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After the phase-out of polybrominated diphenyl ethers, their replacement compounds, organophosphate flame retardants (OPFRs) became ubiquitous in home and work environments. OPFRs, which may act as endocrine disruptors, are detectable in human urine, breast milk, and blood samples collected from pregnant women. However, the effects of perinatal OPFR exposure on offspring homeostasis and gene expression remain largely underexplored. To address this knowledge gap, virgin female mice were mated and dosed with either a sesame oil vehicle or an OPFR mixture (tris(1,3-dichloro-2-propyl)phosphate, tricresyl phosphate, and triphenyl phosphate, 1 mg/kg each) from gestational day (GD) 7 to postnatal day (PND) 14. Hypothalamic and hepatic tissues were collected from one female and one male pup per litter on PND 0 and PND 14. Expression of genes involved in energy homeostasis, reproduction, glucose metabolism, and xenobiotic metabolism were analyzed using quantitative real-time PCR. In the mediobasal hypothalamus, OPFR increased Pdyn, Tac2, Esr1, and Pparg in PND 14 females. In the liver, OPFR increased Pparg and suppressed Insr, G6pc, and Fasn in PND 14 males and increased Esr1, Foxo1, Dgat2, Fasn, and Cyb2b10 in PND 14 females. We also observed striking sex differences in gene expression that were dependent on the age of the pup. Collectively, these data suggest that maternal OPFR exposure alters hypothalamic and hepatic development by influencing neonatal gene expression in a sex-dependent manner. The long-lasting consequences of these changes in expression may disrupt puberty, hormone sensitivity, and metabolism of glucose, fatty acids, and triglycerides in the maturing juvenile.

Previously, sex-dependent alterations in energy homeostasis were reported in adult mice fed a standard chow attributed to exposure to a mixture of organophosphate flame retardants (OPFRs) via estrogen receptors (ERα). In this study, adult male and female mice (C57BL/6J; Taconic) were treated with the same mixture of OPFRs (1 mg/kg each of tricresyl phosphate (TCP), triphenyl phosphate (TPP), and tris(1-3-dichloro-2propyl)phosphate (TDCPP)) for 7 weeks on a low-fat diet (LFD, 10% kcal fat) or a high fat (HFD, 45% kcal fat) in a diet-induced obesity model. Consistent with our previous observations, OPFRs altered weight gain in males, differentially with diet, while females remained unaffected. OPFR treatment also revealed sex-dependent perturbations in metabolic activity. During the night (approximately 0100-0400 hr), males exhibited elevated activity and oxygen consumption, while in females these parameters were decreased, irrespective of diet. OPFR disrupted feeding behavior and abolished diurnal water intake patterns in females while increasing nighttime fluid consumption in males. Despite no marked effect of OPFRs on glucose or insulin tolerance, OPFR treatment altered circulating insulin and leptin in females and ghrelin in males. Data indicate that adult OPFR exposure might influence, and perhaps exacerbate, the effects of diet-induced obesity in adult mice by altering activity, ingestive behavior, and metabolism.

The gut peptide, ghrelin, mediates negative energy homeostasis and the neuroendocrine control of energy homeostasis by acting through its receptor, growth hormone secretagogue receptor (GHSR). GHSR, expressed in hypothalamic Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons in the arcuate (ARC), is well known to regulate energy balance. We have previously shown 17-beta-estradiol (E2) robustly increases Ghsr expression in KNDy neurons, enhancing their sensitivity to ghrelin. We hypothesize that E2-induced increase in GHSR expression augments KNDy sensitivity in a fasting state by elevating ghrelin to reduce energy expenditure in females. We developed a Kiss1-specific GHSR knockout to determine the role of GHSR in ARC KNDy neurons and fed them either a low-fat diet (LFD) or a high-fat diet (HFD). Knockout (experimental) females were resistant to HFD in terms of body weight gain, adiposity, and food intake compared to HFD-fed controls. HFD-fed experimental females also exhibited slower glucose clearance compared to HFD-fed controls. Experimental females, regardless of diet, exhibited elevated fasting (5h) glucose. Metabolic rates (V.O2, V.CO2) and energy expenditure (heat) were not different. Respiratory Exchange Ratio (RER) was elevated in LFD-fed females, indicating the utilization of carbohydrates over fat for energy. Further meal pattern analysis revealed a reduction in meal duration in HFD-fed females, but elevated meal frequency, while HFD-fed experimental females exhibited a reduced meal size. In two separate meal pattern experiments, experimental and control females were fasted for 24h and refed or injected with ghrelin (I.P. 1mg/kg) or saline. We observed a striking delay in refeeding behavior in experimental females compared to controls during the refeeding period after fasting. After injection, control females responded to ghrelin with a rapid and sustained increase in food intake which was blunted in experimentals. Collectively, these data suggest that GHSR activation in KNDy neurons modulates metabolism, glucose homeostasis, and feeding behavior, illustrating a novel mechanism for E2 and ghrelin to synergistically control KNDy neuronal output and their subsequent behavioral and physiological outcomes.

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 benzoate (EB) or oil vehicle and performed whole-cell-patch-clamp 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.

Endocrine disrupting chemicals (EDCs) are chemicals found in our environment that interrupt typical endocrine function. Some flame retardants (FRs) are EDCs as shown in their interaction with steroid and nuclear receptors. Humans are consistently exposed to flame retardants as they are used in everyday items such as plastics, clothing, toys, and electronics. Polybrominated diphenyl ethers were used as the major FR until 2004, when they were replaced by organophosphate flame retardants (OPFRs). Previous research in rodent models utilizing a commercial flame retardant mixture containing OPFRs reported alterations in anxiety-like behavior in the elevated plus maze (EPM) for rodents perinatally exposed to OPFRs. In the present study we utilize wild-type mice maternally exposed (gestational day 7 to postnatal day 14) to either an OPFR mixture of tris(1,3-dichloro-2-propyl), triphenyl phosphate, and tricresyl phosphate or a sesame seed oil vehicle. These mice were evaluated for anxiety-like behavior in adulthood on the open field test (OFT) and the light/dark box (LDB) as well as the EPM. Outcomes from the OFT and LDB indicate that males and females maternally exposed to OPFRs exhibit altered locomotor activity. Results of the EPM were sex-specific as we did not observe an effect in females; however, effects in males differed depending on exposure condition. Males maternally exposed to OPFRs exhibited an anxiolytic-like phenotype in contrast to their vehicle counterparts. This effect in perinatally OPFR-exposed males was not due to alterations in locomotor activity. Our research illustrates that there are sex- and exposure-dependent effects of perinatal OPFR exposure on adult locomotor and anxiety-like behaviors in a mouse model.

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 Gq-coupled membrane ER (Gq-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 Gq-mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS.

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. VIDEO ABSTRACT.

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.