Glucagon-like peptides 1 and 2 and vasoactive intestinal peptide are neuroprotective on cultured and mast cell co-cultured rat myenteric neurons

Ulrikke Voss 0 Elin Sand 0 Per M Hellstrm 1 Eva Ekblad 0 0 Department of Experimental Medical Science, BMC B11, Lund University , SE- 22184 Lund , Sweden 1 Department of Medical Sciences, Uppsala University , SE-75185 Uppsala , Sweden Background: Neuropathy is believed to be a common feature of functional and inflammatory intestinal diseases. Vasoactive intestinal peptide (VIP) is an acknowledged neuroprotective agent in peripheral, including enteric, and central neurons. The proglucagon-like hormones glucagon-like peptide 1 and 2 (GLP1 and GLP2) belong to the secretin/glucagon/VIP superfamily of peptides and GLP1 and GLP2 receptors are expressed in enteric neurons. Possible neuroprotective effects of these peptides were investigated in the present study. Methods: GLP1, GLP2 and VIP were added to cultured myenteric neurons from rat small intestine or to co-cultures of myenteric neurons and rat peritoneal mast cells. Receptor selectivity was tested by the simultaneous presence of a GLP1 receptor antagonist (exendin (9-39) amide) or a VIP receptor antagonist (hybrid of neurotensin 6-11 and VIP 7-28). Neuronal survival was examined using immunocytochemistry and cell counting. Results: GLP1, GLP2 and VIP significantly and concentration-dependently enhanced neuronal survival. In addition the peptides efficiently counteracted mast cell-induced neuronal cell death in a concentration-dependent manner. Exendin(9-39)amide reversed GLP1-induced neuroprotection while GLP2- and VIP-induced enhanced neuronal survival were unaffected. The VIP receptor antagonist reversed GLP1- and VIP-induced neuroprotection while the GLP2-induced effect on neuronal survival was unaffected. Conclusions: By activating separate receptors VIP, GLP1 and GLP2 elicit neuroprotective effects on rat myenteric neurons cultured with or without mast cells. This implies a powerful therapeutic potential of these peptides in enteric neuropathies with a broad spectrum of applications from autoimmunity to functional disorders. - Background The enteric nervous system (ENS) is pivotal in the regulation and coordination of gastrointestinal (GI) motility, secretion and blood flow. GI discomfort such as nausea, bloating, abdominal pain, constipation, diarrhea or delayed gastric emptying are common features in functional bowel diseases affecting, in particular, a large number of diabetic patients [1]. The pathogenesis of GI symptoms in diabetes is not fully understood but autonomic neuropathy and abnormal glucose levels are suggested to be important. In diabetic rats neurodegeneration [2] and apoptosis [3] occur in myenteric neurons. Oxidative stress is considered the most important factor in causing diabetes-induced enteric neuropathy [4,5]. Collectively these studies indicate the occurrence of diabetes-related neurodegenerative processes in the ENS that eventually lead to neuropathy, intestinal dysfunction and GI discomfort. Glucagon-like peptide 1 (GLP1) attracts much attention due to its effects on glucose-stimulated insulin secretion, beta-cell proliferation and food intake. It is currently in clinical use in patients with type II diabetes in order to treat hyperglycemia and a number of beneficial side effects e.g. weight loss due to better satiety control [for a recent review see [6]] and protection of beta-cells against cytokine-mediated apoptosis [7] have been recognized. A number of observations also indicate that GLP1 is neuroprotective in the central nervous system [8] and clinical trials on patients with Parkinsons disease are announced [9]. In the peripheral nervous system GLP1 prevents experimentally induced sensory neuropathy [10,11]. In this context it is of interest to note that GLP1 given to patients with irritable bowel syndrome provides an effective, on demand, relief of acute pain attacks [12]. The incretin GLP1 is encoded within the proglucagon precursor and released, together with glucagonlike peptide 2 (GLP2) in a 1:1 ratio, from L-cells in the ileum and colon in response to food ingestion. GLP2 acts as an intestinotrophic factor mainly by stimulating crypt cell proliferation but it also possesses anti-apoptotic effects and enhances nutrient absorption. Protective effects of GLP2 have been explored in clinical entities like short bowel syndrome, total parenteral nutrition-induced intestinal atrophy and in inflammatory bowel disease (IBD) [for a review see [13]]. In trinitrobenzene sulfonic acid or dextran sodium sulfate models of IBD GLP2 treatment reduces the intestinal inflammation and counteracts inflammation-induced loss of enteric neurons. In addition, GLP2 treatment increases the number of vasoactive intestinal peptide (VIP)-expressing enteric neurons and the possibility that GLP2 effects are mediated via release of VIP is suggested [14,15]. VIP exhibits established neuroprotective properties in peripheral, including enteric, and central neurons [16-20]. Aims of the present study were to investigate possible neuroprotective effects of GLP1, GLP2 and VIP on myenteric neurons from adult rat small intestine. Two different in vitro models were used. First the ability of the three peptides to enhance neuronal survival was tested on myenteric neurons in primary culture. Next neuroprotective effects of the peptides were tested in an in vitro system in which enhanced neuronal cell death was generated by co-culturing myenteric neurons with mast cells. Receptor selectivity was, in both these models, tested by using a GLP1 receptor antagonist (exendin (9-39)amide) and a VIP receptor antagonist (hybrid neurotensin 6-11 and VIP 7-28; hybVIP). GLP1, GLP2 and VIP were in the present study found to efficiently protect myenteric neurons in two different culture systems. Separate receptors, suggested to be neuronally expressed, were activated. These results strongly point towards a powerful therapeutic promise for these three peptides in the prevention of enteric neuropathy in diseases like diabetes, but also in inflammatory and neurodegenerative diseases. Methods Animals Female Sprague-Dawley rats (n = 38, 170-180 g), purchased from Charles River, Sulzfeld, Germany, were used. The rats were allowed to acclimatize to the climate- and light-controlled animal facility for at least 5 days prior execution. Standard rat chow and water were supplied at all times. The experimental design was approved by the animal ethics committee, Lund and Malm, Sweden. Animals were used in accordance with the European Communities Council Directive (86/609/ EEC and 2010/63/EU) and the Swedish Animal Welfare Act (SFS 1988:534). Myenteric neuronal cultures Primary cultures of myenteric neurons were prepared from rat small intestine using a previously described method [21]. In brief, rats were deeply anaesthetized using chloral hydrate (300 mg/kg body weight) and the small intestine was exposed via a midline incision. The longitudinal muscle with attached myenteric ganglia were stripped from approximately 20 cm of the distal (...truncated)