Acetate attenuates hypothalamic pyroptosis in experimentally induced polycystic ovarian syndrome.

This study hypothesized that SCFA, acetate impacts positively on hypothalamic pyroptosis and its related abnormalities in experimentally induced PCOS rat model, possibly through NrF2/HIF1-α modulation. Eight-week-old female Wister rats were divided into groups (n = 5), namely control, PCOS, acetate and PCOS + acetate groups. Induction of PCOS was performed by administering 1 mg/kg body weight of letrozole for 21 days. After PCOS confirmation, the animals were treated with 200 mg/kg of acetate for 6 weeks. Rats with PCOS were characterized with insulin resistance, leptin resistance, increased plasma testosterone as well as degenerated ovarian follicles. There was also a significant increase in hypothalamic triglyceride level, triglyceride-glucose index, inflammatory biomarkers (SDF-1 and NF-kB) and caspase-6 as well as plasma LH and triglyceride. A decrease was observed in plasma adiponectin, GnRH, FSH, and hypothalamic GABA with severe inflammasome expression in PCOS rats. These were accompanied by decreased level of NrF2/HIF1-α, and the alterations were reversed when treated with acetate. Collectively, the present results suggest the therapeutic impact of acetate on hypothalamic pyroptosis and its related comorbidity in PCOS, a beneficial effect that is accompanied by modulation of NrF2/HIF1-α.

Identification of Environmental Compounds That May Trigger Early Female Puberty by Activating Human GnRHR and KISS1R.

There has been an alarming trend toward earlier puberty in girls, suggesting the influence of an environmental factor(s). As the reactivation of the reproductive axis during puberty is thought to be mediated by the hypothalamic neuropeptides kisspeptin and gonadotropin-releasing hormone (GnRH), we asked whether an environmental compound might activate the kisspeptin (KISS1R) or GnRH receptor (GnRHR). We used GnRHR or KISS1R-expressing HEK293 cells to screen the Tox21 10K compound library, a compendium of pharmaceuticals and environmental compounds, for GnRHR and KISS1R activation. Agonists were identified using Ca2+ flux and phosphorylated extracellularly regulated kinase (p-ERK) detection assays. Follow-up studies included measurement of genes known to be upregulated upon receptor activation using relevant murine or human cell lines and molecular docking simulation. Musk ambrette was identified as a KISS1R agonist, and treatment with musk ambrette led to increased expression of Gnrh1 in murine and human hypothalamic cells and expansion of GnRH neuronal area in developing zebrafish larvae. Molecular docking demonstrated that musk ambrette interacts with the His309, Gln122, and Gln123 residues of the KISS1R. A group of cholinergic agonists with structures similar to methacholine was identified as GnRHR agonists. When applied to murine gonadotrope cells, these agonists upregulated Fos, Jun, and/or Egr1. Molecular docking revealed a potential interaction between GnRHR and 5 agonists, with Asn305 constituting the most conservative GnRHR binding site. In summary, using a Tox21 10K compound library screen combined with cellular, molecular, and structural biology techniques, we have identified novel environmental agents that may activate the human KISS1R or GnRHR.

Kynurenic Acid Modulates the Expression of Genes and the Activity of Cellular Antioxidant Enzymes in the Hypothalamus and Hippocampus in Sheep.

Kynurenic acid (KYNA), a tryptophan metabolite, is believed to exert neuromodulatory and neuroprotective effects in the brain. This study aimed to examine KYNA's capacity to modify gene expression and the activity of cellular antioxidant enzymes in specific structures of the sheep brain. Anestrous sheep were infused intracerebroventricularly with two KYNA doses-lower (4 × 5 µg/60 µL/30 min, KYNA20) and higher (4 × 25 µg/60 µL/30 min, KYNA100)-at 30 min intervals. The abundance of superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase 1 (GPx1) mRNA, as well as enzyme activities, were determined in the medial-basal hypothalamus (MBH), the preoptic (POA) area of the hypothalamus, and in the hippocampal CA1 field. Both doses of KYNA caused a decrease (p < 0.01) in the expression of SOD2 and CAT mRNA in all structures examined compared to the control group (except for CAT in the POA at the KYNA100 dose). Furthermore, lower levels of SOD2 mRNA (p < 0.05) and CAT mRNA (p < 0.01) were found in the MBH and POA and in the POA and CA, respectively, in sheep administered with the KYNA20 dose. Different stimulatory effects on GPx1 mRNA expression were observed for both doses (p < 0.05-p < 0.01). KYNA exerted stimulatory but dose-dependent effects on SOD2, CAT, and GPx1 activities (p < 0.05-p < 0.001) in all brain tissues examined. The results indicate that KYNA may influence the level of oxidative stress in individual brain structures in sheep by modulating the expression of genes and the activity of at least SOD2, CAT, and GPx1. The present findings also expand the general knowledge about the potential neuroprotective properties of KYNA in the central nervous system.

