Protective effect of 3-hydroxybutyrate against endoplasmic reticulum stress-associated vascular endothelial cell damage induced by low glucose exposure

Abstract

Aims/hypothesis: The aim of this study was to elucidate the mechanism by which severe hypoglycemia accelerates vascular complications. Furthermore, we assessed the possible protective effect of ketone bodies against the endothelial cell damage caused by glucose deficiency.

Methods: Human umbilical vein endothelial cells (HUVECs) were cultured at a glucose level of either 0.56 or 5.6 mmol/L with or without 3-hydroxybutyrate (3-HB) supplementation. Cell viability was assessed with a CCK-8 assay and a lactate dehydrogenase (LDH) release assay. The activity of caspases was measured using fluorogenic substrates. The expression of genes associated with endothelial cell function and endoplasmic reticulum (ER) stress was evaluated by real-time quantitative PCR. Protein levels of ER stress-related molecules were assessed by Western blotting.

Results: Culture of HUVECs in low-glucose medium for 24 or 48 h resulted in reduction of cell viability accompanied by activation of caspase-3/7 and caspase-8. The addition of a pan caspase inhibitor attenuated the cell death. After incubation in the low-glucose medium, we found reduced mRNA and protein levels of endothelial nitric oxide synthase. ER stress responses mediated by phosphorylation of protein kinase RNA-like ER kinase (PERK) and cleavage of activating transcription factor 6 (ATF6) were augmented, but X-box binding protein 1 (Xbp1) splicing was reduced. Most of these responses to glucose deficiency were significantly attenuated by supplementation with 3-HB.

Conclusions/interpretation: These observations showed that exposure to low glucose induces ER stress, caspase activation, endothelial cell dysfunction and cell death. The beneficial effects of 3-HB shown in this study suggest that hypoketonemic severe hypoglycemia induced by insulin injections or insulin secretagogue administration may be more harmful than hyperketonemic severe hypoglycemia.

Fig 1. Effects of low-glucose exposure on cell viability and protective effects of 3-hydroxybutyrate (3-HB).

Human umbilical vein endothelial cells (HUVECs) were treated with 5.6 or 0.56 mmol/L glucose in the presence of various concentrations of 3-HB (0, 1, 4, 10 mmol/L) for 24 h (A) or 48 h (B). Cell viability was assessed by a CCK-8 assay (A) (B) and an LDH (C) assay. Microscopic images of HUVECs treated for 24 h (D-F). Means and SD, *P<0.05, **P<0.01, ***P<0.001 (n = 4).

Fig 2. Caspase activation by low glucose and suppressive effect of 3-hydroxybutyrate (3-HB).

Caspase-3/7 (A) and caspase-8 (B) activities were measured in HUVECs cultured in low-glucose medium with or without supplementation of 3-HB for 48 h. Results are exppressed as fold increase from control. Means and SD, *P<0.05, **P<0.01, ***P<0.001 (n = 4). Cell nuclei were visualized by Hoechst 33342 staining (blue) (C-G), and caspase-3/7-positive cells were stained green with a fluorogenic substrate (H-L). Bar, 100μm.

Fig 3. Protective effect of a pan caspase inhibitor on HUVECs exposed to low glucose.

HUVECs were treated with 5.6 or 0.56 mmol/L glucose with or without 10 μmol/L zVAD-fmk for 24 h. Cell viability was assessed by the CCK-8 assay (A) and the LDH release assay (B). Means and SD, *P<0.05, **P<0.01, ***P<0.001 (n = 4).

Fig 4. Effect of glucose deprivation on endothelial nitric oxide synthase (eNOS).

HUVECs were treated with 5.6 or 0.56 mmol/L glucose in the presence of various concentrations of 3-hydroxybutyrate (3-HB) for 24 h. mRNA (A) and protein levels (B) of eNOS were assessed by real-time PCR and Western blotting, respectively. A representative image of Western blotting is shown (C). Means and SD, *P<0.05, **P<0.01, ***P<0.001.

Fig 5. Effects of 3-hydroxybutyrate (3-HB) on the endoplasmic reticulum (ER) stress markers.

Expression of ER stress marker genes, Eif2ak3/Perk (A), Ddit3 (B), Ppp1r15a (C), Atf6 (D), Hsppa5 (E), Ire1 (F), XBP1u (G) and XBP1s (H) were assessed in HUVECs treated with 5.6 or 0.56 mmol/L glucose in the presence of various concentrations of 3-HB for 6 h. Means and SD, *P<0.05, **P<0.01, ***P<0.001 (n = 4).

Fig 6. Western blot analysis of ER stress markers.

Protein levels of PERK (A), cleaved ATF6 (cATF6) (B), CHOP (C) and Bip/GRP78 (D) were assessed in HUVECs treated with 5.6 or 0.56 mmol/L glucose in the presence of various concentrations of 3-hydroxybutyrate (3-HB) for 24 h. β-actin was used as a loading control. Representative images of Western blotting are shown. Means and SD, *P<0.05, **P<0.01, ***P<0.001 (n = 3).