PROJECT SUMMARY/ ABSTRACT The long-term goal of my laboratory is to develop a comprehensive understanding of how intracellular metabolic pathways impact bone health. Within this scope the current project aims to explore the osteo- anabolic effects of intermittent parathyroid hormone (iPTH) via its modulation of lipid metabolism on cells within the skeletal niche. Osteoporosis and osteopenia are late-onset diseases affecting a staggering 54 million people in the U.S . In addition to the financial burden, osteoporosis-related fractures often lead to multiple comorbidities which significantly reduce longevity. While anabolic agents that increase bone formation such as PTH or PTH related protein’s (PTHrP) have aided in the management of osteoporosis, patients still experience adverse side-effects. Therefore, continued development of refined therapeutic options is necessary. As such, this project presents dual, complimentary mechanisms whereby PTH signaling up-regulates lipolysis in bone marrow adipocytes (BMAdipo), releasing lipid species into the skeletal niche, while also enhancing fatty acid oxidation in osteoblasts. Given the intimate proximity of BMAdipo to osteoblasts, it stands to reason that PTH treatment acts on both BMAdipo to liberate lipid species while also ‘priming’ the osteoblast to utilize these lipids as a mechanism capable of supporting the energy demanding process of bone formation. Therefore, our overarching hypothesis is that PTH’s bone anabolic actions involve stimulation of lipolysis in BMAdipo and enhanced fatty acid oxidation in osteoblasts. We will test this hypothesis in two specific aims. The first aim (SA1) will determine the ability of PTH-stimulated BMAdipo lipolysis to support PTH-induced bone formation, both in vivo and in vitro. This will be accomplished using a new mouse model, developed by the Co-I, designed to specifically inhibit lipolysis in BMAdipo. These mice will then be ovariectomized (OVX) or sham-operated on and be treated with PTH or vehicle control. Skeletal and metabolic parameters will be assessed at the end of the study. In addition to this experiment, lipid species released from BMAdipo upon PTH treatment will be characterized using liquid chromatography-mass spectrometry (LC-MS). The second aim (SA2) will define PTH’s ability to alter osteoblast lipid metabolism via modulation of cellular bioenergetics. Tracing of 13C18 oleic acid will be performed using LC-MS to determine fatty acid metabolic fate during PTH treatment in osteoblasts. Additionally, osteoblast mitochondrial oxidative phosphorylation substrate dependency will be measured during PTH using Seahorse XFe96 technology. Finally, osteoblast bioenergetics will be monitored using reversed- phase high-performance liquid chromatography (RP-HPLC) to determine adenosine monophosphate (AMP), adenosine diphosphate (ADP), and ATP flux under control conditions and during PTH stimulation. In summary, the goal of this project is to enhance our understan...