Intermittent fasting disrupts hippocampal-dependent memory and norepinephrine content in aged male and female mice.

Intermittent fasting (IMF) is associated with many health benefits in animals and humans. Yet, little is known if an IMF diet affects mood and cognitive processing. We have previously identified that IMF in diet-induced obese males increases norepinephrine and dopamine content in the hypothalamus and increases arcuate neuropeptide Y (NPY) gene expression more than in ad libitum control males. This suggests that IMF may improve cognition through activation of the hindbrain norepinephrine neuronal network and reverse the age-dependent decline in NPY expression. Less is known about the association between anxiety and IMF. Although, in humans, IMF during Ramadan may alleviate anxiety. Here, we address the impact of IMF on anxiety-like behavior using the open field test, hippocampal-dependent memory using the Y-maze and spatial object recognition, and hippocampal-independent memory using novel object recognition in middle-aged male and female (12 mo) and aged male and female (18 mo) mice. Using ELISA, we determined norepinephrine (NE) content in the dorsal hippocampus (DH) and prefrontal cortex (PFC). We also investigated gene expression in the arcuate nucleus (ARC), the lateral hypothalamus (LH), and the locus coeruleus (LC). In IMF-treated females at both ages, we observed an improvement in spatial navigation although an impairment in spatial object orientation. IMF-treated females (12 mo) had a reduction and IMF-treated males (12 mo) displayed an improvement in novel object recognition memory. IMF-treated females (18 mo) exhibited anxiolytic-like behavior and increased locomotion. In the DH, IMF-treated males (12 mo) had a greater amount of NE content and IMF-treated males (18 mo) had a reduction. In the ARC, IMF-treated males (12 mo) exhibited an increase in Agrp and Npy and a decrease in Adr1a. In the ARC, IMF-treated males (18 mo) exhibited an increase in Npy and a decrease in Adr1a; females had a trending decrease in Cart. In the LH at 12 months, IMF-treated males had a decrease in Npy5r, Adr1a, and Adr1b; both males and females had a reduction in Npy1r. In the LH, IMF-treated females (18 mo) had a decrease in Hcrt. In the LC at both ages, mice largely exhibited sex effects. Our findings indicate that IMF produces alterations in mood, cognition, DH NE content, and ARC, LH, and LC gene expression depending on sex and age.

Saturated Fatty Acids and Omega-3 Polyunsaturated Fatty Acids Improve Metabolic Parameters in Ovariectomized Female Mice.

In menopausal and postmenopausal women, the risk for obesity, cardiovascular disease, osteoporosis, and gut dysbiosis are elevated by the depletion of 17ß-estradiol. A diet that is high in omega-6 polyunsaturated fatty acids (PUFAs), particularly linoleic acid (LA), and low in saturated fatty acids (SFAs) found in coconut oil and omega-3 PUFAs may worsen symptoms of estrogen deficiency. To investigate this hypothesis, ovariectomized C57BL/6J and transgenic fat-1 mice, which lower endogenous omega-6 polyunsaturated fatty acids, were treated with either a vehicle or estradiol benzoate (EB) and fed a high-fat diet with a high or low PUFA:SFA ratio for ~15 weeks. EB treatment reversed obesity, glucose intolerance, and bone loss in ovariectomized mice. fat-1 mice fed a 1% LA diet experienced reduced weight gain and adiposity, while those fed a 22.5% LA diet exhibited increased energy expenditure and activity in EB-treated ovariectomized mice. Coconut oil SFAs and omega-3 FAs helped protect against glucose intolerance without EB treatment. Improved insulin sensitivity was observed in wild-type and fat-1 mice fed 1% LA diet with EB treatment, while fat-1 mice fed 22.5% LA diet was protected against insulin resistance without EB treatment. The production of short-chain fatty acids by gut microbial microbiota was linked to omega-3 FAs production and improved energy homeostasis. These findings suggest that a balanced dietary fatty acid profile containing SFAs and a lower ratio of omega-6:omega-3 FAs is more effective in alleviating metabolic disorders during E2 deficiency.

Organophosphate Flame Retardants Excite Arcuate Melanocortin Circuitry and Increase Neuronal Sensitivity to Ghrelin in Adult Mice.

