Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia

Abstract

The current goal of diabetes therapy is to reduce time-averaged mean levels of glycemia, measured as HbA1c, to prevent diabetic complications. However, HbA1c only explains <25% of the variation in risk of developing complications. Because HbA1c does not correlate with glycemic variability when adjusted for mean blood glucose, we hypothesized that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications. We show that transient hyperglycemia induces long-lasting activating epigenetic changes in the promoter of the nuclear factor kappaB (NF-kappaB) subunit p65 in aortic endothelial cells both in vitro and in nondiabetic mice, which cause increased p65 gene expression. Both the epigenetic changes and the gene expression changes persist for at least 6 d of subsequent normal glycemia, as do NF-kappaB-induced increases in monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression. Hyperglycemia-induced epigenetic changes and increased p65 expression are prevented by reducing mitochondrial superoxide production or superoxide-induced alpha-oxoaldehydes. These results highlight the dramatic and long-lasting effects that short-term hyperglycemic spikes can have on vascular cells and suggest that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications.

Figure 1.

Persistent increase in Set7-mediated histone methylation and p65 gene expression caused by transient hyperglycemia. (a) Schematic representation of experimental model. (b) NF-κB p65 subunit mRNA levels in response to transient hyperglycemia in HAECs. (c) Schematic representation of the human NF-κB p65 proximal promoter region. (d) ChIP of NF-κB p65 promoter by Set7 and H3K4me1 after transient hyperglycemia in HAECs. (e) H3K4me1 associated with the p65 promoter after transient hyperglycemia in WT and SET7 knockdown cells. (f) p65 expression after transient hyperglycemia in WT HAECs and SET7 knockdown cells. (g) Effect of p65 and SET7 knockdown on MCP-1 and VCAM-1 expression after transient hyperglycemia. *, P < 0.05 versus LG; ¶, P < 0.05 versus WT group. For B and D–F, n = 3. Error bars show SEM.

Figure 2.

Partial quantitative PCR–based chromosomal walking of the p65 gene by ChIP. (a) Schematic representation of the p65 proximal promoter and downstream sequence mapped. (b) Profiles of H3K4me1 after 16-h exposure to LG, 16-h exposure to HG, and 16-h exposure to HG followed by 6 d of LG. (c) Profiles of H3K4me2 after 16-h exposure to LG, 16-h exposure to HG and 16-h exposure to HG followed by 6 d of LG. (d) Profiles of H3K4me3 after 16-h exposure to LG, 16-h exposure to HG, and 16-h exposure to HG followed by 6 d of LG. *, P < 0.05 versus LG for HG group; #, P < 0.05 versus LG for HG (16 h) + LG (6 d) group. For B–D, n = 3. Error bars show SEM.

Figure 3.

UCP1, MnSOD, and GLO1 prevent persistent increases in Set7-mediated histone methylation and p65 gene expression. (a) p65 mRNA after transient hyperglycemia, measured by quantitative PCR. (b) ChIP analyses for association of Set7 and H3K4m1 with the proximal p65 promoter after transient hyperglycemia. (c) Chromatin remodeling after transient hyperglycemia as determined by susceptibility to Eag1 digestion. Nuclei from treated cells were isolated, digested by Eag1, and the extracted DNA amplified by quantitative PCR over the indicated p65 promoter region. *, P < 0.05 versus LG group. For each graph, n = 3. Error bars show SEM.

Figure 4.

Persistent increases in Set7-mediated histone methylation and p65 gene expression in nondiabetic mice. (a and b) WT mice were exposed to 20 mM glucose for 6 h using a hyperinsulinemic hyperglycemic clamp. Aortas were removed at the indicated times, and aortic endothelial cells were isolated by LCM. (a) cChIP of NF-κB p65 promoter by H3K4m1 antibody (n = 2). (b) NF-κB p65 subunit mRNA levels (n = 3). *, P < 0.05 versus LG. Error bars show SEM. (c and d) Aortic endothelial cells were isolated from UCP-2^+/− mice by LCM. (c) cChIP of NF-κB p65 promoter by H3K4m1 antibody (n = 4). (d) p65, MCP-1, and VCAM-1 mRNA levels (n = 4). *, P < 0.05 versus LG. Error bars show SEM. (e and f) Aortic endothelial cells were isolated from Glo1 KD mice by LCM. (e) cChIP of NF-κB p65 promoter by H3K4m1 antibody (n = 4). (f) p65, MCP-1, and VCAM-1 mRNA levels (n = 4). *, P < 0.05 versus LG. Error bars show SEM.