Elsevier

Cellular Signalling

Volume 25, Issue 12, December 2013, Pages 2348-2361
Cellular Signalling

In vivo activating transcription factor 3 silencing ameliorates the AMPK compensatory effects for ER stress-mediated β-cell dysfunction during the progression of type-2 diabetes

https://doi.org/10.1016/j.cellsig.2013.07.028Get rights and content

Highlights

  • AMPK phosphorylation was remarkably increased at 19 week ZDF rats.

  • ER stress and ATF3/ROS play an important role in β-cell dysfunction.

  • ATF3 knockdown using in vivo-jetPEI attenuated ER stress-mediated β-cell dysfunction.

  • ATF3 plays as a counteracting regulator of AMPK and thus promote β-cell dysfunction.

Abstract

In obese Zucker diabetic fatty (ZDF) rats, ER stress is associated with insulin resistance and pancreatic β-cell dysfunction; however the exact mechanisms by which ER stress drives type-2 diabetes remain uncertain. Here, we investigated the role of ATF3 on the preventive regulation of AMPK against ER stress-mediated β-cell dysfunction during the end-stage progression of hyperglycemia in ZDF rats. The impaired glucose metabolism and β-cell dysfunction were significantly increased in late-diabetic phase 19-week-old ZDF rats. Although AMPK phosphorylation reduced in 6- and 12-week-old ZDF rats was remarkably increased at 19 weeks, the increases of lipogenice genes, ATF3, and ER stress or ROS-mediated β-cell dysfunction were still remained, which were attenuated by in vivo-injection of chemical chaperon tauroursodeoxycholate (TUDCA), chronic AICAR, or antioxidants. ATF3 did not directly affect AMPK phosphorylation, but counteracts the preventive effects of AMPK for high glucose-induced β-cell dysfunction. Moreover, knockdown of ATF3 by delivery of in vivo-jetPEI ATF3 siRNA attenuated ER stress-mediated β-cell dysfunction and enhanced the beneficial effect of AICAR. Our data suggest that ATF3 may play as a counteracting regulator of AMPK and thus promote β-cell dysfunction and the development of type-2 diabetes and could be a potential therapeutic target in treating type-2 diabetes.

Introduction

Type-2 diabetes (T2D) is one of the most prevalent metabolic disorders associated with abnormal lipid and glucose metabolism and it is a principal source of morbidity and mortality worldwide [1], [2]. Obesity is a major underlying pathology associated with the development of T2D, which occurs as pancreatic β-cells fail to compensate for increased insulin demand when the body becomes insulin resistant and hyperglycemia [3]. Several studies have shown that prolonged exposure to fatty acids and high glucose levels, as well as lipid accumulation at sites other than adipose tissue, contribute to insulin resistance and β-cell dysfunction [4], [5]. Specifically, the aforementioned lipid accumulation is caused by insulin secretory defects, as well as a loss of β-cell mass due to apoptosis [6]. Recently, the inhibition of pancreatic β-cell function that occurs in obese ZDF rats, a commonly used T2D animal model [7], was associated with impaired islet lipid homeostasis and glucolipotoxicity, which leads to oxidative stress, ER stress, inflammation, and β-cell apoptosis [8]; however, the underlying mechanisms that lead to β-cell dysfunction are still unclear.

The endoplasmic reticulum (ER) is a specialized cellular organelle that is responsible for the synthesis, packaging, and assembly of secretory and membrane proteins [9]. Because the pancreatic β-cell is the only source of circulating insulin, which is essential for both stimulating peripheral tissue glucose uptake and inhibiting hepatic glucose production, insulin-producing β-cells are very susceptible to changes in ER homeostasis and therefore ER stress, which can result in the accumulation of unfolded and/or aggregated proteins and the activation of the ER stress sensors PERK, IRE1, and ATF6 [10]. Severe or prolonged ER stress promotes lipid accumulation and ROS production, which subsequently lead to β-cell apoptosis, and consequently, diabetes mellitus [11], which were supported by several studies using genetic intervention or chemical chaperones [12], [13]. Although cells elicit a normal insulin secretory response to glucose when the levels are within a homeostatic physiological range, chronically elevated glucose leads to ER stress by the activation of ER stress sensors that alter the expression of downstream gene and increase ROS production from multiple sources, which ultimately leads to β-cell dysfunction and apoptosis [14], [15]. However, the upstream or downstream mediators of the ER stress response that promotes fulminant damage to pancreatic β-cells in obese diabetic animal models are still unclear.