Organophosphate flame retardants (OPFRs) are a class of chemicals that have become near ubiquitous in the modern environment. While OPFRs provide valuable protection against flammability of household items, they are increasingly implicated as an endocrine disrupting chemical (EDC). We previously reported that exposure to a mixture of OPFRs causes sex-dependent disruptions of energy homeostasis through alterations in ingestive behavior and activity in adult mice. Because feeding behavior and energy expenditure are largely coordinated by the hypothalamus, we hypothesized that OPFR disruption of energy homeostasis may occur through EDC action on melanocortin circuitry within the arcuate nucleus. To this end, we exposed male and female transgenic mice expressing green fluorescent protein in either neuropeptide Y (NPY) or proopiomelanocortin (POMC) neurons to a common mixture of OPFRs (triphenyl phosphate, tricresyl phosphate, and tris(1,3-dichloro-2-propyl)phosphate; each 1 mg/kg bodyweight/day) for 4 weeks. We then electrophysiologically examined neuronal properties using whole-cell patch clamp technique. OPFR exposure depolarized the resting membrane of NPY neurons and dampened a hyperpolarizing K+ current known as the M-current within the same neurons from female mice. These neurons were further demonstrated to have increased sensitivity to ghrelin excitation, which more potently reduced the M-current in OPFR-exposed females. POMC neurons from female mice exhibited elevated baseline excitability and are indicated in receiving greater excitatory synaptic input when exposed to OPFRs. Together, these data support a sex-selective effect of OPFRs to increase neuronal output from the melanocortin circuitry governing feeding behavior and energy expenditure, and give reason for further examination of OPFR impact on human health.

Sex- and age-dependent effects of maternal organophosphate flame-retardant exposure on neonatal hypothalamic and hepatic gene expression.

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.

The effects of dietary fatty acids in the physiological outcomes of maternal high-fat diet on offspring energy homeostasis in mice.

The early-life origins of disease hypothesis has been applied to obesity research and modeled through overnutrition, usually with a high-fat diet (HFD). Since the obesity epidemic coincided with societal change in dietary fat consumption, rather than amount, manipulation of fatty acid (FA) profile is an under-investigated area of study. Additionally, the binding of FAs to nuclear receptors may have persistent intergenerational, extranutritive endocrinological effects that interact with the actions of reproductive steroids causing sex-dependent effects. To determine the role of FA type in the effects underlying maternal HFD, we fed wild-type C57BL6/J mating pairs, from preconception through lactation, a HFD with high saturated fat levels from coconut oil or high linoleic acid (LA) levels from vegetable oil. Male and female offspring body weight and food intake were measured weekly for 25 weeks. Assays for glucose metabolism, body composition, and calorimetry were performed at 25 weeks. Plasma metabolic peptides and liver mRNA were measured terminally. Obesity was primarily affected by adult rather than maternal diet in males, yet in females, maternal HFD potentiated the effects of adult HFD. Maternal HFD high in LA impaired glucose disposal in males weaned onto HFD and insulin sensitivity of females. Plasma leptin correlated with adiposity, but insulin and insulin receptor expression in the liver were altered by maternal LA in males. Our results suggest that maternal FA profile is most influential on offspring glucose metabolism and that adult diet is more important than maternal diet for obesity and other parameters of metabolic syndrome.

Interaction of 17ß-estradiol and dietary fatty acids on energy and glucose homeostasis in female mice.

Fatty acid-induced hypothalamic inflammation (HI) is a potential cause of the obesity epidemic. It is unclear whether saturated or n-6 polyunsaturated fat is the primary driver of these effects. Premenopausal women are protected, in part, from obesity and associated comorbidities by circulating 17ß-estradiol (E2). It is unknown how HI interacts with E2, because most studies of HI do not examine females despite the involvement of E2 in hypothalamic energy homeostasis. Our objective is to determine the effects of high-fat diets with varying levels of linoleic acid (LA) and saturated fat on the energy and glucose homeostasis in female mice with and without E2. Female C57BL/6J mice were fed either a control diet or a 45% kilocalories from fat diet with varying levels of LA (1, 15, or 22.5% kilocalories from LA) with or without E2 (300 µg/kg/day orally). After 8 weeks, the oil-treated high-fat groups gained more weight than control groups regardless of fat type. E2 reduced body fat accumulation in all high-fat groups. Glucose clearance from glucose challenge was impaired by LA. Nighttime O2 consumption was increased by E2, regardless of diet and independent of activity. Neuropeptides and HI genes were not affected by LA or SFA content. These data show that fatty acid type does not affect body weight, but does affect glucose metabolism in females, and these effects are not associated with an induction in HI gene expression.

