabetes Complications Group, Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, VIC 3083, Australia. peter.little@rmit.edu.au.

ABSTRACT

Background: Pharmaceutical interventions for diabetes aim to control glycaemia and to prevent the development of complications, such as cardiovascular diseases. Some anti-hyperglycaemic drugs have been found to have adverse cardiovascular effects in their own right, limiting their therapeutic role. Glucokinase activity in the pancreas is critical in enhancing insulin release in response to hyperglycaemia. Glucokinase activators (GKAs) are novel agents for diabetes which act by enhancing the formation of glucose-6-phosphate leading to increased insulin production and subsequent suppression of blood glucose. Little, however, is known about the direct effects of GKAs on cardiovascular cells.

Methods: The effect of the GKAs RO28-1675 and Compound A on glucose utilisation in bovine aortic endothelial cells (BAEC) and rat MIN6 was observed by culturing the cells at high and low glucose concentration in the presence and absence of the GKAs and measuring glucose consumption. The effect of RO28-1675 at various concentrations on glucose-dependent signalling in BAEC was observed by measuring Smad2 phosphorylation by Western blotting. The effect of RO28-1675 on TGF-β stimulated proteoglycan synthesis was measured by 35S-SO4 incorporation and assessment of proteoglycan size by SDS-PAGE. The effects of RO28-1675 on TGF-β mediated Smad2C phosphorylation in BAEC was observed by measurement of pSmad2C levels. The direct actions of RO28-1675 on vascular reactivity were observed by measuring arteriole tone and lumen diameter.

Results: GKAs were demonstrated to increase glucose utilisation in pancreatic but not endothelial cells. Glucose-activated Smad2 phosphorylation was decreased in a dose-dependent fashion in the presence of RO28-1675. No effect of RO28-1675 was observed on TGF-β stimulated proteoglycan production. RO28-1675 caused a modest dilation in arteriole but not contractile sensitivity.

Conclusions: GKA RO28-1675 did not increase glucose consumption in endothelial cells indicating the absence of glucokinase in those cells. No direct deleterious actions, in terms of atherogenic changes or excessive vasoactive effects were seen on cells or vessels of the cardiovascular system in response to GKAs. If reflected in vivo, these drugs are unlikely to have their use compromised by direct cardiovascular toxicity.

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The relationship between the rate of glucose consumption and D-glucose concentration. Endothelial cells treated with 2.5, 5, 15, 25 mM D-glucose −/+GKA and TFG-β. Basal represent normal levels of the rate of glucose consumption mM/well/hour in endothelial cells. In the presence of GKA (10 μM) there was no effect on the rate of glucose consumption. Results are the mean ± SEM from 3 experiments in triplicate **p < 0.01 versus basal, ***p < 0.001 versus basal determined using two-way ANOVA.
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Figure 2: The relationship between the rate of glucose consumption and D-glucose concentration. Endothelial cells treated with 2.5, 5, 15, 25 mM D-glucose −/+GKA and TFG-β. Basal represent normal levels of the rate of glucose consumption mM/well/hour in endothelial cells. In the presence of GKA (10 μM) there was no effect on the rate of glucose consumption. Results are the mean ± SEM from 3 experiments in triplicate **p < 0.01 versus basal, ***p < 0.001 versus basal determined using two-way ANOVA.

Mentions: We then utilised the same assay to assess the effect of GKAs on glucose utilisation by BAEC. BAEC were exposed to different glucose medium concentrations (2.5, 5, 15, 25 mM) +/− GKA (RO28-167 10 μM). Incubation proceeded for different time points (0, 4, 8, 24 h) and glucose levels were measured for each treatment. TGF-β (2 ng/ml), which activates cellular metabolism and increases glucose consumption was used as a positive control for the physiological sensitivity of the assay [34-36]. In the BAEC, the rate of glucose consumption increased with the media glucose concentration reaching a maximum rate of consumption at approximately 30 mM. The rate of glucose consumption was increased in cells treated with TGF-β but was completely unaffected in cells treated with RO28-1675 at the same concentration (10 μM) which stimulated glucose consumption in the MIN6 pancreatic beta cells (compare Figure 1B and Figure 2). The kinetic parameters for the curves are shown in Table 1. The maximum calculated rates of glucose metabolism were 0.14 ± 0.02 mM/well/hour and 0.17 ± 0.02 mM/well/hour (n.s.) in the absence and presence of RO28-1675, respectively and this was increased to 0.21 ± 0.01 mM/well/hour in the presence of TGF-β (Table 1). Half maximally effective concentrations of glucose were not appreciably different amongst the three treatment groups (Table 1). Thus, the GKA RO28-167 did not increase the consumption of glucose by BAECs implying that there is either no GK target or it is expressed at such a low level that its activation by GKAs does not alter glucose utilisation in BAEC.


Atherogenic, fibrotic and glucose utilising actions of glucokinase activators on vascular endothelium and smooth muscle.

Al-aryahi S, Kamato D, Getachew R, Zheng W, Potocnik SJ, Cohen N, Guidone D, Osman N, Little PJ - Cardiovasc Diabetol (2014)

The relationship between the rate of glucose consumption and D-glucose concentration. Endothelial cells treated with 2.5, 5, 15, 25 mM D-glucose −/+GKA and TFG-β. Basal represent normal levels of the rate of glucose consumption mM/well/hour in endothelial cells. In the presence of GKA (10 μM) there was no effect on the rate of glucose consumption. Results are the mean ± SEM from 3 experiments in triplicate **p < 0.01 versus basal, ***p < 0.001 versus basal determined using two-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016772&req=5

Figure 2: The relationship between the rate of glucose consumption and D-glucose concentration. Endothelial cells treated with 2.5, 5, 15, 25 mM D-glucose −/+GKA and TFG-β. Basal represent normal levels of the rate of glucose consumption mM/well/hour in endothelial cells. In the presence of GKA (10 μM) there was no effect on the rate of glucose consumption. Results are the mean ± SEM from 3 experiments in triplicate **p < 0.01 versus basal, ***p < 0.001 versus basal determined using two-way ANOVA.
Mentions: We then utilised the same assay to assess the effect of GKAs on glucose utilisation by BAEC. BAEC were exposed to different glucose medium concentrations (2.5, 5, 15, 25 mM) +/− GKA (RO28-167 10 μM). Incubation proceeded for different time points (0, 4, 8, 24 h) and glucose levels were measured for each treatment. TGF-β (2 ng/ml), which activates cellular metabolism and increases glucose consumption was used as a positive control for the physiological sensitivity of the assay [34-36]. In the BAEC, the rate of glucose consumption increased with the media glucose concentration reaching a maximum rate of consumption at approximately 30 mM. The rate of glucose consumption was increased in cells treated with TGF-β but was completely unaffected in cells treated with RO28-1675 at the same concentration (10 μM) which stimulated glucose consumption in the MIN6 pancreatic beta cells (compare Figure 1B and Figure 2). The kinetic parameters for the curves are shown in Table 1. The maximum calculated rates of glucose metabolism were 0.14 ± 0.02 mM/well/hour and 0.17 ± 0.02 mM/well/hour (n.s.) in the absence and presence of RO28-1675, respectively and this was increased to 0.21 ± 0.01 mM/well/hour in the presence of TGF-β (Table 1). Half maximally effective concentrations of glucose were not appreciably different amongst the three treatment groups (Table 1). Thus, the GKA RO28-167 did not increase the consumption of glucose by BAECs implying that there is either no GK target or it is expressed at such a low level that its activation by GKAs does not alter glucose utilisation in BAEC.

Bottom Line: Some anti-hyperglycaemic drugs have been found to have adverse cardiovascular effects in their own right, limiting their therapeutic role.GKA RO28-1675 did not increase glucose consumption in endothelial cells indicating the absence of glucokinase in those cells.No direct deleterious actions, in terms of atherogenic changes or excessive vasoactive effects were seen on cells or vessels of the cardiovascular system in response to GKAs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Discipline of Pharmacy and Diabetes Complications Group, Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, VIC 3083, Australia. peter.little@rmit.edu.au.

ABSTRACT

Background: Pharmaceutical interventions for diabetes aim to control glycaemia and to prevent the development of complications, such as cardiovascular diseases. Some anti-hyperglycaemic drugs have been found to have adverse cardiovascular effects in their own right, limiting their therapeutic role. Glucokinase activity in the pancreas is critical in enhancing insulin release in response to hyperglycaemia. Glucokinase activators (GKAs) are novel agents for diabetes which act by enhancing the formation of glucose-6-phosphate leading to increased insulin production and subsequent suppression of blood glucose. Little, however, is known about the direct effects of GKAs on cardiovascular cells.

Methods: The effect of the GKAs RO28-1675 and Compound A on glucose utilisation in bovine aortic endothelial cells (BAEC) and rat MIN6 was observed by culturing the cells at high and low glucose concentration in the presence and absence of the GKAs and measuring glucose consumption. The effect of RO28-1675 at various concentrations on glucose-dependent signalling in BAEC was observed by measuring Smad2 phosphorylation by Western blotting. The effect of RO28-1675 on TGF-β stimulated proteoglycan synthesis was measured by 35S-SO4 incorporation and assessment of proteoglycan size by SDS-PAGE. The effects of RO28-1675 on TGF-β mediated Smad2C phosphorylation in BAEC was observed by measurement of pSmad2C levels. The direct actions of RO28-1675 on vascular reactivity were observed by measuring arteriole tone and lumen diameter.

Results: GKAs were demonstrated to increase glucose utilisation in pancreatic but not endothelial cells. Glucose-activated Smad2 phosphorylation was decreased in a dose-dependent fashion in the presence of RO28-1675. No effect of RO28-1675 was observed on TGF-β stimulated proteoglycan production. RO28-1675 caused a modest dilation in arteriole but not contractile sensitivity.

Conclusions: GKA RO28-1675 did not increase glucose consumption in endothelial cells indicating the absence of glucokinase in those cells. No direct deleterious actions, in terms of atherogenic changes or excessive vasoactive effects were seen on cells or vessels of the cardiovascular system in response to GKAs. If reflected in vivo, these drugs are unlikely to have their use compromised by direct cardiovascular toxicity.

Show MeSH
Related in: MedlinePlus