Investigation of Insulin Receptor‐Associated Neuronal Phenotypes in the Subfornical Organ of the Brain
Authors: Jeong Jin Kwon , Simonyan Hayk , Young Colin 
Published: June 2021

The subfornical organ (SFO), a forebrain nucleus that lacks a blood‐brain‐barrier and integrates circulating factors with downstream neural circuits, is strongly implicated in cardiovascular and metabolic physiology. Despite remarkable progress in unraveling the function of the SFO in cardiometabolic control, the underlying cellular phenotypes and signaling mechanisms remain unclear. The hormone insulin acts within the central nervous system to influence cardiometabolic regulation, and alterations in brain insulin signaling have been implicated in metabolic syndrome‐associated conditions including obesity, diabetes and hypertension. We have recently demonstrated that insulin receptors are widely expressed throughout the rostral to caudal extent of the SFO. Building upon this, we investigated insulin receptor‐associated cellular phenotypes and intracellular signaling pathways in the SFO. Immunohistochemistry in male C57Bl/6 mice (n=4) for insulin receptors and the neuronal marker NeuN revealed that SFO insulin receptors are predominately expressed on neurons (80 ± 3% of insulin receptor positive cells). To dissect the neuronal phenotype upon which SFO insulin receptors are expressed, we next utilized a GABA reporter mouse strain (GAD67‐mCherry; n=3). Immunohistochemistry revealed that approximately one third of all SFO insulin receptor positive cells were localized to GABAergic neurons. On the other hand, double immunohistochemistry for insulin receptors and the excitatory neuronal marker Ca 2+ /calmodulin‐dependent protein kinase II (CamKII) demonstrated abundant insulin receptor expression on SFO glutamatergic neurons (77 ± 3% insulin receptor/CAMKII co‐localization, n=3). Importantly, the SFO is critical in mediating angiotensin‐II responses, and within the SFO angiotensin‐II type1a receptor (AT1aR) expressing neurons are exclusively glutamatergic. Therefore, we also examined potential co‐localization of insulin receptors and AT1aR in the SFO using an AT1aR eGFP reporter strain (GENSAT NZ44). Interestingly, insulin receptors were found to be highly expressed on SFO AT1aR‐expressing cells (69 ± 4% insulin receptor/AT1aR co‐localization, n=3). Brain insulin receptors act through two major intracellular signal cascades: mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase (PI3K) pathways. Therefore, we further examined SFO insulin receptor‐associated downstream signaling using immunohistochemistry. Relative to vehicle controls, acute insulin administration (i.p. 5U) resulted in activation of the PI3K pathway, as indicated by phosphorylation of AKT (100 ± 8 vs. 159 ± 7, density/area, saline vs. insulin, p<0.05, n=3–4). SFO phosphorylated ERK1/2, a marker for MAPK pathway activation, was not changed with insulin stimulation (100 ± 21 vs. 114 ± 9, density/area, saline vs. insulin, p=0.5, n=3–4). Collectively, these findings indicate that in the SFO: 1) Insulin receptors are abundantly expressed in inhibitory and excitatory neurons; 2) Insulin may interact with angiotensin‐II; and 3) Insulin appears to predominately utilize PI3K intracellular signaling in the SFO.Support or Funding InformationNIH R01DK117007 & R01HL141393This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.