CREB and ERK Activation by Leptin and Angiotensin in the GT1‐7 Cell Model by Capillary Electrophoresis‐Based Western Blotting

Abstract

Leptin (LEP) increases resting energy expenditure in part by decreasing Agouti‐related peptide (Agrp) within the arcuate nucleus (ARC) of the hypothalamus. Prolonged obesity is associated with hyperleptinemia and development of selective LEP resistance (SLR), referring to the loss of ‘metabolic’ but maintenance of ‘cardiovascular’ sympathetic nervous activity responses to LEP. Previously, we discovered that angiotensin II (ANG) type 1a (Agtr1a) receptors located on Agrp neurons of the ARC are dispensable for blood pressure control but critically required for metabolic responses to LEP. Therefore, we generally hypothesize a role for Agtr1a in Agrp neurons of the ARC in the development of SLR. We used single‐nucleus RNA‐seq followed by Ingenuity Pathway Analysis (IPA) to interrogate the transcriptomes of individual cell types of the ARC from mice after 10 weeks of 45% high fat diet (HFD) or chow. We determined that canonical LEP signaling was strongly under‐represented in Agrp neurons, but not other cell types. Similarly, CREB & ERK pathways, which are implicated in the control of Agrp expression, were significantly under‐represented within Agrp neurons from HFD‐fed mice. To explore changes in the function of these second‐messenger cascades in Agrp neurons, immortalized mouse hypothalamic GT1‐7 cells were examined for expression of relevant genes and activation of signaling cascades in response to various stimuli. First, GT1‐7 cells were confirmed to express Agrp, Lepr, and Agtr1a, but not proopiomelanocortin by qPCR. Second, GT1‐7 cells were treated with LEP (1uM), ANG (200nM), forskolin (30uM), or vehicle for 15 min and lysates were isolated for Western blotting. Capillary electrophoresis methodology (Protein Simple WES; standard matrix (12–230 kDa)/anti‐rabbit detection module) was specifically chosen to enable future studies of ARC Agrp neurons captured after dietary interventions by laser capture microdissection (LCM). Phosphorylated (p) CREB (1:50; CST # 9198), total (t) CREB (1:50; CST #9197), p‐ERK (10ug/ml; R&D Systems # AF1018‐SP), t‐ERK (10ug/ml; R&D Systems # AF1576‐SP) and β‐actin (1:100; Abcam ab8227) protein levels were assessed for all four treatments. Peak signal‐to‐noise (S/N) ratios were well above the threshold value of 10 for all protein samples: p‐CREB (ranging from 560–1500), t‐CREB (25–40), p‐ERK (115–1245) t‐ERK (150–270) and β‐actin (1400–1600). LEP, ANG, and forskolin treatment significantly (p<0.05; n=5–6 each) increased p‐CREB and p‐ERK with no change in t‐CREB or t‐ERK, compared to vehicle. Finally, the viability of protein collection from the ARC was confirmed and optimized by measuring β‐actin from cells isolated by LCM. Ongoing studies are focused on assessing ARC Agrp neuron CREB & ERK responses to acute leptin treatment after prolonged exposure to HFD.Support or Funding InformationHL134850, HL084207