Insulin Activates Parasympathetic Hepatic-related PVN Neurons Through mTOR signaling

Paraventricular nucleus of hypothalamus (PVN) is an important brain region regulating a diversity of autonomic functions, such as hepatic glucose production (HGP). Classical insulin-dependent inhibition of HGP occurs through peripheral actions on liver. However, evidence implicates brain areas, like PVN, in insulin-dependent inhibition of HGP. Although, the precise neuromechanistic circuit(s) responsible for insulin actions in PVN remains unclear. We hypothesized that insulin activates hepatic-related PVN neurons (PVNhepatic) through a parasympathetic pathway. First, the transsynaptic retrograde tracer pseudorabies with an eGFP reporter (PRV-152) was injected into the liver to determine if parasympathetic circuits are utilized by PVNhepatic. We found that the parasympathetic motor output nucleus, dorsal motor nucleus of the vagus (DMV) was labeled at 48h after injection (5±1 neurons, n=4), with increased labeling at 72h (124±36 neurons, n=4, p=0.0337 vs 48h) and 96h (285±44 neurons, n=4, p=0.0001 vs 48h; ANOVA Two-Way with Tukey’s). Congruent with direct PVN→DMV projections, PVN neurons were labeled 24h later than DMV (72h: 111±34 neurons, n=4, vs 48h: 1±1, n=4 neurons, p=0.046; ANOVA Two-Way with Tukey’s). To confirm that PVNhepatic were part of a parasympathetic pathway, a subset of mice underwent a left cervical vagotomy before hepatic PRV-152 injection. Vagotomy abolished labeling in PVN at 72h and 96h (202±41 neurons, n=8, vs vagotomy: 6±1 neurons, n=4; p=0.0001; ANOVA Two-Way with Tukey’s). Finally, to determine if PVNhepatic were responsive to insulin, these neurons were investigated using on-cell patch-clamp technique in the presence of GABAA receptor antagonist, picrotoxin (100μM), and ionotropic glutamate receptor antagonist, kynurenic acid (1mM). Bath application of insulin (1μM) increased action potential (AP) firing frequency of PVN labeled neurons (before: 0.6±0.2 Hz vs insulin: 1.0±0.2 Hz, n=9; p=0.04; Student t test) but failed to alter AP frequency in unlabeled PVN neurons (before: 0.2±0.1 Hz vs insulin: 0.1±0.1 Hz, n=5, p=0.33; Student t test). To evaluate if insulin-dependent increases in hepatic-related PVN neuronal activity are mediated through canonical insulin intracellular signaling pathways, insulin application was repeated in the presence of mammalian target of rapamycin antagonist, rapamycin, (200nM). Pre-treatment with rapamycin occluded the ability of insulin to increase PVNhepatic activity (before: 0.3±0.1 Hz, vs insulin: 0.2±0.1 Hz, n=8, p=0.87; ANOVA Two-Way with Tukey’s). Taken together, these data suggest that canonical mTOR-dependent insulin signaling increases activity of parasympathetic PVNhepatic and this response to insulin is unique to this PVN subpopulation. Therefore, PVNhepatic are likely mediating central insulin-dependent vagally-mediated inhibition of hepatic function. FAPESP #2021/10017-4 to KMS, NIH R01HL157366 to CRB. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.