A hypothalamus-lateral periaqueductal gray GABAergic neural projection facilitates arousal following sevoflurane anesthesia in mice.

BACKGROUND: The lateral hypothalamus (LHA) is an evolutionarily conserved structure that regulates basic functions of an organism, particularly wakefulness. To clarify the function of LHAGABA neurons and their projections on regulating general anesthesia is crucial for understanding the excitatory and inhibitory effects of anesthetics on the brain. The aim of the present study is to investigate whether LHAGABA neurons play either an inhibitory or a facilitatory role in sevoflurane-induced anesthetic arousal regulation. METHODS: We used fiber photometry and immunofluorescence staining to monitor changes in neuronal activity during sevoflurane anesthesia. Opto-/chemogenetic modulations were employed to study the effect of neurocircuit modulations during the anesthesia. Anterograde tracing was used to identify a GABAergic projection from the LHA to a periaqueductal gray (PAG) subregion. RESULTS: c-Fos staining showed that LHAGABA activity was inhibited by induction of sevoflurane anesthesia. Anterograde tracing revealed that LHAGABA neurons project to multiple arousal-associated brain areas, with the lateral periaqueductal gray (LPAG) being one of the dense projection areas. Optogenetic experiments showed that activation of LHAGABA neurons and their downstream target LPAG reduced the burst suppression ratio (BSR) during continuous sevoflurane anesthesia. Chemogenetic experiments showed that activation of LHAGABA and its projection to LPAG neurons prolonged the anesthetic induction time and promoted wakefulness. CONCLUSIONS: In summary, we show that an inhibitory projection from LHAGABA to LPAGGABA neurons promotes arousal from sevoflurane-induced loss of consciousness, suggesting a complex control of wakefulness through intimate interactions between long-range connections.

Hypnotic effect of AR-001 through adenosine A1 receptor.

Insomnia is one of the most common sleep disorders, affecting 10-15% of the global population. Because classical remedies used to treat insomnia have various side effects, new therapeutics for insomnia are attracting attention. In the present study, we found that N2-Ethyl-N4-(furan-2-ylmethyl) quinazoline-2,4-diamine (AR-001) has adenosine A1 receptor agonistic activity and exhibits hypnotic efficacy by decreasing sleep onset latency and increasing total sleep time in a pentobarbital-induced sleep model. This hypnotic effect of AR-001 was significantly inhibited by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). As a result of immunohistochemistry, AR-001 was shown to increase neural activity in the sleep-promoting region, ventrolateral preoptic nucleus (VLPO), and decrease neural activity in the wake-promoting region, basal forebrain (BF), and lateral hypothalamus (LH), and that these effects of AR-001 were significantly inhibited by DPCPX treatment. In addition, AR-001 increased adenosine A1 receptor mRNA levels in the hypothalamus. In conclusion, this study suggests that AR-001 has a hypnotic effect, at least partially, through adenosine A1 receptor and may have therapeutic potential for insomnia.

Sexual dimorphism of hypothalamic serotonin release during systemic inflammation: Role of endothelin-1.