Organophosphate Flame-Retardants Alter Adult Mouse Homeostasis and Gene Expression in a Sex-Dependent Manner Potentially Through Interactions With ERα.

Flame retardants (FRs) such as polybrominated diphenyl ethers and organophosphate FR (OPFR) persist in the environment and interact with multiple nuclear receptors involved in homeostasis, including estrogen receptors (ERs). However, little is known about the effects of FR, especially OPFR, on mammalian neuroendocrine functions. Therefore, we investigated if exposure to FR alters hypothalamic gene expression and whole-animal physiology in adult wild-type (WT) and ERα KO mice. Intact WT and KO males and ovariectomized WT and KO females were orally dosed daily with vehicle (oil), 17α-ethynylestradiol (2.5 µg/kg), 2,2', 4,4-tetrabromodiphenyl ether (BDE-47, 1 or 10 mg/kg), or an OPFR mixture {1 or 10 mg/kg of tris(1, 3-dichloro-2-propyl)phosphate, triphenyl phosphate, and tricresyl phosphate each} for 28 days. Body weight, food intake, body composition, glucose and insulin tolerance, plasma hormone levels, and hypothalamic and liver gene expression were measured. Expression of neuropeptides, receptors, and cation channels was differentially altered between WT males and females. OPFR suppressed body weight and energy intake in males. FR increased fasting glucose levels in males, and BDE-47 augmented glucose clearance in females. Liver gene expression indicated FXR activation by BDE-47 and PXR and CAR activation by OPFR. In males, OPFR increased ghrelin but decreased leptin and insulin independent of body weight. The loss of ERα reduced the effects of both FR on hypothalamic and liver gene expression and plasma hormone levels. The physiological implications are that males are more sensitive than ovariectomized females to OPFR exposure and that these effects are mediated, in part, by ERα.

Perinatal exposure to endocrine disrupting compounds and the control of feeding behavior-An overview.

Endocrine disrupting compounds (EDC) are ubiquitous environmental contaminants that can interact with steroid and nuclear receptors or alter hormone production. Many studies have reported that perinatal exposure to EDC including bisphenol A, PCB, dioxins, and DDT disrupt energy balance, body weight, adiposity, or glucose homeostasis in rodent offspring. However, little information exists on the effects of perinatal EDC exposure on the control of feeding behaviors and meal pattern (size, frequency, duration), which may contribute to their obesogenic properties. Feeding behaviors are controlled centrally through communication between the hindbrain and hypothalamus with inputs from the emotion and reward centers of the brain and modulated by peripheral hormones like ghrelin and leptin. Discrete hypothalamic nuclei (arcuate nucleus, paraventricular nucleus, lateral and dorsomedial hypothalamus, and ventromedial nucleus) project numerous reciprocal neural connections between each other and to other brain regions including the hindbrain (nucleus tractus solitarius and parabrachial nucleus). Most studies on the effects of perinatal EDC exposure examine simple crude food intake over the course of the experiment or for a short period in adult models. In addition, these studies do not examine EDC's impacts on the feeding neurocircuitry of the hypothalamus-hindbrain, the response to peripheral hormones (leptin, ghrelin, cholecystokinin, etc.) after refeeding, or other feeding behavior paradigms. The purpose of this review is to discuss those few studies that report crude food or energy intake after perinatal EDC exposure and to explore the need for deeper investigations in the hypothalamic-hindbrain neurocircuitry and discrete feeding behaviors.

Linoleic acid causes greater weight gain than saturated fat without hypothalamic inflammation in the male mouse.