5′-AMP-activated protein kinase (AMPK), an energy sensor that is sensitive to changes in the AMP/ATP ratio, is considered to be an important regulator of glucose and lipid metabolism in peripheral tissues of humans and rodents with metabolic stress, obesity or diabetes [16]. AMPK activity is generally inhibited as glucose concentrations rise above the physiological range in various cells, which is especially true in islet β-cells [17]. AMPK activation by AICAR inhibits glucose-induced insulin release and impaired glucose or lipid homeostasis in pancreatic β-cells [18], [19]. However, these ideas have been recently challenged, as some studies have shown that the sustained activation of AMPK is associated with the induction of β-cell apoptosis [20], [21]. Despite these discrepancies, an increasing amount of evidence has indicated that AMPK activation is inhibitory to the development of obesity and T2D by suppressing hyperglycemia and thus improving β-cell function [22]. In multiple studies using ZDF rats, which are characterized by progressive β-cell dysfunction, AMPK activation by AICAR prevented the development of hyperglycemia and preserved β-cell mass [23]. Although AMPK has been extensively studied as a potential target for the treatment of hyperglycemia, as well as for the pathogenesis of T2D and obesity [24], [25], [26], the role and regulatory mechanism of AMPK hyper-activation observed in hyperglycemia-mediated fulminant damage to pancreatic β-cells, especially on the post-diabetic phase, are still unknown.

Activating transcription factor 3 (ATF3), a stress-inducible gene that encodes a member of the ATF/cAMP response element-binding (CREB) family of transcription factors, is induced by signals relevant to the pancreatic β-cell dysfunction such as proinflammatory cytokines, nitric oxide, and high concentrations of glucose and free fatty acid [27]. Previously, we demonstrated that ATF3 plays a critical role in β-cell dysfunction and apoptosis through glucokinase (GCK) nitration and downregulation, which could be triggered by enhancing peroxynitrite, iNOS and NO generation [28]. In addition, we reported that ATF3, which consequently induced by ER stress-mediated JNK activation, was involved in the reduction of pancreatic and duodenal homeobox-1 (PDX-1) as well as adiponectin transcription [29], [30]. However, the specific role of ATF3 and associated regulatory mechanism in pancreatic β-cell dysfunction and apoptosis during the development of T2D remain elusive. Therefore, it is necessary to identify how AMPK contributes to impaired glucose metabolism as well as its role in the ER stress-mediated fulminant damage to β-cells during the progressive development of obesity and T2D. In our study, we found that ER stress is a main pathway that promotes the progression of obese T2D in ZDF rats. Furthermore, AMPK activation is greatly increased in end-stage diabetes to serve a protective role against hyperglycemia-induced ER or oxidative stress. However, these compensatory roles in restoring and maintaining cellular homeostasis were overwhelmed by excessive increases of ER stress or ROS production, thereby resulting in β-cell dysfunction and apoptosis. Finally, we can suggest that ATF3 may play as a counteraction molecule for the beneficial effect of AMPK and thus triggers β-cell dysfunction and the development of T2D.

Section snippets

Cell line and animals

Rat INS-1 pancreatic β-cells were cultured in PRMI 1640 containing 11.1 mM glucose supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, 10 mM HEPES, and 100 IU/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Gaithersburg, MD). Male Zucker diabetic fatty(ZDF/Gmi-fa/fa) rats(6, 12, 19 week-old) and their sex- and age-matched Zucker lean control(ZLC/Gmi-+/fa) rats were purchased from Genetic Models(GMI, Indianapolis, IA). Animals were

Pancreatic β-cell dysfunction and apoptosis are correlated with AMPK activation and lipid accumulation in ZDF rats

We have firstly investigated glucose homeostasis at 6, 12, and 19-week-old ZDF rats (Fig. S1). Plasma glucose levels were potently increased with a time in ZDF rats (S1A). Secreted insulin levels peaked at 6 and 12 weeks in the ZDF rats were significantly decreased at 19-week (S1B), accompanied by the progressive development of hypoinsulinemia (not shown) and impaired glucose metabolism leading to overt diabetes (S1C). Compared with ZL rats, the islets of 6- and 12-week-old ZDF rats were

Discussion

In these studies, we demonstrate that ATF3 plays as a counteraction regulator against a preventive response of AMPK for ER stress-mediated pancreatic β-cell fulminant damage during the progression to T2D. ZDF rats serve as a mimic model of human T2D that is characterized by insulin resistance, β-cell defects, and hyperglycemia [43]. Although the animals have early compensatory responses to hyperglycemia-induced fulminant damage to pancreatic β-cells, the exact molecular mechanisms associated

Acknowledgment

We thank Dr. M. Birnbaum and Dr. T. Hai for providing plasmid cDNA (M.B., pcDNA-empty, pcDNA-AMPK-WT, and pcDNA-AMPK-K45R; T.H., ATF3 cDNA).

This work was supported by research grants from the Korean National Institutes of Health (4845-302-210-13).

No potential conflicts of interest relevant to this article were reported.

J.Y.K. and W.H.K. researched data, contributed to discussion, wrote, reviewed, and edited the manuscript. K.J.P., G.H.K, E.A.J., and D.Y.L. researched data. S.S.L., D.J.K.,

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