The hypothalamus receives serotonergic projections from the raphe nucleus in a sex-specific manner. During systemic inflammation, hypothalamic levels of serotonin (5-hydroxytryptamine [5-HT]) decrease in male rats. The present study evaluated the involvement of endothelin-1 (ET-1) in the febrile response, hypolocomotion, and changes in hypothalamic 5-HT levels during systemic inflammation in male and female rats. An intraperitoneal injection of lipopolysaccharide (LPS) induced a febrile response and hypolocomotion in both male and female rats. However, although LPS reduced hypothalamic levels of 5-HT and its metabolite 5-hydroxyindol acetic acid (5-HIAA) in male rats, it increased these levels in female rats. An intracerebroventricular injection of the endothelin-B receptor antagonist BQ788 significantly reduced LPS-induced fever and hypolocomotion and changes in hypothalamic 5-HT and 5-HIAA levels in both male and female rats. The i.c.v. administration of ET-1 induced a significant fever and hypolocomotion, but reduced the hypothalamic levels of 5-HT and 5-HIAA in both males and females. These results suggest an important sexual dimorphism during systemic inflammation regarding the release of 5-HT in the hypothalamus. Moreover, ET-1 arises as an important mediator involved in the changes in hypothalamic 5-HT levels in both male and female rats.

Quercetin alleviates depressive-like behavior by modulating acetyl-H3K9 mediated ferroptosis pathway in hypothalamus of perimenopausal depression rat model.

Perimenopausal depression is a subtype of depression and is prevalent among perimenopausal women, which has brought a heavy burden to family and society. The pathogenesis of perimenopausal depression is still unclear, which affects the prevention and treatment of perimenopausal depression to a certain extent. Quercetin is a flavonoid compound, and has estrogenic activity and pharmacological effects such as antioxidant, anti-inflammatory, and neuroprotective effects. This study investigated whether quercetin improved perimenopausal depression-like behaviors and potential mechanism. The results demonstrated that quercetin could alleviate the depression-like behaviors in perimenopausal depression rat model, inhibit astrocyte activation, improve ferroptosis-associated mitochondrial damage (such as mitochondrial pyknosis and mitochondrial cristae reduction) in hypothalamus, increase the expressions of histone 3 lysine 9 acetylation (acetyl-H3K9), ferroptosis-associated protein including glutathione peroxidase 4 (GPX4) and Xc- antiporter (SLC7A11), and reduce the expressions of endoplasmic reticulum stress-related proteins including inositol-requiring enzyme 1 (IRE1α), phosphorylated IRE1α (p-IRE1α), X-box binding protein 1 (XBP1) and glucose-regulated protein 78 (GRP78) in hypothalamus of perimenopausal depression rat model. Furtherly, in vitro study indicated that quercetin could restore histone acetylase (HAT)/histone deacetylase (HDAC) homeostasis through binding to estrogen receptors and increase the expression of acetyl-H3K9, inhibiting ferroptosis through IRE1α/XBP1 pathway in astrocytes of hypothalamus. Our findings demonstrated that acetyl-H3K9 is a crucial target in development of perimenopausal depression, and quercetin exhibited antidepressant effects through modulating acetyl-H3K9 mediated ferroptosis in perimenopausal depression. Quercetin might be the prevention and adjuvant treatment strategy of perimenopausal depression.

Brain Perception of Different Oils on Appetite Regulation: An Anorectic Gene Expression Pattern in the Hypothalamus Dependent on the Vagus Nerve.

(1) Background: We examined the effect of the acute administration of olive oil (EVOO), linseed oil (GLO), soybean oil (SO), and palm oil (PO) on gastric motility and appetite in rats. (2) Methods: We assessed food intake, gastric retention (GR), and gene expression in all groups. (3) Results: Both EVOO and GLO were found to enhance the rate of stomach retention, leading to a decrease in hunger. On the other hand, the reduction in food intake caused by SO was accompanied by delayed effects on stomach retention. PO caused an alteration in the mRNA expression of NPY, POMC, and CART. Although PO increased stomach retention after 180 min, it did not affect food intake. It was subsequently verified that the absence of an autonomic reaction did not nullify the influence of EVOO in reducing food consumption. Moreover, in the absence of parasympathetic responses, animals that received PO exhibited a significant decrease in food consumption, probably mediated by lower NPY expression. (4) Conclusions: This study discovered that different oils induce various effects on parameters related to food consumption. Specifically, EVOO reduces food consumption primarily through its impact on the gastrointestinal tract, making it a recommended adjunct for weight loss. Conversely, the intake of PO limits food consumption in the absence of an autonomic reaction, but it is not advised due to its contribution to the development of cardiometabolic disorders.

