Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

et al. (2009) Sweet Taste Receptor Expressed in Pancreatic b-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion. PLoS ONE 4(4): e5106. doi:10.1371/journal.pone.0005106 Sweet Taste Receptor Expressed in Pancreatic b-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion Yuko Nakagawa 0 Masahiro Nagasawa 0 Satoko Yamada 0 Akemi Hara 0 Hideo Mogami 0 Viacheslav O. Nikolaev 0 Martin J. Lohse 0 Noriatsu Shigemura 0 Yuzo Ninomiya 0 Itaru Kojima 0 Kathrin Maedler, University of Bremen, Germany 0 1 Institute for Molecular and Cellular Regulation, Gunma University , Maebashi , Japan , 2 Department of Physiology, Hamamatsu Medical School , Hamamatsu , Japan , 3 Institute of Pharmacology and Toxicology, University of Wurzburg , Wurzburg, Germany , 4 Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University , Fukuoka , Japan Background: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. Methodology/Principal Findings: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca2+ ([Ca2+]c) and cAMP ([cAMP]c) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca2+]c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca2+]c response. The effect of sucralose on [Ca2+]c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a Gq inhibitor. Sucralose also induced sustained elevation of [cAMP]c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. Conclusions: Sweet taste receptor is expressed in b-cells, and activation of this receptor induces insulin secretion by Ca2+ and cAMP-dependent mechanisms. - Funding: This work was supported by Grant-in-Aid for Scientific Research from The Ministry of Education, Science, Sport, and Culture of Japan (19659233). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Molecular identification of the sweet taste receptor has provided new and precise insights into our understanding of the taste sensation [14]. The sweet taste receptor is a heterodimer of T1R2 and T1R3, both of which belong to a subclass of G-protein-coupled receptors resembling metabotropic glutamate receptor (mGluR), calcium-sensing receptor and pheromone receptors (V2R). Members of this subclass have large extracellular amino-terminal domains which bind most of the ligands to this region. Based on structural similarity with mGluR1, binding of ligands is thought to stabilize the active form of the sweet receptor by binding them within the cleft. Indeed, the sweet taste receptor is activated by various types of sweet substances including glucose, sucrose, fructose, artificial sweeteners including saccharin and acesulfame-K, and even proteins such as monellin and thaumatin [5,6]. Most of them are small molecules but some are much larger in size. It is thought that various types of sweeteners bind to different portions of the receptor, stabilize by different manners, and activate the bc subunit of the trimeric G protein, which subsequently activates phospholipase C-b (PLC-b) (5, 6). In addition to the taste cells in the tongue, the sweet taste receptor is also expressed in intestinal epithelial cells, in particular, in enteroendocrine cells [7]. Margolskee, et al. [8] recently showed that the sweet taste receptor expressed in enteroendocrine cells regulates the expression of sodium-dependent glucose transporter1 (SGLT1), which is expressed in enterocytes. Activators of the sweet taste receptor including dietary sugar and artificial sweeteners activate the sweet taste receptor expressed in enteroendocrine cells and induce secretion of glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, both of which stimulate the expression of SGLT1 in enterocytes. These observations clearly demonstrate that the sweet taste receptor functions as a sugar sensor in tissues other than taste buds in the tongue. In this regard, pancreatic b-cells are originated from endoderm and resemble enteroendocrine cells in many respects [9,10]. More importantly, these cells respond to fuels, especially sugars, including glucose and secrete insulin, a primary regulator of the glucose metabolism in the body. It is now generally accepted that the glucose-sensing machinery in b-cells is dependent on glucose metabolism [11], and molecules such as glucokinase and ATPsensitive potassium channel are important for glucose sensing. Nevertheless, given that the sweet taste receptor is a key molecule in sugar sensing, it is interesting to address whether or not the sweet taste receptor is expressed in b cells. In the present study, we examined whether or not the sweet taste receptor is expressed in pancreatic b-cells. We also investigated the function of this receptor using MIN6 cells. Expression of the Sweet Taste Receptor in MIN6 Cells We first examined whether or not the sweet taste receptor is expressed in MIN6 cells. As shown in Figure 1A, mRNA for T1R2 and T1R3 was detected by RT-PCR. In addition, mRNA for gustducin was also expressed in MIN6 cells. We then examined the expression of the sweet taste receptor by immunohistochemistry. Immunoreactivities of T1R2 and T1R3 were detected in MIN6 cells (Figure 1B). T1R3 signal was stronger and punctated. Effect of Artificial Sweeteners on Insulin Secretion in MIN6 Cells To determine the function of the sweet taste receptor expressed in MIN6 cells, we examined whether artificial sweeteners affected insulin secretion. As shown in Figure 2A, sucralose stimulated insulin secretion in the presence of a low concentration of glucose. Likewise, saccharin stimulated insulin secretion and acesulfame-K was much more potent. Note that 50 mM mannitol did not affect insulin secretion (data not shown). Saccharin and acesulfame-K also increased insulin secretion induced by a high concentration of glucose. Figure 2B depicts the dose-response relationship of the sucralose effec (...truncated)