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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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Effect of GKA on TGF-β mediated 35S-SO4 incorporation into proteoglycans by endothelial cells. Cells treated with TGF-β (2ng/ml) +/-GKA (RO28-1675) 1-10μM for 24 h and SB431542 (3μM) was used as TβR1 antagonist. Media containing secreted proteoglycans was spotted on chromatography paper and CPC precipitated to assess radiolabel incorporation into proteoglycans. Panel A shows the quantitation by the CPC precipitation method and Panel B shows the resulting SDS-PAGE analysis. Results are mean ±SEM of data normalised to control from three experiments in triplicate, ##p<0.01 versus control and **p<0.01 versus TGF-β using 1-way ANOVA.
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Figure 6: Effect of GKA on TGF-β mediated 35S-SO4 incorporation into proteoglycans by endothelial cells. Cells treated with TGF-β (2ng/ml) +/-GKA (RO28-1675) 1-10μM for 24 h and SB431542 (3μM) was used as TβR1 antagonist. Media containing secreted proteoglycans was spotted on chromatography paper and CPC precipitated to assess radiolabel incorporation into proteoglycans. Panel A shows the quantitation by the CPC precipitation method and Panel B shows the resulting SDS-PAGE analysis. Results are mean ±SEM of data normalised to control from three experiments in triplicate, ##p<0.01 versus control and **p<0.01 versus TGF-β using 1-way ANOVA.

Mentions: The synthesis of proteoglycans (biglycan) in association with increased GAG size is stimulated by the canonical TGF-β Smadphosphorylation pathway in vascular smooth muscle cells and BAEC [38-40]. BAECs were treated with TGF-β (2 ng/ml) to determine the effect on proteoglycan synthesis in the presence and absence of GKA (RO28-1675, 1-10 μM). TGF-β (2 ng/ml) increased 35S-SO4 incorporation into proteoglycans by 78.2% compared to basal (Figure 6). In the presence of GKA there was no effect on TGF-β mediated 35S-SO4 incorporation in BAEC. SB431542 (3 μM) is an inhibitor of the serine/threonine kinase activity of TGF-β receptor type 1 (ALK V) [41]; as expected SB431542 (3 μM) almost completely blocked the stimulation by TGF-β (Figure 6). The assessment of proteoglycan size by SDS-PAGE indicated TGF-β (2 ng/ml) increased proteoglycan size and the GKA had no effect on TGF-β stimulated increase in the size of biglycan a response which occurs due to hyperelongation of the lipid binding glycosaminoglycan (GAG) chains (Figure 6) [42-44]. The band with size of 220 kDa corresponds to biglycan [45].


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)

Effect of GKA on TGF-β mediated 35S-SO4 incorporation into proteoglycans by endothelial cells. Cells treated with TGF-β (2ng/ml) +/-GKA (RO28-1675) 1-10μM for 24 h and SB431542 (3μM) was used as TβR1 antagonist. Media containing secreted proteoglycans was spotted on chromatography paper and CPC precipitated to assess radiolabel incorporation into proteoglycans. Panel A shows the quantitation by the CPC precipitation method and Panel B shows the resulting SDS-PAGE analysis. Results are mean ±SEM of data normalised to control from three experiments in triplicate, ##p<0.01 versus control and **p<0.01 versus TGF-β using 1-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effect of GKA on TGF-β mediated 35S-SO4 incorporation into proteoglycans by endothelial cells. Cells treated with TGF-β (2ng/ml) +/-GKA (RO28-1675) 1-10μM for 24 h and SB431542 (3μM) was used as TβR1 antagonist. Media containing secreted proteoglycans was spotted on chromatography paper and CPC precipitated to assess radiolabel incorporation into proteoglycans. Panel A shows the quantitation by the CPC precipitation method and Panel B shows the resulting SDS-PAGE analysis. Results are mean ±SEM of data normalised to control from three experiments in triplicate, ##p<0.01 versus control and **p<0.01 versus TGF-β using 1-way ANOVA.
Mentions: The synthesis of proteoglycans (biglycan) in association with increased GAG size is stimulated by the canonical TGF-β Smadphosphorylation pathway in vascular smooth muscle cells and BAEC [38-40]. BAECs were treated with TGF-β (2 ng/ml) to determine the effect on proteoglycan synthesis in the presence and absence of GKA (RO28-1675, 1-10 μM). TGF-β (2 ng/ml) increased 35S-SO4 incorporation into proteoglycans by 78.2% compared to basal (Figure 6). In the presence of GKA there was no effect on TGF-β mediated 35S-SO4 incorporation in BAEC. SB431542 (3 μM) is an inhibitor of the serine/threonine kinase activity of TGF-β receptor type 1 (ALK V) [41]; as expected SB431542 (3 μM) almost completely blocked the stimulation by TGF-β (Figure 6). The assessment of proteoglycan size by SDS-PAGE indicated TGF-β (2 ng/ml) increased proteoglycan size and the GKA had no effect on TGF-β stimulated increase in the size of biglycan a response which occurs due to hyperelongation of the lipid binding glycosaminoglycan (GAG) chains (Figure 6) [42-44]. The band with size of 220 kDa corresponds to biglycan [45].

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