Glucocorticoid alleviates hypothalamic nerve injury via remodeling HPA axis homeostasis in stressed rats.

Excessive stress can exceed the adjustment ability of body and cause injury and dysfunction, while elucidation of the mechanism and prevention measures of stress-related injury are still insufficient. The present study was to observe the effect of glucocorticoid (GC) on stress-induced hypothalamic nerve injury and elucidate the potential mechanism. The present study intended to establish a chronic restraint stress rat model for follow-up study. Open field test and elevated plus maze test were used to observe behavioral changes of stress rats; Enzyme-linked immunosorbent assay (ELISA) was used to detect changes in the levels of hypothalamus-pituitary-adrenal (HPA) axis-related hormones and inflammatory factors in hypothalamus; toluidine blue staining was used to observe pathological changes of hypothalamus. The results showed that stress rats showed obvious anxiety-like behaviors, the levels of HPA axis-related hormones and inflammatory factors showed abnormal fluctuations, and morphological results showed significant nerve injury in hypothalamus. Low-dose GC treatment significantly improved behavioral changes, alleviated hypothalamic nerve injury, and restored hypothalamic levels of inflammatory factors, serum levels of GC, corticotropin-releasing hormone (CRH), and adrenocorticotropic hormone (ACTH) and GC level in adrenal cortex of stressed rats, while GC receptor (GR) inhibitor, CRH receptor inhibitor, and adrenalectomy reversed the ameliorative effects of low-dose GC. Our study clarified that low-dose GC can restore stress coping ability by reshaping the homeostasis of the HPA axis, thus alleviating behavioral abnormalities and hypothalamic nerve injury in stressed rats.

The Effects of Testosterone on Hypothalamic and Serum Oxytocin Levels Are Affected by the Estrogen Milieu in Female Rats.

Previous studies have suggested that the effects of androgens on body weight (BW) and appetite are affected by the estrogen milieu in females; however, the mechanism underlying these effects remains unclear. We hypothesized that androgens may affect endogenous oxytocin (OT), which is a hypothalamic anorectic factor, and that these effects of androgens may be altered by the estrogen milieu in females. To investigate this hypothesis, in the present study, we examined the effects of testosterone on peripheral and central OT levels in ovariectomized female rats that did or did not receive estradiol supplementation. Ovariectomized female rats were randomly divided into non-estradiol-supplemented or estradiol-supplemented groups, and half of the rats in each group were concurrently supplemented with testosterone (i.e., rats were divided into four groups, n = 7 per each group). We also measured peripheral and central OT receptor (OTR) gene expression levels. As a result, we found that testosterone increased serum and hypothalamic OT levels and OT receptor mRNA levels in non-estradiol-supplemented rats, whereas it had no effects on these factors in estradiol-supplemented rats. In addition, testosterone reduced food intake, BW gain, and fat weight in non-estradiol-supplemented rats, whereas it did not have any effects on BW, appetite, or fat weight in estradiol-supplemented rats. These findings indicate that the effects of androgens on OT may be affected by the estrogen milieu, and elevated OT levels may be related to the blunting of appetite and prevention of obesity under estrogen-deficient conditions.

Effects of KGM and Degradation Products on Appetite Regulation and Energy Expenditure in High-Fat-Diet Mice via the Adipocyte-Hypothalamus Axis.

