Ali Yasrebi

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 17β-estradiol’s control of energy homeostasis

The steroid 17β-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 E2s 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 E2s suppression of post-ovariectomy body weight gain and its effects on metabolism and activity.

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

Ghrelins 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 17β-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.

Activation of Estrogen Response Element-independent ERα signaling protects female mice from diet-induced obesity

17β-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.

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

17β-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 E2s 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 E2Bs 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.

The interaction of fasting, caloric restriction, and diet-induced obesity with 17β-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 17β-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.

Gq Protein-Coupled Membrane-Initiated Estrogen Signaling Rapidly Excites Corticotropin-Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus in Female Mice

CRH neurons in the hypothalamic paraventricular nucleus (PVN) play a central role in regulating the hypothalamus-pituitary-adrenal (HPA) axis and are directly influenced by 17β-estradiol (E2). Although compelling evidence has suggested the existence of membrane-associated estrogen receptors (mERs) in hypothalamic and other central nervous system neurons, it remains unknown whether E2 impacts CRH neuronal excitability through this mechanism. The purpose of the current study is to examine the existence and function of mER signaling in PVN CRH neurons. Whole-cell recordings were made from CRH neurons identified by Alexa Fluor 594 labeling and post hoc immunostaining in ovariectomized female mice. E2 (100nM) rapidly suppressed the M-current (a voltage-dependent K(+) current) and potentiated glutamatergic excitatory postsynaptic currents. The putative Gq-coupled mER (Gq-mER) characterized in hypothalamic proopiomelanocortin neurons initiates a phospholipase C-protein kinase C-protein kinase A pathway; therefore, we examined the involvement of this pathway using selective inhibitors. Indeed, the ER antagonist ICI 182780 and inhibitors of Gq-phospholipase C-protein kinase C-protein kinase A blocked E2s actions, suggesting dependence on the Gq-mER. Furthermore, STX, a selective ligand for the Gq-mER, mimicked E2s actions. Finally, to examine the in vivo effect of Gq-mER activation, E2 or STX injection increased c-fos expression in CRH neurons in the PVN, suggesting CRH neuronal activation. This corresponded to an increase in plasma corticosterone. We conclude that the Gq-mER plays a critical role in the rapid regulation of CRH neuronal activity and the HPA axis. Our findings provide a potential underlying mechanism for E2s involvement in the pathophysiology of HPA-associated mood disorders.

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. 17β-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.

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.

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.