Elsevier

Metabolism

Volume 63, Issue 4, April 2014, Pages 469-474
Metabolism

Translational
Des-acyl ghrelin protects microvascular endothelial cells from oxidative stress-induced apoptosis through sirtuin 1 signaling pathway

https://doi.org/10.1016/j.metabol.2013.12.011Get rights and content

Abstract

Objective

Ghrelin is a stomach-derived hormone. Acylation of ghrelin has been essential for its biological activities such as stimulating appetite. On the other hand, the function of des-acyl ghrelin (Des-G) has not been fully elucidated. The aim of the present study is to examine the anti-apoptotic effect of Des-G on endothelial cells.

Materials/Methods

After human retinal microvascular endothelial cells (RMECs) were pretreated with or without 100 nmol/L Des-G, apoptosis was induced with 0.1 mmol/L hydrogen peroxide (H2O2). For pharmacological inhibition of surtuin 1 (SIRT1) catalytic activity, the cells were treated with 10 μmol/L Ex-527. Inhibition of SIRT1 with siRNA was also performed. The quantitative estimation of DNA fragmentation was used as a marker of apoptosis. Furthermore, total SIRT activity in nuclear extracts, mRNA and protein levels of SIRT1, manganese superoxide dismutase (MnSOD) and catalase were determined.

Results

Des-G pretreatment protected RMECs from oxidative stress-induced apoptosis and increased SIRTs deacetylase activity in nuclear extracts. On the other hand, both pharmacological and siRNA mediated inhibition of SIRT1 attenuated the anti-apoptotic effect of Des-G. Moreover, Des-G increased mRNA and protein levels of SIRT1 and antioxidant enzymes such as MnSOD and CAT, which are downstream targets of SIRT1. Although the treatment of Ex-527 did not alter mRNA expression levels of SIRT1, it decreased mRNA expression levels of antioxidant enzymes in the cells with Des-G pretreatment.

Conclusions

Our results suggest that SIRT1 signaling pathway contributes to protective effect of Des-G against oxidative stress-induced apoptosis.

Introduction

Ghrelin is a stomach-derived 28-amino-acid peptide hormone that is identified as the ligand for the growth hormone-secretagogue receptor (GHS-R), and stimulates growth hormone release and appetite through GHS-R1a [1]. The Ser at the third amino acid residue is modified by n-octanoic acid and the acylated form (acyl ghrelin) has been essential for ghrelin’s biological activity [2]. In addition to the acyl ghrelin, des-acyl ghrelin (Des-G) is detected in circulation. Des-G, which is the predominant form in circulation, is unable to bind and activate GHS-R1a, therefore Des-G has been considered as the degradation product of acyl ghrelin. However, some reports have shown that Des-G may have a variety of physiological effects through an unknown receptor. Thompson et al. reported that Des-G promoted adipogenesis directly [3] and Delhanty et al. reported that Des-G acutely regulated clusters of genes involved in glucose and lipid metabolism in fat, muscle and liver, consistent with enhancement of insulin sensitivity [4]. Moreover, Benso et al. reported that the intravenous administration of Des-G improved glucose metabolism and inhibited lipolysis in humans [5].

In diabetic patients, vascular disease is the major cause of disability and death. The metabolic abnormalities of diabetes cause mitochondrial superoxide production in endothelial cells of both large and small vessels. Increased reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide (H2O2) and hydroxyl radical, causes apoptosis in vascular endothelial cells. Apoptosis in vascular endothelial cells is thought to be an important step in the development of both microvascular and cardiovascular complications [6]. In addition to the effects of Des-G for metabolism noted above, Baldanzi et al. reported that Des-G inhibited cell death in cardiomyocytes and porcine aortic endothelial cells in vitro [7]. Moreover, Togliatto et al. reported that Des-G, but not acyl ghrelin, systemic administration protected diabetes-induced endothelial progenitor cell damage. However, the role of Des-G on diabetic complications has not been fully elucidated thus far.

The aim of the present study is to examine the effects of Des-G on H2O2-induced apoptosis in human retinal microvascular endothelial cells (RMECs) and to investigate possible mechanisms. Here, we demonstrate that Des-G protects the cells from H2O2-induced apoptosis and the enhancement of sirtuin 1 (SIRT1) signaling pathway is related to the effect.

Section snippets

Cell culture and reagents

Human retinal microvascular endothelial cells (RMECs) were maintained in CSC Complete Medium (DS Pharma Biomedical, Osaka, Japan) containing 10 % FBS at 37 °C in 5% CO2. The expression of GHS-R1a mRNA was examined by RT-PCR with GHS-R1a specific primers (forward: 5’-tcgtgggtgcctcgct-3’, reverse: 5’-acgaggttgcagtactggct-3’). No expression of GHS-R1a was detected in the human RMECs (data not shown). Des-G was bought from Peptide Ins. (Osaka, Japan). H2O2 and Ex-527, a selective inhibitor of SIRT1

Des-G inhibited oxidative stress-induced apoptosis

We first tested whether Des-G pretreatment inhibited oxidative stress-induced apoptosis in human RMECs. The cells were exposed to oxidative stress with 0.1 mmol/L H2O2 for 24 h. As shown in Fig. 1, H2O2 treatment increased apoptosis by 3.43-fold compared to untreated control (p < 0.01). On the other hand, 100 nmol/L Des-G pretreatment for 24 h inhibited H2O2-induced apoptosis (3.43 ± 0.92-fold of control vs. 2.12 ± 0.63-fold of control, p < 0.01).

Des-G increased deacetylase activity of SIRTs in nuclear extracts

As it has been reported that SIRT1 overexpression inhibits

Discussion

In the present study, we demonstrate that Des-G pretreatment protects the human RMECs from oxidative stress-induced apoptosis through SIRT1 signaling pathway.

SIRT1, a nicotinamide adenine dinucleotide dependent histone/protein deacetylase, has been shown to work in a various physiological processes such as induction of cell cycle arrest, inhibition of angiogenesis and reduction of apoptosis [9]. In this study, as one of potential molecules that mediate anti-apoptotic effect of Des-G, we focused

Conflict of interest

The authors have no conflict of interest or financial disclosures to declare.

Author contributions

Takeshi Shimada and Hiroto Furuta designed experiment, performed laboratory work, analyzed data and wrote the manuscript; Asako Doi performed laboratory work and analyzed data; Hiroyuki Ariyasu, Hiromichi Kawashima, Hisao Wakasaki, Masahiro Nishi and Hideyuki Sasaki contributed to discussion. Takashi Akamizu contributed to discussion, reviewed and edited the manuscript.

Acknowledgements

This work was supported in-part by funds from Wakayama Medical University Special Grant-in-Aid for Research Projects; the Ministry of Health, Labour and Welfare of Japan; the Smoking Research Foundation; the Tokyo Biochemical Research Foundation; and the Foundation for Growth Science.

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