Konjac glucomannan (KGM), high-viscosity dietary fiber, is utilized in weight management. Previous investigations on the appetite-suppressing effects of KGM have centered on intestinal responses to nutrients and gastric emptying rates, with less focus on downstream hypothalamic neurons of satiety hormones. In our studies, the molecular mechanisms through which KGM and its degradation products influence energy homeostasis via the adipocyte-hypothalamic axis have been examined. It was found that high-viscosity KGM more effectively stimulates enteroendocrine cells to release glucagon-like peptide-1 (GLP-1) and reduces ghrelin production, thereby activating hypothalamic neurons and moderating short-term satiety. Conversely, low-viscosity DKGM has been shown to exhibit stronger anti-inflammatory properties in the hypothalamus, enhancing hormone sensitivity and lowering the satiety threshold. Notably, both KGM and DKGM significantly reduced leptin signaling and fatty acid signaling in adipose tissue and activated brown adipose tissue thermogenesis to suppress pro-opiomelanocortin (POMC) expression and activate agouti-related protein (AgRP) expression, thereby reducing food intake and increasing energy expenditure. Additionally, high-viscosity KGM has been found to activate the adipocyte-hypothalamus axis more effectively than DKGM, thereby promoting greater daily energy expenditure. These findings provide novel insights into the adipocyte-hypothalamic axis for KGM to suppress appetite and reduce weight.

Gestational bisphenol A exposure alters energy homeostasis and adult hypothalamic neurogenesis in female mice.

Regulation of physiological homeostasis, including energy balance, is thought to be modified by low levels of adult neurogenesis in the hypothalamus. Hormones such as oestradiol can influence both embryonic and adult hypothalamic neurogenic programs, demonstrating a sensitivity of hypothalamic neural progenitor cells to endogenous hormones. Previously we showed that gestational exposure to environmental levels of the xenoestrogen bisphenol A (BPA) changed neural progenitor cell behaviors in the embryo; however, we did not examine if these changes were permanent to affect adult neurogenesis. Here we investigated whether adult neuro- and/or gliogenesis were altered in mice prenatally exposed to BPA and placed on a high-fat diet challenge. Gestationally exposed adult female mice on a standard diet gained less weight than non-BPA controls, whereas gestationally exposed BPA females on a high-fat diet gained more weight than controls. Males exposed to gestational BPA showed no differences in weight gain relative to control males. Concomitantly, adult neurogenesis was increased in the VMH, DMH, and PVN of adult female mice exposed to BPA on standard diet, suggesting that disrupted adult neurogenesis might perturb normal energy balance regulation in females. These results add to growing evidence that low-dose BPA exposure in utero causes changes to adult hypothalamic function.

Intracerebroventricular injection of spexin stimulates the hypothalamic-pituitary-testicular axis and increases the secretion of male reproductive hormones in rats.

BACKGROUND: Male reproductive functions are regulated in the hypothalamic-pituitary-gonadal (HPG) axis. Any problem in this axis would lead to the deterioration of reproductive functions. The present study aimed to investigate the effects of intracerebroventricular (icv) Spexin (SPX) infusion on the HPG axis in detail. METHODS: 40 Wistar albino rats were divided into four groups: control, sham, SPX 30 nmol and SPX 100 nmol (n=10). 30 nmol/1 µl/hour SPX was administered icv to the rats in the SPX 30 nmol group for 7 days, while rats in the SPX 100 nmol group were administered 100 nmol/1 µl/hour SPX. On the 7th day, the rats were decapitated, blood and tissue samples were collected. Serum LH, FSH and testosterone levels were determined with the ELISA method, GnRH mRNA expression level was determined in hypothalamus with the RT-PCR method. Seminiferous tubule diameter and epithelial thickness were determined with the hematoxylin-eosin staining method. RESULTS: SPX infusion was increased GnRH mRNA expression in the hypothalamus tissue independent of the dose (p<0.05). Serum LH, FSH and testosterone levels in the SPX groups were increased when compared to the control and sham groups independent of the dose (p <0.05). Histological analysis revealed that SPX infusion did not lead to any changes in seminiferous epithelial thickness, while the tubule diameter increased in the SPX groups (p<0.05). CONCLUSION: The study findings demonstrated that icv SPX infusion stimulated the HPG axis and increased the secretion of male reproductive hormones.

[Regulating effect of Gegen Decoction on hypothalamic-pituitary-ovarian axis in mice with primary dysmenorrhea].

