br Acknowledgements br Introduction Glucagon a amino acid

Acknowledgements
Introduction Glucagon, a 29-amino Cisplatin peptide, is released from the pancreatic islets, intestine and stomach. Glucagon is released under hypoglycemic conditions and then elevates blood glucose levels, serving as a major counter hormone [1]. The regulation of glucose metabolism by glucagon is mediated by its direct action on the peripheral tissues such as the liver [1] and also by the brain [2], [3]. Glucagon is also released postprandially in a transient manner [4], [5], [6], which was shown to be involved in inhibition of food intake via reduction of meal size [7], [8], [9], [10], [11]. Glucagon and insulin, both released postprandially, elicit an additive inhibition of food intake [12]. Furthermore, glucagon is secreted by cold exposure and plays a role in increasing energy expenditure and thermogenesis via activation of brown adipose tissue (BAT) [8]. Glucagon not only directly regulates BAT, but acts through the sympathetic nervous innervating BAT [8], [13], [14]. Thus, glucagon regulates glucose metabolism, feeding, energy expenditure and thermogenesis at least partly by influencing the brain. However, the route through which peripheral glucagon informs the brain is less defined, and the target site for possible cooperation between glucagon and insulin on feeding is not clear. Peripheral substances influence the brain primarily via (1) entering the brain through blood–brain barrier (BBB) to act on the target cells in the brain and/or via (2) interacting with the vagal afferent nerves that connect peripheral organs to the medulla of brain stem [15], [16]. It has been shown that the transfer of glucagon from the circulation to the brain is strongly restricted by BBB [17]. Among pancreatic islet hormones, insulin and pancreatic polypeptide have been shown to influence the brain functions such as satiety [18], [19], [20] and memory [21], [22], and to activate the nodose ganglion (NG) neurons that compose the afferent vagal nerves [23], [24]. Hence, glucagon could also act on the vagal afferent nerves. In the present study, we examined whether glucagon directly interacts with the vagal afferent NG neurons and whether glucagon and insulin target the common NG neurons in mice. We investigated the direct effect of glucagon on cytosolic Ca2+ concentration ([Ca2+]i) in single neurons isolated from NG. Effect of intraperitoneal (ip) injection of glucagon on NG neurons was assayed by phosphorylation of extracellular signal regulated kinase 1 and 2 (ERK1/2) (also known as mitogen-activated protein kinases), which are activated by membrane depolarization and Ca2+ influx in PC12 cells [25], brain neurons [26], [27], [28] and primary afferent (dorsal root ganglion) neurons [29]. We found that glucagon directly activates vagal afferent NG neurons via the glucagon receptor, and that glucagon and insulin act on the same NG neurons.
Materials and methods
Results
Discussion The present study has demonstrated that glucagon directly interacts with single NG neurons to induce [Ca2+]i signaling via the glucagon receptor. Ip injection of glucagon induced pERK1/2 in NG as early as 15min after injection. The incidence of the NG neurons with pERK1/2 in vivo and that of single NG neurons with [Ca2+]i increases in vitro are comparable (approximately 8%) (Figs. 1C and 2C). These data suggest that the ip glucagon-induced activation of vagal afferents may be due to its direct action on the NG neurons. This finding of glucagon activation of vagal afferent neurons is in accordance with previous report that glucagon injection activates the neurons in the nucleus tractus solitarius (NTS) of the brainstem to which vagal afferents project [34]. In this study, relatively small fraction of NG neurons (8%) responded to glucagon and relatively high concentration of glucagon (10−8M) was required. However, these properties appear to be reasonable in the light of the heterogeneity and role of vagal afferents. The terminals of vagal afferents innervate peripheral organs and sense the local information in/around the organs, including gastrointestinal hormones [16], [35] and pancreatic insulin [24]. Gastrointestinal and pancreatic hormones exist in much higher concentrations in/around the organs releasing these hormones than in the peripheral circulation. In fact, the insulin concentration in the pancreatic vein is approximately 100-fold higher than that in the circulation [24]. Insulin at high concentrations activates the NG neurons innervating the pancreas with much higher incidence than other NG neurons [24]. In the present study, vagal afferent neurons were activated by glucagon at 10−9M or higher (local concentration) but not 10−10M (circulating concentration). Furthermore, the majority of glucagon-responsive NG neurons also responded to another pancreatic hormone, insulin. Taken together, the subpopulations of NG neurons innervating the pancreas and/or portal vein may sense the local glucagon that is present at a higher concentration.