Insulin Activates Vagal Afferent Neurons Including those Innervating Pancreas via Insulin Cascade and Ca 2+ Influx: Its Dysfunction in IRS2-KO Mice with Hyperphagic Obesity Yusaku Iwasaki 1 , Kenju Shimomura 1 , Daisuke Kohno 1 , Katsuya Dezaki 1 , Enkh-Amar Ayush 1 , Hajime Nakabayashi 2 , Naoto Kubota 3 , Takashi Kadowaki 3 , Masafumi Kakei 4 , Masanori Nakata 1 , Toshihiko Yada 1,5 * 1 Division of Integrative Physiology, Department of Physiology, Jichi Medical University School of Medicine, Shimotsuke, Tochigi, Japan, 2 Health Science Service Center, Kanazawa University, Kanazawa, Ishikawa, Japan, 3 Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 4 First Department of Medicine, Saitama Medical Center, Jichi Medical University School of Medicine, Saitama, Saitama, Japan, 5 Department of Developmental Physiology, Division of Adaptation Development, National Institute for Physiological Sciences, Okazaki, Aichi, Japan Abstract Some of insulin’s functions, including glucose/lipid metabolism, satiety and neuroprotection, involve the alteration of brain activities. Insulin could signal to the brain via penetrating through the blood-brain barrier and acting on the vagal afferents, while the latter remains unproved. This study aimed to clarify whether insulin directly regulates the nodose ganglion neurons (NGNs) of vagal afferents in mice. NGs expressed insulin receptor (IR) and insulin receptor substrate-2 (IRS2) mRNA, and some of NGNs were immunoreactive to IR. In patch-clamp and fura-2 microfluorometric studies, insulin (10 212 ,10 26 M) depolarized and increased cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in single NGNs. The insulin-induced [Ca 2+ ] i increases were attenuated by L- and N-type Ca 2+ channel blockers, by phosphatidylinositol 3 kinase (PI3K) inhibitor, and in NGNs from IRS2 knockout mice. Half of the insulin-responsive NGNs contained cocaine- and amphetamine-regulated transcript. Neuronal fibers expressing IRs were distributed in/around pancreatic islets. The NGNs innervating the pancreas, identified by injecting retrograde tracer into the pancreas, responded to insulin with much greater incidence than unlabeled NGNs. Insulin concentrations measured in pancreatic vein was 64-fold higher than that in circulation. Elevation of insulin to 10 27 M recruited a remarkably greater population of NGNs to [Ca 2+ ] i increases. Systemic injection of glibenclamide rapidly released insulin and phosphorylated AKT in NGs. Furthermore, in IRS2 knockout mice, insulin action to suppress [Ca 2+ ] i in orexigenic ghrelin-responsive neurons in hypothalamic arcuate nucleus was intact while insulin action on NGN was markedly attenuated, suggesting a possible link between impaired insulin sensing by NGNs and hyperphagic obese phenotype in IRS2 knockout mice These data demonstrate that insulin directly activates NGNs via IR-IRS2-PI3K-AKT-cascade and depolarization-gated Ca 2+ influx. Pancreas-innervating NGNs may effectively sense dynamic changes of insulin released in response to nutritional states. These interactions could serve to convey the changes in pancreatic and systemic insulin to the brain. Citation: Iwasaki Y, Shimomura K, Kohno D, Dezaki K, Ayush E-A, et al. (2013) Insulin Activates Vagal Afferent Neurons Including those Innervating Pancreas via Insulin Cascade and Ca 2+ Influx: Its Dysfunction in IRS2-KO Mice with Hyperphagic Obesity. PLoS ONE 8(6): e67198. doi:10.1371/journal.pone.0067198 Editor: Yvette Tache, University of California, Los Angeles, United States of America Received February 1, 2013; Accepted May 15, 2013; Published June 26, 2013 Copyright: ß 2013 Iwasaki 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. Funding: This work was supported by Grant-in-Aid for Young Scientist (B) (22790218, 24790221) from Japan Society for the Promotion of Science (JSPS) and Jichi medical university young investigator award to YI. A part of this study was supported by Grant-in-Aid for Scientific Research (B) (20390061, 23390044), for Challenging Exploratory Research (22659044, 24659101) and for Scientific Research on Innovative Areas (23115715) from JSPS, Strategic Research Program for Brain Sciences (10036069) by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), MEXT-Supported Program for the Strategic Research Foundation at Private Universities (2008–2012, 2011–2015), Grants from Japan Diabetes Foundation, Takeda Science Foundation and Uehara Memorial Foundation, and Insulin Research Award from Novo Nordisk and Basic Science Research Award from Sumitomo Foundation to TY. This study was subsidized by JKA through its promotion funds from KEIRIN RACE to TY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: This study was partly funded by Novo Nordisk. There are no other declarations with regards to employment, consultancy, patents, products in development or marketed products. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: tyada@jichi.ac.jp Introduction The vagal afferents, as well as the blood-brain barrier (BBB), serve as the anatomical and functional routes for signaling from the periphery to the brain. It has been shown that the intestinal hormones released upon meal intake, including cholecystokinin (CCK), glucagon-like peptide 1, and peptide YY, act on the vagal afferents to suppress food intake [1]. Insulin, a major hormone released from the pancreas upon food intake, is known to influence peripheral organs and central nerves system (CNS) to regulate a variety of physiological functions, including glucose/lipid metabolism [2,3], reduction of food intake [4,5], and growth and differentiation of the body and brain [6,7]. PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e67198