PROJECT ABSTRACT Obesity develops when food intake chronically exceeds total energy expenditure. Prolonged ingestion of a high-fat diet (HFD) results in obesity. We and others have shown that HFD induces hyperphagia; however, the HFD effect on energy expenditure is not clear. Brown adipose tissue (BAT) effectively dissipates energy and produces heat via BAT-specific mitochondrial uncoupling protein (UCP1). BAT is innervated by stellate ganglion sympathetic nerves (SGN) that modulate BAT thermogenesis. Our pilot studies describe a novel hypothalamic R-spondin 1/POMC neurocircuit that mediates the central actions of insulin and leptin on BAT thermogenesis. Separate studies show HFD reduces SGN response and UCP1 gene expression, contributing to obesity. A higher relative abundance of gram-positive Firmicutes and fewer gram-negative Bacteroides also occurs. Involvement of gut dysbiosis is evident as HFD fails to reduce BAT thermogenesis and cause obesity in germ-free mice. Our objectives are to investigate mechanisms by which HFD reduces BAT thermogenesis. We hypothesize that HFD causes gut dysbiosis, leading to elevated circulating lipoteichoic acid (LTA), a key molecule in gram-positive bacterial cell walls; LTA, in turn, acts via toll-like receptor 2 (TLR2) signaling in the hypothalamus, reducing SGN firing and decreasing BAT thermogenesis. We will test this hypothesis with 3 specific aims: Aim 1: Define neurocircuits and signal transduction pathways in the hypothalamus responsible for insulin and leptin actions on energy expenditure. We hypothesize that the Rspo1-LGR4/POMC/BDNF neurocircuit mediates the central actions of insulin and leptin on sympathetic nerve activity (SNA) and BAT thermogenesis. Chemical phenotyping of POMC neurons and mapping of LGR4 expressing neurons in the ARC will be performed using in situ hybridization and immunofluorescence studies. Trans-synaptic tracing studies will define the Rspo1-POMC-BDNF circuit that regulates the sympathetic outflow to BAT. Aim 2: Investigate the effects of HFD on the hypothalamic Rspo1/POMC/BDNF neurocircuits. We hypothesize HFD alters this neurocircuit, downregulating the actions of insulin and leptin on SNA and BAT thermogenesis, and this is mediated by gut dysbiosis evoked by HFD. Fecal transplant from HFD mice to germ-free mice will be used to investigate the role of HFD induced gut dysbiosis; Aim 3: Examine mechanisms by which gut dysbiosis alters the hypothalamic neurocircuits, impairing SNA and BAT thermogenesis. We hypothesize elevated circulating LTA caused by gram-positive gut dysbiosis acts via hypothalamic TLR2 signaling to release TNFα which results in impaired insulin and leptin signaling and reduced SNA and decreased BAT thermogenesis. Mouse stool microbiota analysis by MiSeq will be accompanied by stool and circulating LTA measurement. TLR2 knockout mice will show TLR2’s critical role in impairing the hypothalamic neurocircuit response to insulin and leptin stimulation followin...