Abstract
High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3β (GSK3β). To determine whether GSK3β affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308: , C249-C263]. Presently, we found that incubation of HAECs with excess nutrients (25mM glucose and 0.4mM palmitate) increased GSK3β activity and impaired lysosome acidification. Suppression of GSK3β in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3β attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3β increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3β inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3β may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis.
Highlights
During hyperglycaemia and dyslipidaemia, dysregulation of macroautophagy [1] and AMPactivated protein kinase (AMPK) [1,2,3] is associated with endothelial cell damage and dysfunction, events that often precede atherogenesis [4,5]
Additional evidence illustrating the important role of autophagy in vascular health can be found in studies using high-fat fed rabbits, in which activation of macrophage autophagy through the use of mammalian target of rapamycin complex 1 inhibitors has been shown to slow progression of Abbreviations: Acetyl-CoA carboxylase (ACC), acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; FOXO1, forkhead box protein O1; GSK3β, glycogen synthase kinase 3β; HAEC, human aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; LC3, microtubule-associated protein light chain 3; MEF, mouse embryonic fibroblast; mTORC1, mammalian target of rapamycin complex 1; PP2A, protein phosphatase 2A; TFEB, transcription factor EB. 1 To whom correspondence should be addressed
High concentrations of glucose and palmitate increase GSK3β activity in HAECs Increased GSK3β activity has been observed in skeletal muscle of humans with diabetes [18] and in epididymal adipose tissue [19] and hearts [20] of diabetic mice
Summary
Dysregulation of macroautophagy [1] (hereafter referred to as ‘autophagy’) and AMPactivated protein kinase (AMPK) [1,2,3] is associated with endothelial cell damage and dysfunction, events that often precede atherogenesis [4,5]. In a human aortic endothelial cell (HAEC) culture model that we developed to mimic the diabetic milieu during early atherogenesis, we observed impairment of autophagosome formation and lysosome enzyme activity [1]. In this model, chronic incubation of the cells in media containing 25 mM glucose, followed by an acute exposure to 0.4 mM palmitate, elicited several pro-atherogenic events in addition to impaired basal autophagy including inflammation, apoptosis and reduced activity of AMPK [1]. We assessed changes in autophagyand AMPK-related signalling in nutrient-laden HAECs subjected to GSK3β inhibition and in aortas of mice treated with the GSK3β inhibitor, CHIR 99021
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