The brainstem preproglucagon system in a non-human primate (Macaca mulatta)

Abstract

The nucleus of the solitary tract (NTS) contains a small population of neurons expressing preproglucagon. In these neurons preproglucagon is processed to the glucagon-like-peptides 1 and 2 (GLP-1 and GLP-2) and oxyntomodulin. Whereas the neuroanatomy of these neurons is well characterized in rodents the location and projection of preproglucagon neurons have never been described in primates. The purpose of the present study was to characterize the location of preproglucagon neurons and their projections in the non-human primate using radioactive in situ hybridization and immunohistochemistry. In situ hybridization revealed preproglucagon mRNA expressing neurons in the caudal nucleus of the solitary tract extending laterally through the intermediate reticular nucleus into the A1 area. Using an antibody raised against rat GLP-2, GLP-2-immunoreactive (− ir) cell bodies were found in the same areas as the preproglucagon mRNA. Only very few GLP-2-ir nerve fibers were observed in the caudal brainstem and mostly in the same areas as the GLP-2-ir cell bodies. The most prominent GLP-2-ir terminal fields were detected in the hypothalamus and rostrally in the bed nucleus of the stria terminalis complex. In the hypothalamus, GLP-2-ir fibers arborized extensively in the paraventricular nucleus of the hypothalamus (PVN), the dorsomedial hypothalamic nucleus (DMH) and the arcuate nucleus (Arc), the latter containing the densest fiber-plexus. The findings indicate that the brainstem preproglucagon neuronal system is highly conserved between rat and non-human primate with the exception of a much denser innervation of the mediobasal hypothalamus in the primate brain.

Introduction

The proglucagon derived peptides Glucagon-like-peptides 1 and 2 (GLP-1 and GLP-2) and oxyntomodulin are all members of the brain–gut peptides that include cholecystokinin (CCK) and peptide YY (PYY). In the periphery, preproglucagon is expressed in pancreatic α-cells where the predominant post-translational form is glucagon and flanking peptides (Holst et al., 1994) and in L-cells of the intestine where post-translational processing gives rise to GLP-1, GLP-2 and smaller amounts of glicentin and oxyntomodulin (Drucker, 1998, Holst, 2007). In response to gut lumen nutrients the endocrine L-cells release a cocktail of peptides including GLP-1, GLP-2, oxyntomodulin and PYY (Tolhurst et al., 2009). Peripherally secreted GLP-1 is believed to bind and activate GLP-1 receptors located in the pancreas, on afferent vagal nerve-endings, and possibly also GLP-1 receptors in the caudal brainstem/hypothalamus, areas playing an important role in the regulation of glucose homeostasis, gastrointestinal motility and appetite (Holst, 2007, Vrang and Larsen, 2010b, Wettergren et al., 1993, Williams et al., 2009).

Preproglucagon expression has also been detected in the brain, but limited to a rather small population of neurons located in the nucleus of the solitary tract and extending laterally into the A1 area (Drucker and Asa, 1988, Jin et al., 1988). These neurons process preproglucagon as L-cells and hence produce GLP-1, GLP-2, oxyntomodulin and glicentin (Larsen et al., 1997). Since preproglucagon expression in the brain is limited to the NTS/A1 area, it has been possible to investigate the efferent projections of these neurons by immunocytochemistry using antibodies raised against proglucagon or proglucagon-derived peptides (Jin et al., 1988, Larsen et al., 1997, Rinaman, 1999a, Vrang et al., 2007). These studies have collectively shown that the brainstem preproglucagon neurons project predominantly rostrally with main terminal fields in hypothalamic areas involved in appetite regulation including the hypothalamic paraventricular (PVN), dorsomedial (DMH) and arcuate (Arc) nuclei (Jin et al., 1988, Larsen et al., 1997, Rinaman, 1999a, Vrang et al., 2007). The areas contain GLP-1 (PVN, DMH and Arc) and GLP-2 (DMH) receptor mRNA (Merchenthaler et al., 1999, Tang-Christensen et al., 2000). In line with the anatomical location of GLP-1 and GLP-2 receptors it has been demonstrated by a number of groups that central administration of either GLP-1, GLP-2 or oxyntomodulin reduces food intake (Dakin et al., 2001, Tang-Christensen et al., 1996, Tang-Christensen et al., 1998, Tang-Christensen et al., 2000, Turton et al., 1996).

Although it is generally accepted that the CNS preproglucagon systems plays a role in appetite regulation, the physiological role is still debated. Located in the NTS the preproglucagon neurons can be activated by vagal afferents. Hence, gastric balloon distension, mimicking gastric distension in response to a meal has been shown to activate preproglucagon neurons in the NTS and adjacent lateral reticular formation (Vrang et al., 2003) and central GLP-1R blockade can block the feeding inhibitory effect of gastric distension (Hayes et al., 2009) suggesting that NTS GLP-1 signalling is engaged by gastric satiation signals. In a recent study in transgenic mice expressing the fluorophore Venus under the control of the preproglucagon promoter,Hisadome et al.(2010) elegantly demonstrated that preproglucagon neurons are activated by vagal afferents and are directly activated by leptin. In further support of vagal-to-preproglucagon-neuron signaling is the findings that interoceptive stressors believed to engage vagal afferent signaling stimulation such as that caused by certain interoceptive stressors (e.g. intraperitoneal administration of LiCl, lipopolysaccharide and high doses of CCK) have been shown to activate central preproglucagon neurons (Rinaman, 1999a). In support of GLP-1 as a mediator of LiCl induced anorexia and taste aversion, both can be significantly blunted by GLP-1 receptor antagonists (Rinaman, 1999b, Seeley et al., 2000).

While the central preproglucagon system has been extensively studied in the rodent, it is unknown if this system is conserved in primates and the presence of a central preproglucagon system has yet to be reported. The purpose of this study was to characterize the distribution of preproglucagon expressing neurons and their projections in a nonhuman primate.