PTEN regulates vagal-insulin signaling to optimize autonomic output determining peripheral inflammatory and metabolic homeostasis

The vagus nerve (VN) is a major component of the parasympathetic nervous system that regulates glucose and energy homeostasis. However, the specific molecular signaling pathways mediated by the VN that regulates homeostasis remain unclear. Here, we show that vagal neuron-specific deletion of phosphatase and tensin homolog (Pten), the endogenous negative regulator of PI3K, led to increased vagal output. Intriguingly, dopaminergic signaling genes were upregulated, correlating with elevated sympathetic nerve density and increased norepinephrine levels in adipose tissue of Pten-deficient mice. These mice were also protected against high-fat diet-induced obesity, insulin resistance, and systemic inflammation. To investigate insulin-specific PI3K signaling within the VN, we generated mice with vagal neuron-specific insulin receptor deletion that resulted in exacerbation of metabolic defects, which was rescued by concomitant Pten deletion. In summary, we show that insulin-PI3K-PTEN axis within vagal neurons is essential in optimizing the autonomic output that determines peripheral inflammatory and metabolic homeostasis. Overall design: Overall experimental design: P-PTEN WT and P-PTEN KO animals were weaned at 4 weeks of age and allowed to acclimatize to newly separated littermates for 2 weeks prior to performing baseline insulin/glucose tolerance tests. Following the 2 weeks, the animals and subsequently separated into chow or high fat diet consisting of 60% fat content cohorots and were maintained on their respective diets for 3 months. Following the 3 months, the animals were assessed for their vagal output , endpoint itt/gtts and metabolic parameters via metabolic caging. Animals were then subsequently euthanized and their adipose, liver and muscle tissue were collected for molecular analysis including western blot, real-time PCR alongside histological analysis Single-nucleus rna-sequencing design: Animals were fed for 2 weeks of HFD after 6 weeks of age where the body weights start to diverge between our P-PTEN WT and P-PTEN KO. Animals were subsequently euthanized and both their left and right cervical vagus nerves which contained the nodose ganglion were collected. 14 vagus nerves collected from 7 animals were then subsequently pooled into a single sample from both cohorts of mice. Nuclei were then extracted per sample and single nucleus rna-sequencing was performed. The Seurat and Harmony programs were used in RStudio version 4.2.2 to annotate and cluster different cell populations and to generate the list of genes enriched within the different cellular populations. The samples are separated into WT and KO data respectively