Hexarelin Protects Rodent Pancreatic Β-Cells Function from Cytotoxic Effects of Streptozotocin Involving Mitochondrial Signalling Pathways In Vivo and In Vitro

RESEARCH ARTICLE Hexarelin Protects Rodent Pancreatic Β-Cells Function from Cytotoxic Effects of Streptozotocin Involving Mitochondrial Signalling Pathways In Vivo and In Vitro Yan Zhao1,2,3☯, Xinli Zhang3☯, Jiezhong Chen3, Chao Lin3, Renfu Shao4, Chunxia Yan2, Chen Chen3* a11111 1 Institute of Basic Medicine Science, Xi'an Medical University, Xi'an, China, 2 Department of Forensic Science, School of Medicine, Xi’an Jiaotong University, Xi’an, China, 3 School of Biomedical Sciences, The University of Queensland, St Lucia, QLD, Australia, 4 Gene Cology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore, QLD, Australia ☯ These authors contributed equally to this work. * OPEN ACCESS Citation: Zhao Y, Zhang X, Chen J, Lin C, Shao R, Yan C, et al. (2016) Hexarelin Protects Rodent Pancreatic Β-Cells Function from Cytotoxic Effects of Streptozotocin Involving Mitochondrial Signalling Pathways In Vivo and In Vitro. PLoS ONE 11(2): e0149730. doi:10.1371/journal.pone.0149730 Editor: Irina V Lebedeva, Columbia University, UNITED STATES Received: July 20, 2015 Accepted: February 4, 2016 Published: February 26, 2016 Copyright: © 2016 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: Research work was supported by NHMRC, the University of Queensland, the National Nature Science Foundation of China (NSFC grant no. 81501175) and the Leading Disciplines Development Government Foundation of Shaanxi, China. YZ is a recipient of overseas postgraduate research scholarship from the China Scholarship Council (CSC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Mitochondrial functions are crucial for pancreatic β-cell survival and glucose-induced insulin secretion. Hexarelin (Hex) is a synthetic small peptide ghrelin analogue, which has been shown to protect cardiomyocytes from the ischemia-reperfusion process. In this study, we used in vitro and in vivo models of streptozotocin (STZ)-induced β-cell damage to study the protective effect of Hex and the associated mechanisms. We found that STZ produced a cytotoxic effect in a dose- and time-dependent manner in MIN6 cells (a mouse β-cell line). Hex (1.0 μM) decreased the STZ-induced damage in β-cells. Rhodamine 123 assay and superoxide DHE production assay revealed that Hex ameliorated STZ-induced mitochondrial damage and excessive superoxide activity in β-cells. In addition, Hex significantly reduced STZ-induced expression of cleaved Caspases-3, Caspases-9 and the ratio of proapoptotic protein Bax to anti-apoptotic protein Bcl-2 in MIN6 cells. We further examined the in vivo effect of Hex in a rat model of type 1 diabetes induced by STZ injection. Hex ameliorated STZ-induced decrease in plasma insulin and protected the structure of islets from STZ-induced disruption. Hex also ameliorated STZ-induced expression of cleaved Caspase-9 and the Bax in β-cells. In conclusion, our data indicate that Hex is able to protects βcell mass from STZ-caused cytotoxic effects involving mitochondrial pathways in vitro and in vivo. Hex may serve as a potential protective agent for the management of diabetes. Introduction Diabetes mellitus, a global public health problem that affects in excess of 350 million people worldwide [1], is now emerging as a pandemic and by the year 2025, three quarters of the PLOS ONE | DOI:10.1371/journal.pone.0149730 February 26, 2016 1 / 15 Hexarelin Protects β-Cell Function from STZ-Induced Dysfunction Competing Interests: The authors have declared that no competing interests exist. world’s 300 million adults with diabetes will be in non-industrialized countries, and almost a third in India and China alone [2]. There are two classical main types of diabetes. (1) Type 1 diabetes (T1DM) is an autoimmune disease that results in the loss of pancreatic β-cell function and hence the loss of insulin production, and accounts for about 5–10% of the population diagnosed with diabetes [3]. (2) Type 2 diabetes (T2DM) is the most common form of diabetes, accounting for >90% of the cases which result from the development of insulin resistance. T2DM, although once associated with adults and hence the older terminology of “adult onset diabetes”, is now frequently also seen in adolescents and children, and the incidence has dramatically increased worldwide [4]. The common feature of both T1DM and T2DM is the loss of glycaemic control and β-cell dysfunction [5]. Moreover, the progressive worsening of T2DM in humans is thought to result from a gradual loss of functional β-cell mass [6]. Thus, there is strong interest in dissecting the molecular pathways that lead to the decline in mass and function of β-cells in diabetes, especially as the disease remains a serious public health challenge with limited numbers of effective therapies to reverse the pathology [7]. Streptozotocin (STZ) is a widely used chemical for the induction of experimental diabetes in rodents [8, 9]. It has been used alone or in combination with other chemicals or with dietary manipulations for induction of either T1DM or T2DM [10, 11]. All these STZ-involved diabetic animal models have been very useful in elucidating the mechanisms of diabetic pathogenesis and in screening chemicals, and pharmacological agents that are potentially capable of lowering blood glucose levels [12, 13]. STZ contains a glucose molecule (in deoxy form) that is linked to a highly reactive methylnitrosourea moiety that is thought to exert STZ’s cytotoxic effects, while the glucose moiety directs the chemical to the pancreatic β-cells [14]. STZ recognizes the GLUT2 (Slc2A2) receptor which is much more abundant on β-cell plasma membranes than on other cell types (liver, kidney, small intestine) [9]. Therefore, pancreatic β-cell is accepted as a specific target of STZ. Once transported into the cell through Slc2A2 transporters, STZ kills cells by forming a toxic DNA adduct and cuntributes to mitochondrial glucotoxicity in β-cells [15, 16]. Mitochondria are essential for β-cell survival and glucose-induced insulin secretion [17–19]. Production of ATP by the mitochondria stimulates insulin exocytosis in β-cells [20]. It has also been reported that environmental and industrial pollutants can cause the death of β-cells through mitochondrial impairment [21, 22]. Thus, protection against mitochondrial dysfunction in β-cells might serve as a target in the development of novel therapeutic strategies for diabetes [23–25]. Ghrelin is a hormone mainly produced in the stomach, but also in other organs such as pancreas, which has both central and peripheral effects [26]. It acts to incre (...truncated)