The loss of ERE-dependent ERα signaling potentiates the effects of maternal high-fat diet on energy homeostasis in female offspring fed an obesogenic diet.

Maternal high-fat diet (HFD) alters hypothalamic programming and disrupts offspring energy homeostasis in rodents. We previously reported that the loss of ERα signaling partially blocks the effects of maternal HFD in female offspring fed a standard chow diet. In a companion study, we determined if the effects of maternal HFD were magnified by an adult obesogenic diet in our transgenic mouse models. Heterozygous ERα knockout (wild-type (WT)/KO) dams were fed a control breeder chow diet (25% fat) or a semipurified HFD (45% fat) 4 weeks prior to mating with heterozygous males (WT/KO or WT/ knockin) to produce WT, ERα KO, or ERα knockin/knockout (KIKO) (no estrogen response element (ERE) binding) female offspring, which were fed HFD for 20 weeks. Maternal HFD potentiated the effects of adult HFD on KIKO and KO body weight due to increased adiposity and decreased activity. Maternal HFD also produced KIKO females that exhibit KO-like insulin intolerance and impaired glucose homeostasis. Maternal HFD increased plasma interleukin 6 and monocyte chemoattractant protein 1 levels and G6pc and Pepck liver expression only in WT mice. Insulin and tumor necrosis factor α levels were higher in KO offspring from HFD-fed dams. Arcuate and liver expression of Esr1 was altered in KIKO and WT, respectively. These data suggest that loss of ERE-dependent ERα signaling, and not total ERα signaling, sensitizes females to the deleterious influence of maternal HFD on offspring energy and glucose potentially through the control of peripheral inflammation and hypothalamic and liver gene expression. Future studies will interrogate the tissue-specific mechanisms of maternal HFD programming through ERα signaling.

Stress rapidly suppresses in vivo LH pulses and increases activation of RFRP-3 neurons in male mice

Restraint stress is a psychosocial stressor that suppresses reproductive status, including LH pulsatile secretion, but the neuroendocrine mechanisms underlying this inhibition remains unclear. Reproductive neural populations upstream of gonadotropin-releasing hormone (GnRH) neurons, such as kisspeptin, neurokinin B and RFRP-3 (GnIH) neurons, are possible targets for psychosocial stress to inhibit LH pulses, but this has not been well examined, especially in mice in which prior technical limitations prevented assessment of in vivo LH pulse secretion dynamics. Here, we examined whether one-time acute restraint stress alters in vivo LH pulsatility and reproductive neural populations in male mice, and what the time-course is for such alterations. We found that endogenous LH pulses in castrated male mice are robustly and rapidly suppressed by one-time, acute restraint stress, with suppression observed as quickly as 12­18 min. This rapid LH suppression parallels with increased in vivo corticosterone levels within 15 min of restraint stress. Although Kiss1, Tac2 and Rfrp gene expression in the hypothalamus did not significantly change after 90 or 180 min restraint stress, arcuate Kiss1 neural activation was significantly decreased after 180 min. Interestingly, hypothalamic Rfrp neuronal activation was strongly increased at early times after restraint stress initiation, but was attenuated to levels lower than controls by 180 min of restraint stress. Thus, the male neuroendocrine reproductive axis is quite sensitive to short-term stress exposure, with significantly decreased pulsatile LH secretion and increased hypothalamic Rfrp neuronal activation occurring rapidly, within minutes, and decreased Kiss1 neuronal activation also occurring after longer stress durations.

Estradiol-Dependent and -Independent Stimulation of Kiss1 Expression in the Amygdala, BNST, and Lateral Septum of Mice.

