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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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Effects of GKA (RO28-1675) on TGF-β mediated Smad2C phosphorylation in endothelial cells. A. Western blot of whole cell lysates from BAECS treated with TGF-β (2 ng/ml) for 5 min in the presence and absence of GKA (RO28-1675) (1-10 μM). SB431542 (3 μM) was used as a positive control. Cell lysates were resolved on SDS-PAGE and transferred onto a PVDF membrane. This membrane was probed with anti-phosphoSmad2(Ser465/467) Also reprobed for anti-Smad2 and anti-GAPDH. Chemiluminescence was used to detect the proteins of interest. B. Histogram is a densitometric quantitation of 3 Western blots.
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Figure 7: Effects of GKA (RO28-1675) on TGF-β mediated Smad2C phosphorylation in endothelial cells. A. Western blot of whole cell lysates from BAECS treated with TGF-β (2 ng/ml) for 5 min in the presence and absence of GKA (RO28-1675) (1-10 μM). SB431542 (3 μM) was used as a positive control. Cell lysates were resolved on SDS-PAGE and transferred onto a PVDF membrane. This membrane was probed with anti-phosphoSmad2(Ser465/467) Also reprobed for anti-Smad2 and anti-GAPDH. Chemiluminescence was used to detect the proteins of interest. B. Histogram is a densitometric quantitation of 3 Western blots.

Mentions: The canonical TGF-β signalling pathway leads to the rapid generation of phosphoSmad2C. To test if GKA had any effects on upstream TGF-β signalling, BAEC were treated with TGF-β (2 ng/ml) for 5 min in the presence of GKA (RO28-1675) (1-10 μM). We observed no effect of GKA on TGF-β stimulated phosphoSmad2 levels (Figure 7). These data indicate that RO28-1657, which blocks glucose dependent Smad signalling does not directly block TGF-β activated Smad signalling in BAECs.


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)

Effects of GKA (RO28-1675) on TGF-β mediated Smad2C phosphorylation in endothelial cells. A. Western blot of whole cell lysates from BAECS treated with TGF-β (2 ng/ml) for 5 min in the presence and absence of GKA (RO28-1675) (1-10 μM). SB431542 (3 μM) was used as a positive control. Cell lysates were resolved on SDS-PAGE and transferred onto a PVDF membrane. This membrane was probed with anti-phosphoSmad2(Ser465/467) Also reprobed for anti-Smad2 and anti-GAPDH. Chemiluminescence was used to detect the proteins of interest. B. Histogram is a densitometric quantitation of 3 Western blots.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effects of GKA (RO28-1675) on TGF-β mediated Smad2C phosphorylation in endothelial cells. A. Western blot of whole cell lysates from BAECS treated with TGF-β (2 ng/ml) for 5 min in the presence and absence of GKA (RO28-1675) (1-10 μM). SB431542 (3 μM) was used as a positive control. Cell lysates were resolved on SDS-PAGE and transferred onto a PVDF membrane. This membrane was probed with anti-phosphoSmad2(Ser465/467) Also reprobed for anti-Smad2 and anti-GAPDH. Chemiluminescence was used to detect the proteins of interest. B. Histogram is a densitometric quantitation of 3 Western blots.
Mentions: The canonical TGF-β signalling pathway leads to the rapid generation of phosphoSmad2C. To test if GKA had any effects on upstream TGF-β signalling, BAEC were treated with TGF-β (2 ng/ml) for 5 min in the presence of GKA (RO28-1675) (1-10 μM). We observed no effect of GKA on TGF-β stimulated phosphoSmad2 levels (Figure 7). These data indicate that RO28-1657, which blocks glucose dependent Smad signalling does not directly block TGF-β activated Smad signalling in BAECs.

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