Studies in this proposal will define the role of this cholinergic innervation of the liver in controlling glucose and lipid metabolism. The dorsal motor nucleus of the vagus (DMV) contains parasympathetic cholinergic neurons. Although there is a contradictory finding of the lack of cholinergic innervation to the mouse liver, prior studies with the retrograde neuronal tracers such as cholera toxin B, pseudorabies virus, and AAV encoding a Cre-inducible reporter protein strongly support that the mouse liver receives DMV cholinergic innervation. Furthermore, we have recently published peer reviewed work demonstrating that hepatocytes receive direct DMV cholinergic input and express muscarinic acetylcholine receptors, suggesting that the hepatic cholinergic system is critical for proper liver function. In our recent studies, optogenetic excitation of cholinergic fibers innervating the liver reduces blood glucose, consistent with the ability of parasympathetic efferent outflow to the liver to suppress hepatic glucose output. Conversely, optogenetic silencing of liver-projecting cholinergic nerves elevates blood glucose levels and also stimulates the expression of key hepatic gluconeogenic genes. As there is no change in plasma glucagon that drives hepatic glucose production, this effect appears to be pancreatic hormone-independent. In contrast to the traditional view that the activation of sympathetic nerves promotes, while the parasympathetic innervation suppresses, hepatic glucose output, our recently published work strongly supports the significant contribution of DMV cholinergic neurons to hepatic glucose output in lean mice. Importantly, our pilot studies reveal that ablation of liver-projecting cholinergic neurons increases hepatic lipolysis and insulin sensitivity in mice fed a high-fat diet. As hepatic gluconeogenesis and intrahepatic lipolysis are closely linked to each other, studies in Aim 1 will identify the necessity of these neurons in the control of ingestive behaviors and hepatic energy metabolism in lean and diet-induced obese mice. Despite the importance of parasympathetic cholinergic neurons to the regulation of hepatic glucose output via both insulin-dependent and -independent ways, it is not known that glucose sensing is an important physiological trigger for this DMV- liver neural circuit. To address this major gap, studies in Aim 2 will determine if glucose-sensing by DMV liver- projecting cholinergic neurons controls cholinergic tone to the liver. While high-fat feeding elevates hepatic sympathetic nerve activity, diet-induced obesity (DIO) reduces action potential firing of parasympathetic motor neurons. Thus, we have begun to probe if the hepatic cholinergic system is subject to modulation by the nutrient status. Our pilot studies reveal that high-fat feeding upregulates Gi-coupled muscarinic acetylcholine receptor type 2 (M2R) and 4 (M4R) expression in the liver. Accordingly, studies in Aim 3 will test the hypothesis that diet-induced alterations in the hepatic cholinergic system disrupt whole- body energy homeostasis and hepatic energy metabolism, causing insulin resistance and hepatic steatosis in DIO.

Narrative: Understanding the autonomic regulation of the liver and manipulating the brain-liver circuit have the potential to improve hepatic glucose and lipid metabolism in patients with diabetes. The potential to target specific populations of neurons to restore metabolic homeostasis will provide novel therapeutic targets for the treatment of type 1 and type 2 diabetes.