Kisspeptin, encoded by Kiss1, activates reproduction by stimulating GnRH neurons. Although most Kiss1 neurons are located in the hypothalamus, smaller Kiss1 populations also reside in the medial amygdala (MeA), bed nucleus of the stria terminalis (BnST), and lateral septum (LS). However, very little is known about the regulation and function of these extra-hypothalamic Kiss1 neurons. This study focused on the roles and interactions of two signaling factors, estradiol (E2) and GABA, known to stimulate and inhibit, respectively, extra-hypothalamic Kiss1 expression. First, using estrogen receptor (ER)α knockout (KO) and ßERKO mice, we demonstrated that Kiss1 in both the BnST and LS is stimulated by E2, as occurs in the MeA, and that this E2 upregulation occurs via ERα, but not ERß. Second, using GABABR KO and wild-type mice, we determined that whereas E2 normally increases extra-hypothalamic Kiss1 levels, such upregulation by E2 is further enhanced by the concurrent absence of GABABR signaling in the MeA and LS, but not the BnST. Third, we demonstrated that when GABABR signaling is absent, the additional removal of gonadal sex steroids does not abolish Kiss1 expression in the MeA and BnST, and in some cases the LS. Thus, Kiss1 expression in these extra-hypothalamic regions is not solely dependent on E2 stimulation. Finally, we demonstrated a significant positive correlation between Kiss1 levels in the MeA, BnST, and LS, but not between these regions and the hypothalamus (anteroventral periventricular nucleus/periventricular nucleus). Collectively, our findings indicate that both E2 and GABA independently regulate all three extra-hypothalamic Kiss1 populations, but their regulatory interactions may vary by brain region and additional yet-to-be-identified factors are likely involved.

Acute Psychosocial Stress Inhibits LH Pulsatility and Kiss1 Neuronal Activation in Female Mice.

Psychosocial stress, such as isolation and restraint, disrupts reproductive neuroendocrine activity. Here we investigate the impact of psychosocial stress on luteinizing hormone (LH) pulses and gene expression and neuronal activation within Rfrp and Kiss1 cells in female mice. Mice were ovariectomized (OVX) and handled daily to habituate to the tail-tip blood collection procedure. Blood was collected every 5 minutes for 180 minutes for measurement of LH. After 90 minutes, stress animals were placed into restraint devices and isolated to new cages. No-stress control animals remained in their home cages. LH pulses occurred at regular intervals during the entire 180-minute sampling period in controls. In contrast, stress induced a rapid and robust suppression of pulsatile LH secretion. Stress reduced the frequency of pulses by 60% and diminished basal LH levels by 40%; pulse amplitude was unaffected. In a separate cohort of OVX females, brains were collected after 45, 90, or 180 minutes of stress or in no-stress controls. At all time points, stress induced a potent decrease in arcuate Kiss1 neuronal activation, using cfos induction as a marker, with a 50% to 60% suppression vs control levels, whereas Rfrp and cfos coexpression in the dorsal-medial nucleus was elevated after 45 minutes of stress. Although arcuate Kiss1 gene expression remained stable, Rfrp expression was elevated 20% after 180 minutes of stress. These findings demonstrate rapid suppression of LH pulsatile secretion by psychosocial stress, associated with reduced cfos induction in Kiss1 neurons and time-dependent increases in Rfrp neuronal activation and messenger RNA.

Loss of ERα partially reverses the effects of maternal high-fat diet on energy homeostasis in female mice.

Maternal high-fat diet (HFD) alters hypothalamic developmental programming and disrupts offspring energy homeostasis in rodents. 17ß-estradiol (E2) also influences hypothalamic programming through estrogen receptor (ER) α. Therefore, we hypothesized that females lacking ERα would be more susceptible to maternal HFD. To address this question, heterozygous ERα knockout (WT/KO) dams were fed a control breeder chow diet (25% fat) or a semi-purified HFD (45% fat) 4 weeks prior to mating with WT/KO males or heterozygous males with an ERα DNA-binding domain mutation knocked in (WT/KI) to produce WT, ERα KO, or ERα KIKO females lacking ERE-dependent ERα signaling. Maternal HFD increased body weight in WT and KIKO, in part, due to increased adiposity and daytime carbohydrate utilization in WT and KIKO, while increasing nighttime fat utilization in KO. Maternal HFD also increased plasma leptin, IL-6, and MCP-1 in WT and increased arcuate expression of Kiss1 and Esr1 (ERα) and liver expression of G6pc and Pepck in WT and KIKO. Contrary to our hypothesis, these data suggest that loss of ERα signaling blocks the influence of maternal HFD on energy homeostasis, inflammation, and hypothalamic and liver gene expression and that restoration of ERE-independent ERα signaling partially reestablishes susceptibility to maternal HFD.

