Assessing the Effects of Leptin and Angiotensin II Upon Expression of Agouti‐Related Peptide in the GT1‐7 Cell Model

Abstract

Leptin acts at its receptor (Lepr) within the arcuate nucleus of the hypothalamus (ARC) to reduce feeding behavior and increase resting metabolic rate (RMR) in part through the inhibition of Agouti‐related peptide (Agrp) signaling. Previously we demonstrated that a unique subset of Agrp neurons express the angiotensin II (ANG) type 1a receptor (Agtr1a). Genetic deletion of Agtr1a from Agrp neurons of mice was sufficient to disinhibit expression of Agrp within the ARC, and to attenuate adipose sympathetic nerve activity responses to leptin. In contrast, RMR responses to brain renin‐angiotensin system (RAS) stimulation are intact in the dysfunctional Lepr db/db mouse model, and acute leptin administration does not additively exaggerate RMR activation in a transgenic mouse model of increased brain RAS activity. New unpublished data also indicate that ANG signaling within the ARC stimulates RMR through suppression of Agrp expression and Agrp neuron electrical activity, and thereby disinhibition of melanocortin Mc4r receptor signaling. To further dissect the interaction of Lepr and Agtr1a within Agrp neurons, here we examined the effects of leptin and ANG stimulation of the “GT1‐7” immortalized cell culture model of mouse Agrp‐expressing cells. We confirmed that cultured GT1‐7 cells express critical markers of the Agtr1a‐positive Agrp neuron subtype, including Agrp, Agtr1a, and Neuropeptide‐Y (Npy), but not pro‐opiomelanocortin (Pomc). Dose‐ and time‐course experiments were performed to identify mid‐range doses and time courses for Agrp gene expression responses to acute application of leptin and ANG. Subsequently, Agrp expression responses to low doses of leptin (100 nM) and ANG (100 nM), which would allow detection of any additive or synergistic effect, were assessed after 1 and 2–2.5 hours to evaluate the hypothesis that leptin and ANG signaling are non‐additive for the suppression of Agrp expression. After 1 hour, ANG (0.66 (0.49–0.91), fold of vehicle (± 1 sem), n=0.5) and leptin (0.61 (0.44–0.84), n=6) had similar effects on Agrp mRNA expression compared to vehicle (1.00 (0.86–1.17), n=7), but co‐administration of both peptides had no synergistic effect (0.96 (0.78–1.18), n=5). Likewise after 2–2.5 hours, compared to vehicle (1.00 (0.78–1.28), n=6), ANG (0.56 (0.34–0.94), n=6) and leptin (0.69 (0.57–0.85), n=6) had similar effects that were not additive (0.81 (0.67–0.98), n=6). These findings support the preliminary conclusion that the effects of leptin and ANG upon Agrp expression are not additive in the GT1‐7 cell culture model of Agrp neurons. These findings support our working model in which leptin action at the ARC results in increased Agtr1a signaling to suppress Agrp, ultimately to disinhibit Mc4r signaling and thus increase resting energy expenditure.Support or Funding InformationHL134850, HL084207