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