The Arcuate Estrogen-Regulated Transcriptome: Estrogen Response Element-Dependent and -Independent Signaling of ERα in Female Mice.

To influence energy homeostasis and reproduction, 17ß-estradiol (E2) controls the arcuate nucleus (ARC) through multiple receptor-mediated mechanisms, but primarily via estrogen receptor (ER) α, which signals through both estrogen response element (ERE)-dependent and -independent mechanisms. To determine ERα-mediated, ERE-dependent, and ERE-independent E2 signaling in the ARC, we examined the differential regulation of the mouse arcuate transcriptome by E2 using three mice genotypes: (1) wild-type, (2) ERα knock-in/knockout (ERE-independent mechanisms), and (3) total ERα knockout (ERα-independent mechanisms). Females were ovariectomized and injected with oil or E2, and RNA sequencing on the ARC was used to identify E2-regulated genes in each genotype. Our results show that E2 regulates numerous genes involved in cell signaling, cytoskeleton structure, inflammation, neurotransmission, neuropeptide production, and transcription. Furthermore, ERE-independent signaling regulates ARC genes expressed in kisspeptin neurons and transcription factors that control the hypothalamic/pituitary/gonadal axis. Interestingly, a few genes involved in mitochondrial oxidative respiration were regulated by E2 through ERα-independent signaling. A comparison within oil- and E2-treated females across the three genotypes suggests that genes involved in cell growth and proliferation, extracellular matrices, neuropeptides, receptors, and transcription are differentially expressed across the genotypes. Comparing with previously published chromatin immunoprecipitation sequencing analysis, we found that ERE-independent regulation in the ARC is mainly mediated by tethering of ERα, which is consistent with previous findings. We conclude that the mouse arcuate estrogen-regulated transcriptome is regulated by multiple receptor-mediated mechanisms to modulate the central control of energy homeostasis and reproduction, including novel E2-responsive pathways.

Activation of Estrogen Response Element-Independent ERα Signaling Protects Female Mice From Diet-Induced Obesity.

17ß-estradiol (E2) regulates central and peripheral mechanisms that control energy and glucose homeostasis predominantly through estrogen receptor α (ERα) acting via receptor binding to estrogen response elements (EREs). ERα signaling is also involved in mediating the effects of E2 on diet-induced obesity (DIO), although the roles of ERE-dependent and -independent ERα signaling in reducing the effects of DIO remain largely unknown. We hypothesize that ERE-dependent ERα signaling is necessary to ameliorate the effects of DIO. We addressed this question using ERα knockout (KO) and ERα knockin/knockout (KIKO) female mice, the latter expressing an ERα that lacks a functional ERE binding domain. Female mice were ovariectomized, fed a low-fat diet (LFD) or a high-fat diet (HFD), and orally dosed with vehicle or estradiol benzoate (EB) (300 µg/kg). After 9 weeks, body composition, glucose and insulin tolerance, peptide hormone and inflammatory cytokine levels, and hypothalamic arcuate nucleus and liver gene expression were assessed. EB reduced body weight and body fat in wild-type (WT) female mice, regardless of diet, and in HFD-fed KIKO female mice, in part by reducing energy intake and feeding efficiency. EB reduced fasting glucose levels in KIKO mice fed both diets but augmented glucose tolerance only in HFD-fed KIKO female mice. Plasma insulin and interleukin 6 were elevated in KIKO and KO female mice compared with LFD-fed WT female mice. Expression of arcuate neuropeptide and receptor genes and liver fatty acid biosynthesis genes was altered by HFD and by EB through ERE-dependent and -independent mechanisms. Therefore, ERE-independent signaling mechanisms in both the brain and peripheral organs mediate, in part, the effects of E2 during DIO.

The interaction of fasting, caloric restriction, and diet-induced obesity with 17ß-estradiol on the expression of KNDy neuropeptides and their receptors in the female mouse.

