Rat glucagon receptor mRNA is directly regulated by glucose through transactivation of the carbohydrate response element binding protein Katsumi Iizuka a,b,⇑ , Reiko Tomita a , Jun Takeda a , Yukio Horikawa a a Department of Diabetes and Endocrinology, Graduate School of Medicine, Gifu University, Gifu 501-1194, Japan b Gifu University, University Hospital Center for Nutritional Support and Infection Control, Gifu 501-1194, Japan article info Article history: Received 6 December 2011 Available online 16 December 2011 Keywords: Carbohydrate response element binding protein (ChREBP) Glucagon receptor cAMP G-box Carbohydrate response element (ChoRE) Glucagon abstract The glucagon receptor (Gcgr) is essential for maintaining glucose homeostasis in the liver and for stimulat- ing insulin secretion in pancreatic b-cells. Glucose induces rat Gcgr mRNA expression; however, the precise mechanism remains unknown. We previously have studied the role of the carbohydrate response element binding protein (ChREBP), a glucose-activated transcription factor, in the regulation of glucose-stimulated gene expression. The G-box has previously been reported to be responsible for glucose regulation of Gcgr mRNA expression. The G-box comprises two E-boxes separated by 3 bp, which distinguishes it from the carbohydrate response element (ChoRE), which has 5-bp spacing between the two E-boxes. In the rat Gcgr promoter, a putative ChoRE (À554 bp/À538 bp) is localized near the G-box (À543 bp/À529 bp). In rat INS-1E insulinoma cells, deletion studies of the rat Gcgr promoter show that ChoRE is a minimal glucose response element. Moreover, reporter assays using a pGL3 promoter vector, which harbors ChoRE and chromatin immunoprecipitation assays reveal that ChoRE is a functional glucose response element in the rat Gcgr promoter. Furthermore, In contrast, glucagon partly suppresses glucose- induced expression of Gcgr mRNA. Thus, ChREBP directly regulates rat Gcgr expression in INS-1E cells. In addition, negative feedback looping between ChREBP and GCGR may further contribute to the regulation of glucose-induced gene expression. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Type 2 diabetes mellitus (T2DM) has become a serious health problem worldwide. T2DM is characterized by a decrease in insulin secretion caused by b-cell dysfunction and death and an increase in insulin resistance [1]. The role of glucagon in this process is the focus of much attention in current research [2–4]. In the liver, the actions elicited by glucagon are essential for maintaining a euglycemic state under normal physiological conditions [2–4]. On the other hand, hyperglucagonemia is associated with hyperglycemia and diabetes under pathophysiological conditions [2–4]. A state of chronic hyperg- lucagonemia is correlated with excess hepatic glucose production and hyperglycemia in diabetic patients [2–4]. Indeed, experimental suppression of hyperglucagonemia has been shown to correct post- prandial hyperglycemia in diabetic patients [4]. Similarly, antago- nism of the glucagon receptor gene (Gcgr) and its deletion improve glucose tolerance in genetically obese mice [5–7]. Interestingly, Gcgr mRNA expression is positively regulated by glucose both in vitro and in vivo [8,9]. Elucidation of the mechanisms underlying glucose-in- duced expression of Gcgr mRNA in the liver and pancreatic islets should be of significant value in broadening the approaches to improving effective glycemic control in patients with T2DM [2–4]. We have previously studied the role of the carbohydrate re- sponse element binding protein (ChREBP), a glucose-activated transcription factor, in the regulation of glucose-induced gene expression in the liver [10–17]. Chrebp mRNA and Gcgr mRNA are generally expressed in the same tissues, including liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart, and pancre- atic islet b-cells [10,18]. ChREBP binds to the carbohydrate re- sponse element (ChoRE) to induce lipogenic gene expression [11–13,16]. ChoRE is composed of two tandem E-boxes separated by 5 bp [10,19–21]. Two CACGTG motifs, separated only by 5 bp, can induce glucose-stimulated gene transcription [19]. It has also been reported that a G-box composed of two E-box motifs sepa- rated by 3 bp forms a glucose response element in the rat Gcgr pro- moter [8,22,23]. However, whether this G-box is functional is questionable for the following reasons: (1) the rat G-box is com- posed of two E-boxes separated by only 3 bp [22,23], (2) the se- quence of the rat G-box differs from that of the mouse G-box [22–23], and (3) deletion of one E-box does not affect luciferase activities [22,23]. Since glucagon suppresses ChREBP transactivity through the cAMP-dependent protein kinase (PKA) pathway in the liver [10,24], we considered that ChREBP directly regulates 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.042 ⇑ Corresponding author. Address: Department of Diabetes and Endocrinology, Graduate School of Medicine, Gifu University, Gifu 501-1194, Japan. Fax: +81 58 230 6376. E-mail address: kiizuka@gifu-u.ac.jp (K. Iizuka). Biochemical and Biophysical Research Communications 417 (2012) 1107–1112 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc