Knockdown of GLP-1 receptors in vagal afferents affects normal food

17 Nutrient stimulation of enteroendocrine L-cells induces the release of the incretin and 18 satiating peptide glucagon-like peptide-1 (GLP-1). The vagus nerve innervates visceral organs 19 and may contribute to the mediation of gut-derived GLP-1’s effects on food intake and 20 glycemic control. To test the hypothesis that vagal afferent neuron (VAN) GLP-1 receptors 21 (GLP-1R) are necessary for the effect of endogenous GLP-1 on eating and energy balance, we 22 established a novel bilateral nodose ganglia (NG) injection technique to deliver a lentiviral 23 vector and to knock down VAN GLP-1R in male Sprague Dawley rats. We found that a full 24 expression of VAN GLP-1R is not necessary for the maintenance of long-term energy balance 25 in normal eating conditions. VAN GLP-1R knockdown (kd) did, however, increase meal size 26 and accelerated gastric emptying. Moreover, post-meal glycemia was elevated and insulin 27 release was blunted in GLP-1R kd rats, suggesting that VAN GLP-1R are physiological 28 contributors to the neuroincretin effect after a meal. Collectively, our results highlight a 29 crucial role for the VAN in mediating the effects of endogenous GLP-1 on food intake and 30 glycemia and may promote the further development of GLP-1-based therapies. 31 Diabetes Page of 30 2 INTRODUCTION 32 Glucagon-like peptide-1 (GLP-1) is an incretin and satiating hormone that has provided new 33 tools for the pharmacotherapy of obesity and diabetes (1,2). Yet, despite the clinical 34 effectiveness of GLP-1-based drugs in ameliorating the symptoms of type 2 diabetes, the role 35 of endogenous GLP-1 in the control of energy intake and glucose homeostasis is not fully 36 understood. Vagal afferent neurons (VAN) express GLP-1R (3,4) and terminate in the lamina 37 propria of the intestinal mucosa as well as in the wall of the hepatic portal vein (HPV) (5). 38 VAN may therefore relay the gut GLP-1-derived signals to the brain and, hence, mediate 39 satiating and glucoregulatory responses. Previous studies using lesioning approaches have 40 implicated the vagus nerve in the effects of peripherally administered GLP-1 on food intake 41 and glycemia (see (6) and (7) for review). In more recent studies, sudiaphragmatic vagal 42 deafferentation (SDA) in rats clearly attenuated the acute eating-inhibitory effect of 43 intraperitoneally (IP) infused GLP-1 (8) and exendin-4 (Ex-4, a GLP-1R agonist) (9). 44 Moreover, unlike Sham-operated rats, SDA rats failed to show a GLP-1R-mediated incretin 45 response (10). Based on these findings, it is reasonable to hypothesize that endogenous gut46 derived GLP-1 could activate GLP-1R on VAN in a paracrine-like fashion to reduce food 47 intake, limit gastric emptying and trigger a neural component of the incretin effect. Disruption 48 of this endogenous GLP-1 signaling mechanism in the VAN due to genetic or environmental 49 factors may contribute to the pathophysiology of obesity and diabetes. Hence, we examined 50 the physiological role of VAN GLP-1R in the control of food intake and regulation of glucose 51 homeostasis by generating a specific knockdown (kd) of VAN GLP-1R expression in rats. 52 Our approach is based on the delivery of a shRNA construct targeting the GLP-1R mRNA 53 transcript by injecting a lentiviral vector bilaterally into the nodose ganglia (NG) of rats. 54 Using RNA interference to manipulate gene expression in a tissue specific manner, we report 55 that VAN GLP-1R 1) are required for the physiological control of meal size and gastric 56 Diabetes Page of 30 3 emptying, but not for the regulation of long-term energy intake and body weight; 2) are 57 necessary for the full effects of acute IP GLP-1 and Ex-4 administration on eating and gastric 58 emptying; 3) mediate a neural component of GLP-1’s incretin effect that is physiologically 59 relevant for the post-prandial control of blood glucose. 60 Collectively, our findings establish the VAN as a major mediator of endogenous GLP-1’s 61 short-term effects on eating, gastric emptying and glycemia. They also indicate, however, that 62 the reduction of GLP-1R expression in VAN is not sufficient to promote obesity under normal 63 eating conditions. 64 65 66 MATERIAL AND METHODS 67 Animals and housing 68 Male Sprague Dawley rats (Charles River) were individually housed (21±1°C, 55±5% HR) 69 with a 12/12 h dark/light cycle. Unless otherwise noted, animals had ad libitum access to 70 water and standard chow (Kliba 3433, energy density: 3.13 kcal/g). All experimental 71 procedures were approved by the Zürich Cantonal Veterinary Office. 72 73 Lentivirus-mediated short hairpin RNA interference 74 pLKO.1-puro vectors expressing turboGFP and the U6 promoter-driven shRNA sequence 75 targeting the rat GLP-1R mRNA or a non-target shRNA sequence were obtained from Sigma76 Aldrich. Efficiency of the GLP-1R-targeting shRNA construct was verified in vitro in INS-1E 77 cells (Prof. Maechler and Prof. Wollheim, Geneva University). GLP1-R-targeting or control 78 lentiviral particles were produced in HEK 293T cells using the pMD2.G and psPAX2 79 plasmids (Prof. Trono, Addgene #12259 and 12260) and concentrated to 10 10 particles/mL 80 using 8% PEG6000 (Millipore) and resuspended in PBS. 81 Diabetes Page of 30 4