Arcuate neurons that coexpress kisspeptin (Kiss1), neurokinin B (Tac2), and dynorphin (Pdyn) mediate negative feedback of 17ß-estradiol (E2) on the HPG axis. Previous studies report that fasting and caloric restriction reduce arcuate Kiss1 expression. The objective of this study was to determine the interactions of E2 with fasting, caloric restriction, and diet-induced obesity on KNDy gene and receptor expression. Ovariectomized female mice were separated into control and estradiol benzoate (E2B)-treated groups. E2B decreased Kiss1 and the tachykinin 2 receptor, Tac3r, in ARC tissue and Tac2 in Tac2 neurons. Diet-induced obesity decreased Kiss1 in oil-treated animals and the kisspeptin receptor, Kiss1r and Tac3r in the ARC of E2B-treated animals. Chronic caloric (30%) restriction reduced all three neuropeptides in oil-treated females and Kiss1r by E2B in CR animals. Taken together, our experiments suggest that steroidal environment and energy state negatively regulate KNDy gene expression in both ARC and Tac2 neurons.

Regulation of arcuate genes by developmental exposures to endocrine-disrupting compounds in female rats.

Developmental exposure to endocrine-disrupting compounds (EDCs) alters reproduction and energy homeostasis, both of which are regulated by the arcuate nucleus (ARC). Little is known about the effects of EDC on ARC gene expression. In Experiment #1, pregnant dams were treated with either two doses of bisphenol A (BPA) or oil from embryonic day (E)18-21. Neonates were injected from postnatal day (PND)0-7. Vaginal opening, body weights, and ARC gene expression were measured. Chrm3 (muscarinic receptor 3) and Adipor1 (adiponectin receptor 1) were decreased by BPA. Bdnf (brain-derived neurotropic factor), Igf1 (insulin-like growth factor 1), Htr2c (5-hydroxytryptamine receptor), and Cck2r (cholescystokinin 2 receptor) were impacted. In Experiment #2, females were exposed to BPA, diethylstilbestrol (DES), di(2-ethylhexyl)phthalate, or methoxychlor (MXC) during E11-PND7. MXC and DES advanced the age of vaginal opening and ARC gene expression was impacted. These data indicate that EDCs alter ARC genes involved in reproduction and energy homeostasis in females.

Regulation of gene expression by 17ß-estradiol in the arcuate nucleus of the mouse through ERE-dependent and ERE-independent mechanisms.

17ß-Estradiol (E2) modulates gene expression in the hypothalamic arcuate nucleus (ARC) to control homeostatic functions. In the ARC, estrogen receptor (ER) α is highly expressed and is an important contributor to E2's actions, controlling gene expression through estrogen response element (ERE)-dependent and -independent mechanisms. The objective of this study was to determine if known E2-regulated genes are regulated through these mechanisms. The selected genes have been shown to regulate homeostasis and have been separated into three subsections: channels, receptors, and neuropeptides. To determine if ERE-dependent or ERE-independent mechanisms regulate gene expression, two transgenic mouse models, an ERα knock-out (ERKO) and an ERα knock-in/knock-out (KIKO), which lacks a functional ERE binding domain, were used in addition to their wild-type littermates. Females of all genotypes were ovariectomized and injected with oil or estradiol benzoate (E2B). Our results suggest that E2B regulates multiple genes through these mechanisms. Of note, Cacna1g and Kcnmb1 channel expression was increased by E2B in WT females only, suggesting an ERE-dependent regulation. Furthermore, the NKB receptor, Tac3r, was suppressed by E2B in WT and KIKO females but not ERKO females, suggesting that ERα-dependent, ERE-independent signaling is necessary for Tac3r regulation. The adrenergic receptor Adra1b was suppressed by E2B in all genotypes indicating that ERα is not the primary receptor for E2B's actions. The neuropeptide Tac2 was suppressed by E2B through ERE-dependent mechanisms. These results indicate that E2B activates both ERα-dependent and independent signaling in the ARC through ERE-dependent and ERE-independent mechanisms to control gene expression.

Differential gene regulation of GHSR signaling pathway in the arcuate nucleus and NPY neurons by fasting, diet-induced obesity, and 17ß-estradiol.

