Insulin resistance is at the core of the metabolic syndrome, and men exhibit a higher incidence of metabolic syndrome than women in early adult life, but this sex advantage diminishes sharply when women reach the postmenopausal state. Because 17β-estradiol (E2) augments the excitability of the anorexigenic proopiomelanocortin (POMC) neurons, we investigated the neuroprotective effects of E2 against insulin resistance in POMC neurons from diet-induced obese (DIO) female and male mice. The efficacy of insulin to activate canonical transient receptor potential 5 (TRPC5) channels and depolarize POMC neurons was significantly reduced in DIO male mice but not in DIO female mice. However, the insulin response in POMC neurons was abrogated in ovariectomized DIO females but restored with E2 replacement. E2 increased T-type calcium channel Cav3.1 messenger RNA (mRNA) expression and whole-cell currents but downregulated stromal-interaction molecule 1 mRNA, which rendered POMC neurons more excitable and responsive to insulin-mediated TRPC5 channel activation. Moreover, E2 prevented the increase in suppressor of cytokine signaling-3 mRNA expression with DIO as seen in DIO males. As proof of principle, insulin [intracerebroventricular injection into the third ventricle (ICV)] decreased food intake and increased metabolism in female but not male guinea pigs fed a high-fat diet. The uncoupling of the insulin receptor from its downstream effector system was corroborated by the reduced expression of phosphorylated protein kinase B in the arcuate nucleus of male but not female guinea pigs following insulin. Therefore, E2 protects female POMC neurons from insulin resistance by enhancing POMC neuronal excitability and the coupling of insulin receptor to TRPC5 channel activation.

Orexigenic mediators can impact the hypothalamic feeding circuitry via the activation of AMP-dependent protein kinase (AMPK). Given that testosterone is an orexigenic hormone, we hypothesized that androgenic changes in energy balance are due to enhanced cannabinoid-induced inhibition of anorexigenic proopiomelanocortin (POMC) neurons via activation of AMPK. To this end, whole animal experiments were carried out in gonadectomized male guinea pigs treated subcutaneously with either testosterone propionate (TP; 400 μg) or its sesame oil vehicle (0.1 ml). TP-treated animals displayed increases in energy intake associated with increases in meal size. TP also increased several indices of energy expenditure as well as the p-AMPK/AMPK ratio in the arcuate nucleus (ARC) measured 2 and 24 h posttreatment. Subcutaneous administration of the CB1 receptor antagonist AM251 (3 mg/kg) rapidly blocked the hyperphagic effect of TP. This was mimicked largely upon third ventricular administration of AM251 (10 μg). Electrophysiological studies revealed that TP potentiated the ability of the cannabinoid receptor agonist WIN 55,212-2 to decrease the frequency of miniature excitatory postsynaptic currents in ARC neurons. TP also increased the basal frequency of miniature inhibitory postsynaptic currents. In addition, depolarization-induced suppression (DSE) is potentiated in cells from TP-treated animals and blocked by AM251. The AMPK inhibitor compound C attenuated DSE from TP-treated animals, whereas the AMPK activator metformin enhanced DSE from vehicle-treated animals. These effects occurred in a sizable number of identified POMC neurons. Collectively, these results indicate that the androgen-induced increases in energy intake are mediated via an AMPK-dependent augmentation in endocannabinoid tone onto POMC neurons.

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.

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.

The appetite suppressant actions of estradiol are due to its ability to attenuate orexigenic signals and potentiate anorexigenic signals. The work from my laboratory has shown that male guinea pigs are more sensitive to the hyperphagic and hypothermic effects of cannabinoids than their female counterparts. Cannabinoid sensitivity is further dampened by the activational effects of estradiol. This occurs via the hypothalamic feeding circuitry, where estradiol rapidly attenuates the cannabinoid CB1 receptor-mediated presynaptic inhibition of glutamatergic input onto anorexigenic proopiomelanocortin (POMC) neurons in the arcuate nucleus. This disruption is blocked by the estrogen receptor antagonist ICI 182,780, and associated with increased expression of phosphatidylinositol-3-kinase (PI3K). Moreover, the ability of estradiol to reduce both the cannabinoid-induced hyperphagia and glutamate release onto POMC neurons is abrogated by the PI3K inhibitor PI 828. The peptide orphanin FQ/nociceptin (OFQ/N) activates opioid receptor-like (ORL)1 receptors to hyperpolarize and inhibit POMC neurons via the activation of postsynaptic G protein-gated, inwardly-rectifying (GIRK) channels. We have demonstrated that the fasting-induced hyperphagia observed in ORL1-null mice is blunted compared to wild type controls. In addition, the ORL1 receptor-mediated activation of GIRK channels in POMC neurons from ovariectomized female rats is markedly impaired by estradiol. The estrogenic attenuation of presynaptic CB1 and postsynaptic ORL1 receptor function may be part of a more generalized mechanism through which anorexigenic hormones suppress orexigenic signaling. Indeed, we have found that leptin robustly suppresses the OFQ/N-induced activation of GIRK channels in POMC neurons. Furthermore, its ability to augment excitatory input onto POMC neurons is blocked by PI 828. Thus, estradiol and other hormones like leptin reduce energy intake at least partly by activating PI3K to disrupt the pleiotropic functions of Gi/o-coupled receptors that inhibit anorexigenic POMC neurons.