Parasympathetic Innervation and Function of Endocrine Pancreas Requires the Glial Cell Line–Derived Factor Family Receptor 2 (GFR2) Jari Rossi, 1 Paavo Santama ¨ ki, 1 Matti S. Airaksinen, 1 and Karl-Heinz Herzig 2 Vagal parasympathetic input to the islets of Langerhans is a regulator of islet hormone secretion, but factors promoting parasympathetic islet innervation are un- known. Neurturin signaling via glial cell line– derived neurotrophic factor family receptor 2 (GFR2) has been demonstrated to be essential for the development of subsets of parasympathetic and enteric neurons. Here, we show that the parasympathetic nerve fibers and glial cells within and around the islets express GFR2 and that islet parasympathetic innervation in GFR2 knockout (KO) mice is reduced profoundly. In wild-type mice, neuroglucopenic stress produced a ro- bust increase in plasma levels of islet hormones. In the GFR2-KO mice, however, pancreatic polypeptide and insulin responses were completely lost and glucagon response was markedly impaired. Islet morphology and sympathetic innervation, as well as basal secretions of the islet hormones, were unaffected. Moreover, a glu- cose tolerance test failed to reveal differences between the genotypes, indicating that direct glucose-stimulated insulin secretion was not affected by GFR2 deficiency. These results show that GFR2 signaling is needed for development of the parasympathetic islet innervation that is critical for vagally induced hormone secretion. The GFR2-KO mouse represents a useful model to study the role of parasympathetic innervation of the endocrine pancreas in glucose homeostasis. Diabetes 54:1324 –1330, 2005 E ndocrine cells in the islets of Langerhans are well innervated by sympathetic, parasympa- thetic, and sensory nerve fibers. The parasym- pathetic branch is thought to be a regulator of the physiological islet hormone secretion (1– 4). The para- sympathetic fibers in the endocrine pancreas originate from neurons in the intrapancreatic ganglia (5) that re- ceive preganglionic input from the brainstem via the vagus nerve, as well as direct input from enteric (6) and other intrapancreatic neurons (7). Activation of the vagus nerve is known to stimulate the secretion of insulin and other pancreatic hormones, although the relative contributions of noncholinergic parasympathetic neurotransmitters and the enteropancreatic projection to islet hormone secretion remain elusive (3). Meal-induced insulin secretion is tra- ditionally divided into a preabsorptive or cephalic phase that is vagally mediated (3,4) and a subsequent and much larger postabsorptive or postprandial phase that is thought to be mainly controlled by circulating glucose levels. However, several studies (8 –10) suggest that parasympa- thetic regulation of postprandial insulin secretion may be more important than previously believed. Sympathetic innervation of the pancreatic islets is pro- moted by the nerve growth factor (11,12). Factors that control the development and maintenance of islet para- sympathetic innervation, by contrast, are poorly known. Neurturin, a member of the glial cell line– derived factor family, signals through glial cell line– derived factor family receptor 2 (GFR2) and has been found to be essential for the development of enteric and parasympathetic inner- vation of several target tissues (13). GFR2 knockout (KO) mice have various neuronal deficits in cholinergic innervation along the alimentary tract, including the sali- vary and exocrine pancreatic glands and the small intes- tine (14). Here, we have combined immunohistochemical analysis with physiological tests to study the in vivo role of GFR2 signaling in endocrine pancreas innervation and islet cell function. RESEARCH DESIGN AND METHODS Immunohistological analysis of islet innervation. GFR2-KO and wild- type littermates in an F1 hybrid background (C57BL/6  129S2) were obtained and genotyped as described earlier (14,15). All animal experiments were approved by the Animal Research Ethics Committee at the University of Helsinki. Adult wild-type and GFR2-KO littermate mice were anesthetized with chloral hydrate and perfused transcardially with 4% paraformaldehyde in PBS, pH 7.5. The pancreas was removed and postfixed at 4°C for 2–3 h or overnight (depending on antibody used), cryoprotected in sucrose, and cut into 10- to 20-m sections that were stained using standard immunofluores- cence techniques. Primary polyclonal antibodies were against pancreatic polypeptide (PP) (guinea pig; Linco Research), somatostatin and glucagon (rabbit; Affiniti), insulin (guinea pig; Abcam), GFR2 (goat; R&D Systems), vesicular acetylcholine transporter (VAChT) (goat; Chemicon, or rabbit; Phoenix Pharmaceuticals, Mountain View, CA), tyrosine hydroxylase (TH) (rabbit and sheep; Chemicon), S100 (rabbit; Swant), and vasoactive intesti- nal peptide (VIP) (rabbit; Progen). Donkey secondary antibodies were from From the 1 Neuroscience Center, University of Helsinki, Helsinki, Finland; and the 2 Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, Department of Internal Medicine, University of Kuopio, Kuopio, Finland. Address correspondence and reprint requests to Dr. Jari Rossi or Matti S. Airaksinen, Neuroscience Center, P.O. Box 56 (Viikinkaari 4), 00014 Univer- sity of Helsinki, Finland. E-mail: jari.rossi@helsinki.fi or matti.airaksinen@ helsinki.fi. Received for publication 27 October 2004 and accepted in revised form 16 February 2005. Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. 2-DG, 2-deoxyglucose; GFR2, glial cell line– derived factor family receptor 2; PP, pancreatic polypeptide; TH, tyrosine hydroxylase; VAChT, vesicular acetylcholine transporter; VIP, vasoactive intestinal peptide. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1324 DIABETES, VOL. 54, MAY 2005