Abstract P190: The Angiotensin AT 1A Receptor Couples to G-alpha-i inAgouti-Related Peptide-ExpressingNeurons to Control Resting Metabolic Rate

Abstract

We recently demonstrated that leptin stimulates resting metabolic rate (RMR) through a mechanism that requires angiotensin (ANG) type 1A receptors (AT 1A ) localized to neurons of the arcuate nucleus (ARC) which express Agouti-related peptide (AgRP). Genetic disruption of AT 1A in AgRP neurons results in the loss of RMR responses to leptin, high fat diet, and various other stimuli which correlates with the disinhibition of AgRP, neuropeptide Y (NPY), and production enzymes and transporters (GAD1, GAD2, VGAT) for γ-aminobutyric acid (GABA) within the ARC. We hypothesize that AT 1A activation in AgRP neurons causes disinhibition of AgRP, NPY, GAD1, GAD2 and VGAT expression and thus increased inhibitory neurotransmission to pre-autonomic nuclei, and we seek to understand the second-messenger network activated by AT 1A which mediates transcriptional control of these genes. Intracellular calcium ([Ca 2+ ] i , determined by FURA-2 fluorescence), cyclic AMP (cAMP, by LANCE assay), receptor surface localization (by HiBiT-tagging), and gene expression responses (by qPCR) to ANG were examined in immortalized mouse hypothalamic cells (N43/5 and N39 cells) that express typical markers of AgRP neurons. ANG (0.1 and 1 μM, n = 3) had no effect to modulate [Ca 2+ ] i , but caused a dose-dependent reduction in forskolin-stimulated cAMP (EC 50 = 43 nM) which could be blocked by losartan (1 μM). ANG increased GTP-bound Gαi (PBS vehicle = 0.57±0.08, ANG 1 μM = 0.94±0.07 ratio vs IgG, p < 0.05, n = 3), reduced cell-surface localization of HiBiT-tagged AT 1A (PBS vehicle = 0.22±0.02, ANG 0.1 μM = 0.15±0.01, p < 0.05, n = 4), and significantly (p < 0.05, n = 6) reduced AgRP and GAD1 expression by 43% and 27%, respectively. Lastly, preliminary studies suggest that pretreatment with pertussis toxin (PTX, 100 ng/mL), an inhibitor of Gαi, abrogated AgRP and GAD1 suppression by ANG (0.01 μM). Collectively these findings support the novel concept that within immortalized cells that express markers of AgRP neurons, AT 1A couples to Gαi to reduce cAMP, which suppresses AgRP, NPY and GABA. This should disinhibit pre-motor, pre-autonomic circuits within the hypothalamus, which results in increased thermogenic sympathetic nerve activity, ultimately increasing RMR and energy expenditure.