Ghrelin's receptor, growth hormone secretagogue receptor (GHSR), is highly expressed in the arcuate nucleus (ARC) and in neuropeptide Y (NPY) neurons. Fasting, diet-induced obesity (DIO), and 17ß-estradiol (E2) influence ARC Ghsr expression. It is unknown if these effects occur in NPY neurons. Therefore, we examined the expression of Npy, Agrp, and GHSR signaling pathway genes after fasting, DIO, and E2 replacement in ARC and pools of NPY neurons. In males, fasting increased ARC Ghsr and NPY Foxo1 but decreased NPY Ucp2. In males, DIO decreased ARC and NPY Ghsr and Cpt1c. In fed females, E2 increased Agrp, Ghsr, Cpt1c, and Foxo1 in ARC. In NPY pools, E2 decreased Foxo1 in fed females but increased Foxo1 in fasted females. DIO in females suppressed Agrp and augmented Cpt1c in NPY neurons. In summary, genes involved in GHSR signaling are differentially regulated between the ARC and NPY neurons in a sex-dependent manner.

Intermittent Fasting Promotes Fat Loss With Lean Mass Retention, Increased Hypothalamic Norepinephrine Content, and Increased Neuropeptide Y Gene Expression in Diet-Induced Obese Male Mice.

Clinical studies indicate alternate-day, intermittent fasting (IMF) protocols result in meaningful weight loss in obese individuals. To further understand the mechanisms sustaining weight loss by IMF, we investigated the metabolic and neural alterations of IMF in obese mice. Male C57/BL6 mice were fed a high-fat diet (HFD; 45% fat) ad libitum for 8 weeks to promote an obese phenotype. Mice were divided into four groups and either maintained on ad libitum HFD, received alternate-day access to HFD (IMF-HFD), and switched to ad libitum low-fat diet (LFD; 10% fat) or received IMF of LFD (IMF-LFD). After 4 weeks, IMF-HFD (∼13%) and IMF-LFD (∼18%) had significantly lower body weights than the HFD. Body fat was also lower (∼40%-52%) in all diet interventions. Lean mass was increased in the IMF-LFD (∼12%-13%) compared with the HFD and IMF-HFD groups. Oral glucose tolerance area under the curve was lower in the IMF-HFD (∼50%), whereas the insulin tolerance area under the curve was reduced in all diet interventions (∼22%-42%). HPLC measurements of hypothalamic tissue homogenates indicated higher (∼55%-60%) norepinephrine (NE) content in the anterior regions of the medial hypothalamus of IMF compared with the ad libitum-fed groups, whereas NE content was higher (∼19%-32%) in posterior regions in the IMF-LFD group only. Relative gene expression of Npy in the arcuate nucleus was increased (∼65%-75%) in IMF groups. Our novel findings indicate that intermittent fasting produces alterations in hypothalamic NE and neuropeptide Y, suggesting the counterregulatory processes of short-term weight loss are associated with an IMF dietary strategy.

Estrogen response element-independent signaling partially restores post-ovariectomy body weight gain but is not sufficient for 17ß-estradiol's control of energy homeostasis.

The steroid 17ß-estradiol (E2) modulates energy homeostasis by reducing feeding behavior and increasing energy expenditure primarily through estrogen receptor α (ERα)-mediated mechanisms. Intact ERαKO female mice develop obesity as adults exhibiting decreased energy expenditure and increased fat deposition. However, intact transgenic female mice expressing a DNA-binding-deficient ERα (KIKO) are not obese and have similar energy expenditure, activity and fat deposition as to wild type (WT) females, suggesting that non-estrogen response element (ERE)-mediated signaling is important in E2 regulation of energy homeostasis. Initial reports did not examine the effects of ovariectomy on energy homeostasis or E2's attenuation of post-ovariectomy body weight gain. Therefore, we sought to determine if low physiological doses of E2 (250 ng QOD) known to suppress post-ovariectomy body weight gain in WT females would suppress body weight gain in ovariectomized KIKO females. We observed that the post-ovariectomy increase in body weight was significantly greater in WT females than in KIKO females. Furthermore, E2 did not significantly attenuate the body weight gain in KIKO females as it did in WT females. E2 replacement suppressed food intake and fat accumulation while increasing nighttime oxygen consumption and activity only in WT females. E2 replacement also increased arcuate POMC gene expression in WT females only. These data suggest that in the intact female, ERE-independent mechanisms are sufficient to maintain normal energy homeostasis and to partially restore the normal response to ovariectomy. However, they are not sufficient for E2's suppression of post-ovariectomy body weight gain and its effects on metabolism and activity.