This study aimed to explore the regulating effect of Gegen Decoction(GGD) on the hypothalamic-pituitary-ovarian axis(HPOA) dysfunction in the mouse model of primary dysmenorrhea(PD). The mouse model of PD with periodic characteristics was established by administration of estradiol benzoate and oxytocin. Mice were randomized into control, model, GGD, and ibuprofen groups. The writhing response, hypothalamus index, pituitary index, ovary index, and uterus index were observed and determined. The serum levels of prostaglandin F_(2α)(PGF_(2α)), gonadotropin-releasing hormone(GnRH), follicle-stimulating hormone(FSH), luteinizing hormone(LH), and estrogen(E_2) levels were measured by ELISA kits. Western blot and qPCR were employed to determine the protein and mRNA levels, respectively, of gonadotropin-releasing hormone receptor(GnRH-R) in the pituitary tissue, follicle-stimulating hormone receptor(FSHR) and luteinizing hormone receptor(LHR) in the ovarian tissue, and estrogen receptor(ER) in the uterine tissue. The results showed that the writhing response, serum levels of PGF_(2α), GnRH, FSH, LH, and E_2, ovarian and uterine indexes, the protein and mRNA levels of GnRH-R in the pituitary tissue, FSHR and LHR in the ovarian tissue, and ER in the uterine tissue were significantly increased in the model group compared with those in the control group. GGD inhibited the writhing response, reduced the serum levels of PGF_(2α), GnRH, FSH, LH, and E_2, decreased the ovarian and uterine indexes, and down-regulated the protein and mRNA levels of GnRH-R in the pituitary tissue, FSHR and LHR in the ovarian tissue, and ER in the uterine tissue. The data suggested that GGD can regulate the HPOA and inhibit E_2 generation in the mice experiencing recurrent PD by down-regulating the expression of proteins and genes related to HPOA axis, thus exerting the therapeutic effect on PD.

Effect of kynurenic acid on enzymatic activity of the DNA base excision repair pathway in specific areas of the sheep brain.

Relatively low levels of antioxidant enzymes coupled with high oxygen metabolism result in the formation of numerous oxidative DNA damages in the tissues of the central nervous system. Recently, kynurenic acid (KYNA), knowns for its neuroprotective properties, has gained increasing attention in this context. Therefore, our hypothesis assumed that increased KYNA levels in the brain would positively influence mRNA expression of selected enzymes of the base excision repair pathway as well as enhance their efficiency in excising damaged nucleobases in specific areas of the sheep brain. The study was conducted on adult anestrous sheep (n = 18), in which two different doses of KYNA (20 and 100 µg/day) were infused into the third brain ventricle for three days. Molecular and biochemical analysis included the hypothalamus (preoptic and mediol-basal areas), hippocampus (CA3 field) and amygdala (central amygdaloid nucleus), dissected from the brain of sheep euthanized immediately after the last infusion. The results revealed a significant increase P < 0.001) in the relative mRNA abundance of N-methylpurine DNA glycosylase (MPG) following administration of both dose of KYNA across all examined tissues. The transcription of thymine-DNA glycosylase (TDG) increased significantly (P < 0.001) in all tissues in response to the lower KYNA dose compared to the control group. Moreover, 8-oxoguanine (8-oxoG) DNA glycosylase (OGG1) mRNA levels were also higher in both animal groups (P < 0.001). In addition, in the hypothalamus, hippocampus and amygdala, AP endonuclease 1 (APE1) mRNA expression increased under both doses of KYNA. Moreover, the both dose of KYNA significantly stimulated the efficiency of 8-oxoG excision in hypothalamus and amygdala (P < 0.05-0.001). The lower and higher doses of KYNA significantly influenced the effectiveness of εA and εC in all structures (P < 0.01-0.001). In conclusion, the favorable effect of KYNA in the brain may include the protection of genetic material in nerve and glial cells by stimulating the expression and efficiency of BER pathway enzymes.

Glioprotective Effects of Sulforaphane in Hypothalamus: Focus on Aging Brain.

