Investigating the Metabolic role of PTEN in Parasympathetic Phox2B neurons Title: Investigating the Metabolic role of PTEN in Parasympathetic Phox2B neurons Authors: Y Li1,2, J Rapps1,2, E Tahiri2, M Woo1,2,3 1University of Toronto, Institute of Medical Science 2Toronto General Hospital Research Institute 3Division of Endocrinology and Metabolism, Department of Medicine, UHN/SHS, University of Toronto Background: Insulin resistance and deficiency are two critical factors that contribute to the development of type 2 diabetes (T2D). One major negative regulator of insulin signalling is phosphatase and tensin homolog (PTEN) which antagonizes the insulin-mediated phosphoinositol 3-kinase pathway, the main enzymatic pathway that is activated in response to insulin. We and others have shown that loss of Pten in multiple tissues leads to improved systemic insulin sensitivity and glucose tolerance, suggesting that selective targeting of Pten may protect against T2D. Previously, our lab has shown that selective Pten knockout (KO) in rat insulin-2 (RIP2) promoter expressing neurons resulted in the activation of cholinergic anti-inflammatory reflex and improved peripheral insulin sensitivity (Wang et al Nat Med). As RIP2 promoter is expressed non-specifically in the brain, we proceeded to investigate the essential role of PTEN exclusively in parasympathetic cholinergic paired like homeobox 2B (Phox2B) neurons in the dorsal vagal complex. Approach and Results: To assess the metabolic effects of PTEN in Phox2B neurons, we generated Phox2B-PTEN KO mice using the Cre-Loxp system. Phox2B-PTEN KO mice and wildtype (WT) littermate controls were subsequently fed with a high fat diet (HFD) consisting of 60% fat content for 3 months from 6 weeks of age, and in vivo intra-peritoneal insulin and glucose tolerance tests were performed to assess glucose homeostasis. Notably, we found that deletion of PTEN in Phox2B neurons resulted in improved glucose tolerance and insulin sensitivity when compared to WT littermate controls following 3 months of HFD and gained less weight. Phox2B-PTEN KO mice had decreased liver weights and liver histological sections stained with hematoxylin and eosin or oil-red-o showed decreased liver lipid droplets. Liver triglyceride content was also decreased in Phox2B-PTEN KO mice compared to WT controls. qPCR performed on liver samples of Phox2B-PTEN KO mice showed increased expression of anti-inflammatory and decreased expression of lipogenic, fibrotic and inflammatory genes compared to controls. Phox2B-PTEN KO mice also had significantly reduced visceral white adipose tissue (vWAT) mass and histological sections revealed decreased adipocyte size and increased number compared to WT controls. qPCR analysis on vWAT samples from Phox2B-PTEN mice showed increased expression of lipolytic and adipogenesis genes with a decrease in leptin gene expression. To assess for potential mechanism by which Phox2B-PTEN KO mice were metabolically protected, we tested the role of acetylcholine by injecting mecamylamine (MEC) IP, to antagonize nicotinic receptor to ablate the effects of peripheral acetylcholine. Interestingly, MEC abolished the protective effects observed in Phox2B-PTEN KO mice including the improved insulin sensitivity, low weight gain on HFD, and decreased adiposity. Furthermore, MEC abolished the differences in gene expression of vWAT samples of Phox2B-PTEN KO mice. Similarly, vagotomy performed on Phox2B-PTEN KO mice ablated their protective metabolic effects compared to sham group. Conclusion: Our results suggest that Pten in PHOX2B neurons have a critical role in regulating energy and glucose homeostasis with alterations in liver and adipose fat content as well as inflammation. Thus, PTEN in the PNS may have an important role in regulating peripheral insulin sensitivity and inflammation.