SUMMARY Glucagon Like Peptide 1 (GLP-1)-expressing neurons in the hindbrain send robust projections to the paraventricular nucleus of the hypothalamus (PVN), which is involved in the regulation of food intake. Here, we describe that stimulation of GLP-1 afferent fibers within the PVN is sufficient to suppress food intake independent of glutamate release. We also show that GLP-1 receptor (GLP-1R) activation augments excitatory synaptic strength in PVN corticotropin-releasing hormone (CRH) neurons, with GLP-1R activation promoting a protein kinase A (PKA) dependent signaling cascade leading to phosphorylation of serine S845 on GluA1 AMPA receptors and their trafficking to the plasma membrane. Finally, we show that postnatal depletion of GLP-1R in the PVN increases food intake and causes obesity. This study provides a comprehensive multi-level (circuit, synaptic, and molecular) explanation of how food intake behavior and body weight are regulated by endogenous central GLP-1.

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Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiological recordings were generated in ARH neurons held at or near -60 mV, and neuronal phenotype was determined post hoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via G protein-gated, inwardly rectifying K⁺ (GIRK) channels that were attenuated by pretreatment with either 17-β estradiol (E₂) or E₂ conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and mimicked by the G_q-coupled membrane ER (G_q-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E₂ inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate L-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E₂, an effect which is stereoselective and membrane initiated via G_q-mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS.

We tested the hypotheses that steroidogenic factor (SF)-1 neurons in the hypothalamic ventromedial nucleus (VMN) provide sexually disparate, endocannabinoid (EC)- and diet-sensitive glutamatergic input onto proopiomelanocortin (POMC) neurons. Electrophysiological recordings were performed in hypothalamic slices from intact and castrated guinea pigs, along with in vitro optogenetic experiments in intact male as well as cycling and ovariectomized female NR5A1-Cre mice. In slices from castrated male and female guinea pigs, depolarized-induced suppression of excitation (DSE) time-dependently reduced the amplitude of evoked excitatory postsynaptic currents (eEPSCs) in POMC neurons generated by electrically stimulating the dorsomedial VMN. Androgen stimulation rapidly enhanced this DSE, which was also found in insulin-resistant, high-fat diet (HFD)-fed males. By contrast, retrograde signaling at VMN/ARC POMC synapses was markedly attenuated in periovulatory females. HFD potentiated central cannabinoid-induced hyperphagia in both males and females, but exerted differential influences on cannabinoid-induced increases in energy expenditure. In NR5A1-Cre mice, the reduction in light-evoked EPSC amplitude caused by postsynaptic depolarization in cycling females was modest in comparison to that seen in intact males. Estradiol attenuated the DSE in light-evoked EPSC amplitude in slices from ovariectomized females. Moreover, the retrograde inhibition of transmission was further accentuated in HFD-fed males. Chemogenetic activation of SF-1 neurons suppressed appetite and increased energy expenditure in males, effects which were attenuated by HFD. Conversely, energy expenditure was increased in estradiol- but not vehicle-treated ovariectomized females. Together with our previous studies indicating that DSE in POMC neurons is EC-mediated, these findings indicate that VMN SF-1/ARC POMC synapses represent a sexually differentiated, EC- and diet-sensitive anorexigenic component within the hypothalamic energy balance circuitry.

Testosterone exerts profound effects on reproduction and energy homeostasis. Like other orexigenic hormones, it increases endocannabinoid tone within the hypothalamic feeding circuitry. Therefore, we tested the hypothesis that testosterone upregulates the expression of diacylglycerol lipase (DAGL)α in the hypothalamic arcuate nucleus (ARC) to increase energy intake via enhanced endocannabinoid-mediated retrograde inhibition of anorexigenic proopiomelanocortin (POMC) neurons. Energy intake, meal patterns, and energy expenditure were evaluated in orchidectomized, male guinea pigs treated subcutaneously with testosterone propionate (TP; 400 μg) or its sesame oil vehicle (0.1 mL). TP rapidly increased energy intake, meal size, O₂ consumption, CO₂ production, and metabolic heat production, all of which were antagonized by prior administration of the DAGL inhibitor orlistat (3 μg) into the third ventricle. These orlistat-sensitive, TP-induced increases in energy intake and expenditure were temporally associated with a significant elevation in ARC DAGLα expression. Electrophysiological recordings in hypothalamic slices revealed that TP potentiated depolarization-induced suppression of excitatory glutamatergic input onto identified ARC POMC neurons, which was also abolished by orlistat (3 μM), the CB₁ receptor antagonist AM251 (1 μM), and the AMP-activated protein kinase inhibitor compound C (30 μM) and simulated by transient bath application of the dihydrotestosterone mimetic Cl-4AS-1 (100 nM) and testosterone-conjugated bovine serum albumin (100 nM). Thus, testosterone boosts DAGLα expression to augment retrograde, presynaptic inhibition of glutamate release onto ARC POMC neurons that, in turn, increases energy intake and expenditure. These studies advance our understanding of how androgens work within the hypothalamic feeding circuitry to affect changes in energy balance.

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Obesity and anorexia result in dysregulation of the hypothalamic-pituitary-gonadal axis, negatively impacting reproduction. Ghrelin, secreted from the stomach, potentially mediates negative energy states and neuroendocrine control of reproduction by acting through the growth hormone secretagogue receptor (GHSR). GHSR is expressed in hypothalamic arcuate (ARC) Kisspeptin/Neurokinin B (Tac2)/Dynorphin (KNDy) neurons. Ghrelin is known to inhibit the M-current produced by KCNQ channels in other ARC neurons. In addition, we have shown 17β-estradiol (E2) increases Ghsr expression in KNDy neurons 6-fold and increases the M-current in NPY neurons. We hypothesize that E2 increases GHSR expression in KNDy neurons to increase ghrelin sensitivity during negative energy states. Furthermore, we suspect ghrelin targets the M-current in KNDy neurons to control reproduction and energy homeostasis. We utilized ovariectomized Tac2-EGFP adult female mice, pretreated with estradiol ben-zoate (EB) or oil vehicle and performed whole-cell-patchclamp recordings to elicit the M-current in KNDy neurons using standard activation protocols in voltage-clamp. Using the selective KCNQ channel blocker XE-991 (40 µM) to target the M-current, oil-and EB-treated mice showed a decrease in the maximum peak current by 75.7 ± 13.8 pA (n = 10) and 68.0 ± 14.7 pA (n = 11), respectively. To determine the actions of ghrelin on the M-current, ghrelin was perfused (100 nM) in oil-and EB-treated mice resulting in the suppression of the maximum peak current by 58.5 ± 15.8 pA (n = 9) and 59.2 ± 11.9 pA (n = 9), respectively. KNDy neurons appeared more sensitive to ghrelin when pretreated with EB, revealing that ARC KNDy neurons are more sensitive to ghrelin during states of high E2.