AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance

AMPK and TOR: the Yin and Yang of cellular nutrient sensing and growth control

Mechanisms of Cardiovascular Benefits of Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitors: A State-of-the-Art Review

Mechanisms of Cardiorenal Effects of Sodium-Glucose Cotransporter 2 Inhibitors: JACC State-of-the-Art Review

Effects of SGLT2 inhibitors on systemic and tissue low-grade inflammation: The potential contribution to diabetes complications and cardiovascular disease.

Recent clinical trials of the hypoglycaemic sodium-glucose co-transporter-2 (SGLT2) inhibitors, which inhibit renal glucose reabsorption, have reported beneficial cardiovascular outcomes. Whether SGLT2 inhibitors directly affect cardiovascular tissues, however, remains unclear. We have previously reported that the SGLT2 inhibitor canagliflozin activates AMP-activated protein kinase (AMPK) in immortalised cell lines and murine hepatocytes. As AMPK has anti-inflammatory actions in vascular cells, we examined whether SGLT2 inhibitors attenuated inflammatory signalling in cultured human endothelial cells. Incubation with clinically-relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin activated AMPK and inhibited IL-1β-stimulated adhesion of pro-monocytic U937 cells and secretion of IL-6 and monocyte chemoattractant protein-1 (MCP-1). Inhibition of MCP-1 secretion was attenuated by expression of dominant-negative AMPK and was mimicked by the direct AMPK activator, A769662. Stimulation of cells with either canagliflozin or A769662 had no effect on IL-1β-stimulated cell surface levels of adhesion molecules or nuclear factor-κB signalling. Despite these identical effects of canagliflozin and A769662, IL-1β-stimulated IL-6/MCP-1 mRNA was inhibited by canagliflozin, but not A769662, whereas IL-1β-stimulated c-jun N-terminal kinase phosphorylation was inhibited by A769662, but not canagliflozin. These data indicate that clinically-relevant canagliflozin concentrations directly inhibit endothelial pro-inflammatory chemokine/cytokine secretion by AMPK-dependent and -independent mechanisms without affecting early IL-1β signalling.

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Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms.

The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated 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

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Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

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.

https://www.mdpi.com/1422-0067/19/12/3886/pdf

A769662 Inhibits Insulin-Stimulated Akt Activation in Human Macrovascular Endothelial Cells Independent of AMP-Activated Protein Kinase.

Abstract The counterregulatory response to hypoglycemia that restores normal blood glucose levels is an essential physiological function. It is initiated, in large part, by incompletely characterized brain hypoglycemia sensing neurons that trigger the secretion of counterregulatory hormones, in particular glucagon, to stimulate hepatic glucose production. In a genetic screen of recombinant inbred BXD mice we previously identified Agpat5 as a candidate regulator of hypoglycemia-induced glucagon secretion. Here, using genetic mouse models, we demonstrate that Agpat5 expressed in agouti-related peptide neurons is required for their activation by hypoglycemia, for hypoglycemia-induced vagal nerve activity, and glucagon secretion. We find that inactivation of Agpat5 leads to increased fatty acid oxidation and ATP production and that suppressing Cpt1a -dependent fatty acid import into mitochondria restores hypoglycemia sensing. Collectively, our data show that AgRP neurons are involved in the control of glucagon secretion and that Agpat5 , by partitioning fatty acyl-CoAs away from mitochondrial fatty acid oxidation and ATP generation, ensures that the fall in intracellular ATP, which triggers neuronal firing, faithfully reflects changes in glycemia. 

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Anastasiya Strembitska

Papers

Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms.

Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

A769662 Inhibits Insulin-Stimulated Akt Activation in Human Macrovascular Endothelial Cells Independent of AMP-Activated Protein Kinase.

Lipid biosynthesis enzyme Agpat5 in AgRP-neurons is required for insulin-induced hypoglycemia sensing and glucagon secretion