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. 2016 Nov;59(3):174-181.
doi: 10.3164/jcbn.16-26. Epub 2016 Sep 8.

Exendin-4 induces extracellular-superoxide dismutase through histone H3 acetylation in human retinal endothelial cells

Hiroyuki Yasuda  1 , Atsuko Ohashi  1 , Shohei Nishida  2 , Tetsuro Kamiya  1 , Tetsuya Suwa  3 , Hirokazu Hara  1 , Jun Takeda  3 , Yoshinori Itoh  2 , Tetsuo Adachi  1
Affiliations

Exendin-4 induces extracellular-superoxide dismutase through histone H3 acetylation in human retinal endothelial cells

Hiroyuki Yasuda et al. J Clin Biochem Nutr. 2016 Nov.

Abstract

Extracellular-superoxide dismutase (genetic name SOD3) is a secreted anti-oxidative enzyme, and its presence in vascular walls may play an important role in protecting the vascular system against oxidative stress. Oxidative stress has been implicated in the pathogenesis of diabetic retinopathy; therefore, increases in extracellular-superoxide dismutase have been suggested to inhibit the progression of diabetic retinopathy. Incretin-based drugs such as glucagon-like peptide-1 receptor agonists are used in the treatment of type 2 diabetes. Glucagon-like peptide-1 receptor agonists are expected to function as extrapancreatic agents because the glucagon-like peptide-1 receptor is expressed not only in pancreatic tissues, but also in many other tissue types. We herein demonstrated that exendin-4, a glucagon-like peptide-1 receptor agonist, induced the expression of extracellular-superoxide dismutase in human retinal microvascular endothelial cells through epigenetic regulation. The results of the present study demonstrated that exendin-4 induced the expression of extracellular-superoxide dismutase through histone H3 acetylation at the SOD3 proximal promoter region. Moreover, plasma extracellular-superoxide dismutase concentrations in diabetic patients were elevated by incretin-based therapies. Therefore, incretin-based therapies may exert direct extrapancreatic effects in order to protect blood vessels by enhancing anti-oxidative activity.

Keywords: diabetic retinopathy; epigenetics; exendin-4; extracellular-superoxide dismutase; incretin-based therapy.

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Conflict of interest statement

No potential conflicts of interest were disclosed.

Figures

Fig. 1
Fig. 1
Effects of Ex4 on EC-SOD expression through binding to the GLP-1 receptor. (A) HRECs were treated with the indicated concentrations of Ex4 for 24 h, followed by the measurement of SOD mRNA levels. Data are shown as the mean ± SD (n = 3). **p<0.01 vs vehicle. (B) HRECs were pretreated with (+) or without (–) 200 nM Ex9-39 for 1 h, and then treated with (+) or without (–) 100 nM Ex4 for 24 h. Real-time RT-PCR data were normalized using 18S rRNA levels. Data are shown as the mean ± SD (n = 3). **p<0.01 vs vehicle, ##p<0.01 vs Ex4-treated cells. (C) Cells were treated with or without 100 nM Ex4 for 24 h, followed by the measurement of EC-SOD mRNA level. Data are shown as the mean ± SD (n = 3). *p<0.05, **p<0.01 vs vehicle.
Fig. 2
Fig. 2
Involvement of Ex4 in DNA demethylation within the SOD3 promoter region in HRECs. cDNA or genomic DNA from HRECs and fibroblasts were purified, and followed by RT-PCR (A) or methylation-specific PCR (MSP) (B). MSP was carried out with methylation (M) and unmethylation (U) site primers after these genomic DNA were treated with bisulfite. (C) HRECs were treated with the indicated concentrations of 5-azacytidine (Aza) for 72 h, followed by the measurement of EC-SOD mRNA levels. Real-time RT-PCR data were normalized using 18S rRNA levels. Data are shown as the mean ± SD (n = 3). (D) HRECs were treated with 100 nM Ex4 for 24 h. After the treatment, the genomic DNA of HRECs was purified and treated with bisulfite, and this was followed by MSP amplification.
Fig. 3
Fig. 3
Involvement of Ex4 in histone acetylation within the SOD3 promoter region in HRECs. (A) HRECs were treated with the indicated concentrations of TSA or VPA for 24 h, followed by the measurement of EC-SOD mRNA levels. Real-time RT-PCR data were normalized using 18S rRNA levels. Data are shown as the mean ± SD (n = 3). *p<0.05, **p<0.01 vs vehicle. (B) HRECs were treated with the indicated concentrations of TSA for 24 h. After the treatment, the histones of HRECs were extracted, and this was followed by a Western blotting analysis for acetylated histone H3 (AcH3) and acetylated histone H4 (AcH4). The loading of histones was monitored by Coomassie staining. (C) HRECs were treated with the indicated concentrations of Ex4 for 24 h. After the treatment, the histones of HRECs were extracted, and this was followed by a Western blotting analysis for AcH3 and AcH4. The loading of histones was monitored by Coomassie staining. (D) HRECs were treated with 100 nM Ex4 for 24 h. After the treatment, a ChIP assay was performed as described in the Materials and methods section. Relative binding to the promoter region is expressed as the percentage amount over input (%). *p<0.05 vs vehicle (AcH3).
Fig. 4
Fig. 4
Expression and activity of HDACs by the treatment with Ex4. (A) HRECs were treated with 100 nM Ex4 for 24 h. After the treatment, real-time RT-PCR was carried out. Real-time RT-PCR data were normalized using 18S rRNA levels. Data are shown as the mean ± SD (n = 3). (B) HRECs were treated with the indicated concentrations of Ex4. After the treatment, HDAC activities were measured. Data are shown as the mean ± SD (n = 3). *p<0.05 vs vehicle.
Fig. 5
Fig. 5
Changes in plasma EC-SOD, LDL-C, and TG levels by incretin-based therapies. Data are presented as the mean ± SD of plasma levels in patients with incretin-based therapies (n = 12) pre-treatment (pre) and post-treatment (post). Significant differences (p<0.05) before and after treatments were analyzed.
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