In the ARC NeuroHet project, I planned to combine the new Dre/rox recombinase technology with the well-established Cre/loxP technology to study the heterogeneity of the AgRP population. I planned to use the novel AgRP-Dre mouse line, engineered in the host laboratory “Neuronal Control of Metabolism” (NCM) group, at the Max Planck Institute for Metabolism Research (MPI-MR) in Cologne, Germany. By breeding AgRP-Dre mouse with the P2Y6-Cre mouse line, I can directly express a specific transgene (fluorescent reporter, Ca2+ reporter, etc.) only in neurons that express AgRP and P2Y6 markers. By doing this, I will delineate the AgRP, P2Y6 subpopulation localization, axonal projections and their response to physiological activators to unravel its specific role in energy homeostasis. Additionally, I will obtain their transcriptional profile and investigate for possible new druggable targets that affect this subpopulation exclusively. Unexpectedly, the validation of the AgRP-Dre mouse line revealed that Dre expression only reached 25-30% of the total AgRP population. This low expression level made the mouse model not suitable for the proposed experiments. Evaluation of the risk assessment, which was based in successful preliminary data obtained from the POMC-Dre transgenic line created at the host laboratory, confirmed that we underestimate an important critical risk.
Then it was decided to start an alternative research project based in the same premises as the original project: combine the Dre/rox and Cre/LoxP technologies to further explore the regulation of energy homeostasis by AgRP and POMC neurons. For this alternative project, instead of dissect neuronal subpopulations, I focused into the study of the combined net effects of AgRP and POMC activated neurons into the overall energy homeostasis. AgRP and POMC are considered to have antagonic effect in the regulation of energy homeostasis. Nevertheless, they project their axonal projections to the same areas, suggesting they can connect to similar downstream neurons. For the new project, I combine the POMC-Dre line with the well establish AgRP-ires-Cre line (obtained from B. Lowell lab) and with already available DREADDs technology using a carefully breeding strategy. By this, I will simultaneously activate and inhibited AgRP and POMC neurons, respectively. Using different experimental paradigms, I will investigate the total net effect over the regulation of food intake, glucose homeostasis and goal-oriented behaviors (Figure 1). Additionally, I will collect whole hypothalamus to analyze the transcriptional profiling of downstream neuronal networks affected by AgRP and POMC neurons. This analysis will revealed new markers of downstream neurons that participate in the regulation of body weight and glucose homeostasis. Results obtained had discover an unexpected interaction between AgRP and POMC neurons in the regulation of overall insulin sensitivity. In addition, preliminary results suggest that peripheral organs modify their sensitivity to insulin depending of the net effect composed by AgRP and POMC activated states.