A significant change in the Western diet, concurrent with the obesity epidemic, was a substitution of saturated fatty acids with polyunsaturated, specifically linoleic acid (LA). Despite increasing investigation on type as well as amount of fat, it is unclear which fatty acids are most obesogenic. The objective of this study was to determine the obesogenic potency of LA vs. saturated fatty acids and the involvement of hypothalamic inflammation. Forty-eight mice were divided into four groups: low-fat or three high-fat diets (HFDs, 45% kcals from fat) with LA comprising 1%, 15% and 22.5% of kilocalories, the balance being saturated fatty acids. Over 12 weeks, bodyweight, body composition, food intake, calorimetry, and glycemia assays were performed. Arcuate nucleus and blood were collected for mRNA and protein analysis. All HFD-fed mice were heavier and less glucose tolerant than control. The diet with 22.5% LA caused greater bodyweight gain, decreased activity, and insulin resistance compared to control and 1% LA. All HFDs elevated leptin and decreased ghrelin in plasma. Neuropeptides gene expression was higher in 22.5% HFD. The inflammatory gene Ikk was suppressed in 1% and 22.5% LA. No consistent pattern of inflammatory gene expression was observed, with suppression and augmentation of genes by one or all of the HFDs relative to control. These data indicate that, in male mice, LA induces obesity and insulin resistance and reduces activity more than saturated fat, supporting the hypothesis that increased LA intake may be a contributor to the obesity epidemic.

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.

Physiological consequences of membrane-initiated estrogen signaling in the brain.

Many of the actions of 17beta-estradiol (E2) in the central nervous system (CNS) are mediated via the classical nuclear steroid receptors, ER(alpha) and ERbeta, which interact with the estrogen response element to modulate gene expression. In addition to the nuclear-initiated estrogen signaling, E2 signaling in the brain can occur rapidly within minutes prior to any sufficient effects on transcription of relevant genes. These rapid, membrane-initiated E2 signaling mechanisms have now been characterized in many brain regions, most importantly in neurons of the hypothalamus and hippocampus. Furthermore, our understanding of the physiological effects of membrane-initiated pathways is now a major field of interest in the hypothalamic control of reproduction, energy balance, thermoregulation and other homeostatic functions as well as the effects of E2 on physiological and pathophysiological functions of the hippocampus. Membrane signaling pathways impact neuronal excitability, signal transduction, cell death, neurotransmitter release and gene expression. This review will summarize recent findings on membrane-initiated E2 signaling in the hypothalamus and hippocampus and its contribution to the control of physiological and behavioral functions.

Estrogen regulation of genes important for K+ channel signaling in the arcuate nucleus.

Estrogen affects the electrophysiological properties of a number of hypothalamic neurons by modulating K(+) channels via rapid membrane actions and/or changes in gene expression. The interaction between these pathways (membrane vs. transcription) ultimately determines the effects of estrogen on hypothalamic functions. Using suppression subtractive hybridization, we produced a cDNA library of estrogen-regulated, brain-specific guinea pig genes, which included subunits from three prominent K+ channels (KCNQ5, Kir2.4, Kv4.1, and Kvbeta(1)) and signaling molecules that impact channel function including phosphatidylinositol 3-kinase (PI3K), protein kinase Cepsilon (PKCepsilon), cAMP-dependent protein kinase (PKA), A-kinase anchor protein (AKAP), phospholipase C (PLC), and calmodulin. Based on these findings, we dissected the arcuate nucleus from ovariectomized guinea pigs treated with estradiol benzoate (EB) or vehicle and analyzed mRNA expression using quantitative real-time PCR. We found that EB significantly increased the expression of KCNQ5 and Kv4.1 and decreased expression of KCNQ3 and AKAP in the rostral arcuate. In the caudal arcuate, EB increased KCNQ5, Kir2.4, Kv4.1, calmodulin, PKCepsilon, PLCbeta(4), and PI3Kp55gamma expression and decreased Kvbeta(1). The effects of estrogen could be mediated by estrogen receptor-alpha, which we found to be highly expressed in the guinea pig arcuate nucleus and, in particular, proopiomelanocortin neurons. In addition, single-cell RT-PCR analysis revealed that about 50% of proopiomelanocortin and neuropeptide Y neurons expressed KCNQ5, about 40% expressed Kir2.4, and about 60% expressed Kv4.1. Therefore, it is evident that the diverse effects of estrogen on arcuate neurons are mediated in part by regulation of K(+) channel expression, which has the potential to affect profoundly neuronal excitability and homeostatic functions, especially when coupled with the rapid effects of estrogen on K(+) channel function.