A769662 inhibits insulin-stimulated akt activation in human macrovascular endothelial cells independent of AMP-activated protein kinase

Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation

Inducing energetic switching using Klotho improves vascular smooth muscle cell phenotype

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival

Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

The key metabolic regulator, AMP-activated protein kinase (AMPK) is reported to be downregulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491 respectively as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues

The effects of experimental hyperglycaemia and hyperinsulinaemia on macrovascular endothelial cell function

Endothelial dysfunction, characterised by decreased NO synthesis, is associated with an increased risk of cardiovascular complications in patients with type 1 diabetes mellitus, type 2 diabetes mellitus (T2DM) and insulin resistance. The dysfunctional endothelium exhibits impaired insulin-stimulated NO generation and vasodilatation, as well as increased pro-inflammatory and pro-atherogenic signalling, which promote the development of atherosclerosis and hypertension. Despite cardiovascular complications being the leading cause of mortality and morbidity in people with diabetes and insulin resistance, the mechanism of underlying macrovascular endothelial dysfunction is poorly understood. In this study, the effects of experimental hyperglycaemia (eHG) and hyperinsulinaemia (eHI) were examined on endothelial cell phenotype and NO production in cultured human arterial endothelial cells. eHG markedly impaired ionomycin-stimulated NO production and insulin-stimulated eNOS activation and NO production in human aortic endothelial cells (HAECs), having similar effects in human coronary artery endothelial cells (HCAECs). IR/Akt/eNOS pathway activation and calcium signalling were preserved in eHG, suggesting that eHG targets eNOS directly, decreasing enzymatic activity. In addition to diminished eNOS function, eHG decreased mitochondrial activity in HAECs while preserving total mitochondrial mass and morphology. Intriguingly, inhibition of insulin-stimulated NO production and mitochondrial function by eHG were rescued by an AMP-activated protein kinase (AMPK) activator, AICAR, yet, the AMPK-dependence of these effects remains to be investigated. Ionomycin-stimulated NO production was impaired and insulin-stimulated NO production tended to reduce in HAECs cultured in eHI media. eHI decreased insulin-stimulated endothelial NO synthase (eNOS) activation, suggesting that the decrease in NO bioavailability in eHI is due to impaired eNOS activation. Furthermore, eHI abolished acute insulin-stimulated NO synthesis and phosphorylation of protein kinase B (Akt) Ser473 and eNOS Ser1177 in HAECs, and also tended to increase basal Akt Ser473, eNOS Ser1177 and G-protein-coupled receptor kinase interacting protein 1 (GIT1) Tyr554 phosphorylation, implying an impairment of insulin signalling. The inhibition of insulin-stimulated NO production by eHI was restored completely by GIT1 downregulation with siRNA and partially restored by inhibiting c-Src-dependent GIT1 Tyr554 phosphorylation. Desensitisation of the insulin receptor (IR)/Akt/eNOS pathway and altered GIT1-eNOS binding dynamics may therefore contribute to the decreased eNOS activity in eHI. Neither eHI nor eHG had any marked effect on reactive oxygen species generation or proinflammatory signalling yet von Willebrand factor levels and secretion were augmented in eHI-treated HAECs. Therefore, chronic insulin treatment may increase the risk of thrombosis by promoting coagulation. The effects of eHI and eHG were observed to partially replicate phenotypical changes observed in HAECs from donors with T2DM. HAECs from people with T2DM exhibited impaired insulin- and ionomycin-stimulated NO generation, decreased insulin-stimulated GIT1-eNOS binding and reduced eNOS Ser1177 phosphorylation. Unlike eHI and eHG, HAECs from people with T2DM also exhibited increased H2O2 generation and mitochondrial network fragmentation and loss of endothelial cell identity, suggesting that T2DM HAECs represent a more profound state of diabetic endothelial dysfunction. Overall, the results presented in this thesis demonstrate that eHG and eHI impair eNOS function and NO production in cultured HAECs, mimicking in part the phenotype observed in T2DM HAECs. Moreover, altered GIT1-eNOS binding and diminished mitochondrial function may contribute to diabetic endothelial dysfunction, yet, further investigation of GIT1 and mitochondrial function in eNOS regulation is necessary to design an effective therapeutic strategy

Osteoprotegerin regulates vascular function through syndecan-1 and NADPH oxidase-derived reactive oxygen species

Osteogenic factors, such as osteoprotegerin (OPG), are protective against vascular calcification. However, OPG is also positively associated with cardiovascular damage, particularly in pulmonary hypertension, possibly through processes beyond effects on calcification. In the present study, we focused on calcification-independent vascular effects of OPG through activation of syndecan-1 and NADPH oxidases (Noxs) 1 and 4. Isolated resistance arteries from Wistar–Kyoto (WKY) rats, exposed to exogenous OPG, studied by myography exhibited endothelial and smooth muscle dysfunction. OPG decreased nitric oxide (NO) production, eNOS activation and increased reactive oxygen species (ROS) production in endothelial cells. In VSMCs, OPG increased ROS production, H2O2/peroxynitrite levels and activation of Rho kinase and myosin light chain. OPG vascular and redox effects were also inhibited by the syndecan-1 inhibitor synstatin (SSNT). Additionally, heparinase and chondroitinase abolished OPG effects on VSMCs-ROS production, confirming syndecan-1 as OPG molecular partner and suggesting that OPG binds to heparan/chondroitin sulphate chains of syndecan-1. OPG-induced ROS production was abrogated by NoxA1ds (Nox1 inhibitor) and GKT137831 (dual Nox1/Nox4 inhibitor). Tempol (SOD mimetic) inhibited vascular dysfunction induced by OPG. In addition, we studied arteries from Nox1 and Nox4 knockout (KO) mice. Nox1 and Nox4 KO abrogated OPG-induced vascular dysfunction. Vascular dysfunction elicited by OPG is mediated by a complex signalling cascade involving syndecan-1, Nox1 and Nox4. Our data identify novel molecular mechanisms beyond calcification for OPG, which may underlie vascular injurious effects of osteogenic factors in conditions such as hypertension and/or diabetes