Novel Anti-inflammatory Effects of Canagliflozin Involving Hexokinase II in Lipopolysaccharide-Stimulated Human Coronary Artery Endothelial Cells

Abstract

Purpose Vascular inflammation and disturbed metabolism are observed in heart failure and type 2 diabetes mellitus. Glycolytic enzyme hexokinase II (HKII) is upregulated by inflammation. We hypothesized that SGLT2 inhibitors Canagliflozin (Cana), Empagliflozin (Empa) or Dapagliflozin (Dapa) reduces inflammation via HKII in endothelial cells, and that HKII-dependent inflammation is determined by ERK1/2, NF-κB. and/or AMPK activity in lipopolysaccharide (LPS)-stimulated human coronary artery endothelial cells (HCAECs). Methods HCAECs were pre-incubated with 3 μM or 10 μM Cana, 1 μM, 3 μM or 10 μM Empa or 0.5 μM, 3 μM or 10 μM Dapa (16 h) and subjected to 3 h LPS (1 μg/mL). HKII was silenced via siRNA transfection. Interleukin-6 (IL-6) release was measured by ELISA. Protein levels of HK I and II, ERK1/2, AMPK and NF-κB were detected using infra-red western blot. Results LPS increased IL-6 release and ERK1/2 phosphorylation; Cana prevented these pro-inflammatory responses (IL-6: pg/ml, control 46 ± 2, LPS 280 ± 154 p < 0.01 vs. control, LPS + Cana 96 ± 40, p < 0.05 vs. LPS). Cana reduced HKII expression (HKII/GAPDH, control 0.91 ± 0.16, Cana 0.71 ± 0.13 p < 0.05 vs. control, LPS 1.02 ± 0.25, LPS + Cana 0.82 ± 0.24 p < 0.05 vs. LPS). Empa and Dapa were without effect on IL-6 release and HKII expression in the model used. Knockdown of HKII by 37% resulted caused partial loss of Cana-mediated IL-6 reduction (pg/ml, control 35 ± 5, LPS 188 ± 115 p < 0.05 vs. control, LPS + Cana 124 ± 75) and ERK1/2 activation by LPS. In LPS-stimulated HCAECs, Cana, but not Empa or Dapa, activated AMPK. AMPK activator A769662 reduced IL-6 release. Conclusion Cana conveys anti-inflammatory actions in LPS-treated HCAECs through 1) reductions in HKII and ERK1/2 phosphorylation and 2) AMPK activation. These data suggest a novel anti-inflammatory mechanism of Cana through HKII.

Sodium Glucose Co-Transporter 2 Inhibitors Ameliorate Endothelium Barrier Dysfunction Induced by Cyclic Stretch through Inhibition of Reactive Oxygen Species

“…In vivo and in vitro studies have shown that pathological mechanical forces stimulate cytokine production and expression of adhesion molecules, leading to stiffness of vessels and monocyte adhesion [ 17 , 44 ]. Interestingly, none of the three SGLT-2i’s inhibited stretch-induced interleukin secretion ( Figure 5 ), which is in contrast to previous data showing that SGLT-2i’s inhibit interleukin production of endothelial cells from humans, as well as from ApoE (-/-) mice [ 45 , 46 ]. One potential explanation might be that the stretch-induced interleukin secretion of HCAECs is much milder than that caused by lipopolysaccharide (1.5–2 fold vs. 6–7 fold) [ 46 ], making it a less optimal model to investigate the potential anti-inflammatory effects of SGLT-2i’s.…”

“…However, the involvement of AMPK phosphorylation is still under discussion. In the previous studies of Uthman et al, AMPK activation was not observed in EMPA or DAPA, but only in CANA treated cells [ 46 ].…”

“…HCAECs (PCS-100-020 from ATCC, Manassas, VA, USA; C-12221 from PromoCell, Heidelberg, Germany) were used for cell culture with vascular cell growth medium (ATCC) containing supplements as previously described [ 46 ].…”

Anti-inflammatory Effects of Empagliflozin and Gemigliptin on LPS-Stimulated Macrophage via the IKK/NF-κB, MKK7/JNK, and JAK2/STAT1 Signalling Pathways

“…According to other study groups [57], canagliflzoin reduced proinflammatory cytokines by lowering hexose kinase II (HKII) and blocking ERK phosphorylation, but not affecting NF-κB in LPS-stimulated human coronary artery endothelial cells (HCAECs). Dapagliflozin also suppressed iNOS, TNF-α, IL-1β, and IL-6 mRNA expression by attenuating the NF-κB transcription factor in dietinduced atherosclerosis in rat aortic arteries [58].…”

Bioinformatics identification of hub genes and signaling pathways regulated by intravenous immunoglobulin treatment in acute Kawasaki disease

“…The HCAECs were seeded in each well of a 12-well culture plate at a density of 5x104 cells and incubated for 18 h at 37˚C in 5% CO 2 and 95% humidified air. The HCAECs were then subjected to 1 µg/ml lipopolysaccharide (LPS; Merck KGaA) challenge for 3 h (22). Subsequently, the HCAECs were treated with IVIGs (25 mg/ml) (Tonrol Bio-pharmaceutical Co., Ltd.) and vehicle for 18 h (23).…”