AICAR potentiates ROS production induced by chronic high glucose: Roles of AMPK in pancreatic β-cell apoptosis

Abstract

We previously demonstrated that chronic high glucose (33.3 mM) induced β-cell dysfunction and apoptosis through glucokinase (GCK) downregulation, but the exact mechanisms involved remain unclear. Here, we show that prolonged exposure of 5-aminoimidazole-4-carboxamide (AICA)-riboside potentiated apoptosis induced by high glucose in MIN6N8 pancreatic β-cells, correlating with enhanced GCK downregulation and decreased production of ATP and insulin. These events are potentiated in AMPK-overexpressing cells, but are prevented in cells transfected with mutant dominant-negative AMPK (AMPK-K45R). Furthermore, AMPK activation increases production of reactive oxygen species (ROS) and loss of mitochondria membrane potential induced by high glucose, which is significantly inhibited by treatment with compound C or by AMPK-K45R overexpression. Overexpression of GCK prevents apoptosis; decreased cellular ATP and insulin secretion, and ROS production enhanced by AICAR, but does not affect AMPK activation. Similar results are obtained using isolated primary islet cells. Collectively, these data demonstrate that AMPK activation potentiates β-cell apoptosis induced by chronic high glucose through augmented GCK downregulation mediated by enhanced ROS production.

Introduction

Inadequate adaptation of functional pancreatic β-cell in the face of insulin resistance contributes to the development of type-2 and type-1 diabetes [1], [2]. Previously, we have demonstrated that β-cell apoptosis by chronic high glucose occurs in relation to lowered glucokinase (GCK) expression and decreased mitochondrial association [3]. However, the exact metabolic mechanism(s) by which chronic high glucose induces GCK downregulation and apoptosis is still poorly understood.

In cells stressed by conditions such as glucose deprivation, heat shock, hypoxia, and ischemia [4], [5], [6], [7], AMPK acts as an intracellular energy sensor, stimulated by increased intracellular AMP/ATP ratios. AMPK is also activated by the adenosine analogue, 5-aminoimidazole-4-carboxamide (AICA)-riboside (AICAR), in liver cells, pancreatic β-cell lines, and in the heart and skeletal muscle [8], [9]. Initially identified as an important regulator of fatty acid oxidation in the heart and skeletal muscles, AMPK has also emerged as an important mediator of glucose metabolism, particularly glucose uptake [10], [11], [12]. Activated AMPK phosphorylates and inactivates metabolic enzymes involved in cholesterol (hydroxymethylglutaryl-CoA reductase, HMG-CoA reductase) and fatty acid (acetyl-CoA carboxylase, ACC) synthesis, thus reducing cellular ATP consumption during metabolic stress [13], [14]. At low glucose concentrations, AMPK activation inhibits glucose metabolism in pancreatic islet β-cells, and subsequently, inhibits insulin secretion [15]. Although these results have been challenged recently, it is now generally accepted that the effect of AMPK activation differs depending on duration of exposure to triggers. It has been shown that short-term activation of AMPK by AICAR inhibits glucose-induced insulin release in insulin-producing cell lines and pancreatic islets [16], [17] and that sustained activation by AICAR induces apoptosis in rat β-cells and insulin-producing MIN6N8 cells [9], [18]. Conversely, acute increases in glucose concentrations or amino acids have been shown to inhibit AMPK activity in islet β-cells [8], and that sustained hyperglycemia or chronic exposure to high glucose impairs glucose-induced insulin release and ATP production, resulting in β-cell apoptosis or glucose toxicity [19], [20], [21]. While AMPK activation is an important component for glucose metabolism in pancreatic β-cells, the precise role of AMPK activation on β-cell dysfunction, including impaired insulin secretion and apoptosis, induced by chronic exposure to high glucose remains controversial.

Mutations in GCK have been associated with maturity-onset type-2 diabetes of the young (MODY-2) [22], a disease characterized by early onset and persistent hyperglycemia. This was recently confirmed by a study demonstrating that GCK and Glut2 downregulation increased blood glucose and duration of hyperglycemia in hyperglycemia-induced rat islets [3], [22], [23]. Recently, it was suggested that activated AMPK is a major regulator of both glucose and lipid metabolism activated by oxidative stress [6]. In the complex network of glucose metabolism, ATP synthesis by oxidative phosphorylation, cellular energy metabolism (ATP/ADP ratio), redox status, ROS production, membrane potential, and substrate transport across the mitochondrial membrane are known to be involved at various steps. Although the effects of ROS on GCK expression varies depending on exposure to different drugs, hyperglycemia-induced β-cell apoptosis from ROS production is associated with reduced GCK and Glut-2 expression [24], [25]. Thus it cannot be excluded that AMPK activation may be involved in β-cell apoptosis through downregulation of GCK expression, which plays an important role in glucose metabolism, particularly glucose-stimulated insulin production and/or ROS production.

Although the role of AMPK on gene regulation is controversial and differs depending on tissue type, AMPK activation is considered responsible for transcriptional and/or translational regulation of glucose-responsive genes. Recently, it was demonstrated that long-term AMPK activation with AICAR increased expression of Glut4 in heart and skeletal muscles [26], [27], leading to increased glucose transport. Whether AMPK regulates expression of GCK and Glut2 in pancreatic β-cells, and in what manner, is still unknown however. Activated by AICAR, AMPK dephosphorylates and inactivates the mammalian target of rapamycin (mTOR) signaling pathway, which regulates multiple cellular functions, including protein translation [28]. The mTOR pathway has also been suggested to serve as an ATP sensor in the phosphorylation of p70 ribosomal protein S6 kinase (p70S6K) and eukaryotic initiation factor-4E binding protein 1 (4E-BP1), the downstream effectors in the mTOR translational pathway and upstream regulators of translational effectors [29], [30], [31], [32]. Modulation of these translational events allows for more immediate control of protein synthesis in response to changes associated with acute or chronic glucose metabolism interactions.

In this investigation, we find that AMPK activation potentiated high glucose-induced β-cell apoptosis by reducing mTOR/p70S6/4E-BP1 activation, thereby downregulating GCK expression, increasing ROS production, and decreasing intracellular insulin and ATP levels. These finding suggest that AMPK may be an important regulator of GCK expression and β-cell apoptosis by glucotoxicity. The data also suggest a novel approach to prevent and treat diabetes by mediating AMPK activation and GCK signaling.