1,25(OH)2D3 inhibits oxidative stress and monocyte adhesion by mediating the upregulation of GCLC and GSH in endothelial cells treated with acetoacetate (ketosis)

Abstract

Background: There is a significantly higher incidence of cardiovascular disease (CVD) among type 1 diabetic (T1D) patients than among non-diabetic subjects. T1D is associated with hyperketonemia, a condition with elevated blood levels of ketones, in addition to hyperglycemia. The biochemical mechanism by which vitamin D (VD) may reduce the risk of CVD is not known. This study examines whether VD can be beneficial in reducing hyperketonemia (acetoacetate, AA) induced oxidative stress in endothelial cells.

Methods: HUVEC were pretreated with 1,25(OH)2D3, and later exposed to the ketone body acetoacetate.

Results: The increases in ROS production, ICAM-1 expression, MCP-1 secretion, and monocyte adhesion in HUVEC treated with AA were significantly reduced following treatment with 1,25(OH)2D3. Interestingly, an increase in glutathione (GSH) levels was also observed with 1,25(OH)2D3 in ketone treated cells. The effects of 1,25(OH)2D3 on GSH, ROS, and monocyte-endothelial adhesion were prevented in GCLC knockdown HUVEC. This suggests that 1,25(OH)2D3 inhibits ROS, MCP-1, ICAM-1, and adherence of monocytes mediated by the upregulation of GCLC and GSH.

Conclusion: This study provides evidence for the biochemical mechanism through which VD supplementation may reduce the excess monocyte adhesion to endothelium and inflammation associated with T1D.

Keywords: 1,25(OH)(2)D3; Endothelium; Ketones; Oxidative stress; Type 1 diabetes; Vitamin D.

Figure 1. Effect of 1,25(OH)₂D₃ supplementation on inhibition of monocyte adhesion to endothelial cells

Adherence of monocytes to HUVEC was determined after both cell lines were pretreated with 1,25(OH)₂VD followed by treatment with AA (4 mM). The difference between C (untreated control cells) vs. * (AA treated cells) is significant at a p value <0.05. The difference between * (AA treated cells) vs. ** (AA+1,25(OH)₂VD treated cells) is significant at a p value <0.05. Values are mean±SE (n=3).

Figure 2. Effect of 1,25(OH)₂D₃ supplementation on oxidative stress inhibition in ketone treated endothelial cells

Panel A shows the ROS levels in HUVEC pretreated with 1,25(OH)₂VD for 24 h and then treated with AA (4 mM) for another 24 h. Panel B shows a phospho-NF-κB blot and its quantification. Panel C shows ICAM-1 protein expression in cells pretreated with 1,25(OH)₂VD (25 nM) for 24 h and then exposed to AA (4 mM) for a period of 24 h. MCP-1 secretion is shown in panel D. The difference between # (untreated control cells) vs. * (AA treated cells) is significant at a p value <0.05. The difference between * (AA treated cells) vs. ** (AA+1,25(OH)₂VD treated cells) is significant at a p value <0.05. Values are mean±SE (n=3).

Figure 3. Effect of 1,25(OH)₂D₃ supplementation on increases in glutathione levels

Panel A shows the expression of GCLC in AA treated and 1,25(OH)₂VD supplemented HUVEC. The bar graph in panel B shows the cellular levels of GSH. The difference between # (untreated control cells) vs. * (AA treated cells) is significant at a p value <0.05 in panel A. The difference between * (AA treated cells) vs. ** (AA+1,25(OH)₂VD treated cells) is significant at a p value <0.05. Values are mean±SE (n=3).

Figure 4. Effect of 1,25(OH)₂D₃ supplementation on ROS and monocyte adhesion to endothelial cells in glutathione-deficient HUVEC

The ROS levels in GCLC knockdown HUVEC treated with 1,25(OH)₂VD and AA are shown in panel A. The knockdown efficiency of GCLC siRNA along with ICAM-1 protein expression is shown in panel B. The percentage of monocyte adhesion to the GCLC knockdown HUVEC is shown in panel C. Panel D shows the mRNA levels of GCLC in HUVEC. p<0.05 when untreated control cells (#) were compared with GCLC knockdown AA treated or AA+1,25(OH)₂VD treated cells (*). 1,25(OH)₂D₃ supplementation has no beneficial effects in glutathione deficient HUVEC. Values are mean±SE (n=3).

Figure 5. Effect of 1,25(OH)₂D₃ supplementation on GCLC levels in vitamin D receptor-deficient HUVEC

Panel A shows the expression levels of VDR and GCLC in VDR knockdown cells treated with 1,25(OH)₂VD and ketones. The mRNA levels of VDR after knockdown are shown in panel B. p<0.05 when control siRNA cells (#) were compared with VDR knockdown cells (*). Values are mean±SE (n=3).

Figure 6. Proposed mechanism by which vitamin D supplementation can reduce hyperketonemia induced endothelial cell dysfunction

This schematic diagram represents the events that follow diabetic hyperketonemia, eventually leading to monocyte adherence to the endothelium that may progress into atherosclerosis. The various steps at which VD supplementation can have an effect in reducing ROS and the development of cardiovascular disease in T1D are indicated.