Sulforaphane is a natural compound with neuroprotective activity, but its effects on hypothalamus remain unknown. In line with this, astrocytes are critical cells to maintain brain homeostasis, and hypothalamic astrocytes are fundamental for sensing and responding to environmental changes involved in a variety of homeostatic functions. Changes in brain functionality, particularly associated with hypothalamic astrocytes, can contribute to age-related neurochemical alterations and, consequently, neurodegenerative diseases. Thus, here, we investigated the glioprotective effects of sulforaphane on hypothalamic astrocyte cultures and hypothalamic cell suspension obtained from aged Wistar rats (24 months old). Sulforaphane showed anti-inflammatory and antioxidant properties, as well as modulated the mRNA expression of astroglial markers, such as aldehyde dehydrogenase 1 family member L1, aquaporin 4, and vascular endothelial growth factor. In addition, it increased the expression and extracellular levels of trophic factors, such as glia-derived neurotrophic factor and nerve growth factor, as well as the release of brain-derived neurotrophic factor and the mRNA of TrkA, which is a receptor associated with trophic factors. Sulforaphane also modulated the expression of classical pathways associated with glioprotection, including nuclear factor erythroid-derived 2-like 2, heme oxygenase-1, nuclear factor kappa B p65 subunit, and AMP-activated protein kinase. Finally, a cell suspension with neurons and glial cells was used to confirm the predominant effect of sulforaphane in glial cells. In summary, this study indicated the anti-aging and glioprotective activities of sulforaphane in aged astrocytes.

Chlorpyrifos induces autophagy by suppressing the mTOR pathway in immortalized GnRH neurons.

Chlorpyrifos (CPF) is a widely used pesticide inducing adverse neurodevelopmental and reproductive effects. However, knowledge of the underlying mechanisms is limited, particularly in the hypothalamus. We investigated the mode of action of CPF at human relevant concentrations (1 nM-100 nM) in immortalized mouse hypothalamic GnRH neurons (GT1-7), an elective model for studying disruption of the hypothalamus-pituitary-gonads (HPG) axis. We firstly examined cell vitality, proliferation, and apoptosis/necrosis. At not-cytotoxic concentrations, we evaluated neuron functionality, gene expression, Transmission Electron Microscopy (TEM) and proteomics profiles, validating results by immunofluorescence and western blotting (WB). CPF decreased cell vitality with a dose-response but did not affect cell proliferation. At 100 nM, CPF inhibited gene expression and secretion of GnRH; in addition, CPF reduced the immunoreactivity of the neuronal marker Map2 in a dose-dependent manner. The gene expression of Estrogen Receptor α and ß (Erα, Erß), Androgen Receptor (Ar), aromatase and oxytocin receptor was induced by CPF with different trends. Functional analysis of differentially expressed proteins identified Autophagy, mTOR signaling and Neutrophil extracellular traps (NETs) formation as significant pathways affected at all concentrations. This finding was phenotypically supported by the TEM analysis, showing marked autophagy and damage of mitochondria, as well as by protein analysis demonstrating a dose-dependent decrease of mTOR and its direct target pUlk1 (Ser 757). The bioinformatics network analysis identified a core module of interacting proteins, including Erα, Ar, mTOR and Sirt1, whose down-regulation was confirmed by WB analysis. Overall, our results demonstrate that CPF is an inhibitor of the mTOR pathway leading to autophagy in GnRH neurons; a possible involvement of the Erα/Ar signaling is also suggested. The evidence for adverse effects of CPF in the hypothalamus in the nanomolar range, as occurs in human exposure, increases concern on potential adverse outcomes induced by this pesticide on the HPG axis.

Comprehensive Glycomic and Proteomic Analysis of Mouse Striatum and Lateral Hypothalamus Following Repeated Exposures to Cocaine or Methamphetamine.

Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry-based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.

The short-chain fatty acid acetate modulates orexin/hypocretin neurons: A novel mechanism in gut-brain axis regulation of energy homeostasis and feeding.

The short-chain fatty acids (SCFAs) acetate, propionate and butyrate, the major products of intestinal microbial fermentation of dietary fibres, are involved in fine-tuning brain functions via the gut-brain axis. However, the effects of SCFAs in the hypothalamic neuronal network regulating several autonomic-brain functions are still unknown. Using NMR spectroscopy, we detected a reduction in brain acetate concentrations in the hypothalamus of obese leptin knockout ob/ob mice compared to lean wild-type littermates. Therefore, we investigated the effect of acetate on orexin/hypocretin neurons (hereafter referred as OX or OX-A neurons), a subset of hypothalamic neurons regulating energy homeostasis, which we have characterized in previous studies to be over-activated by the lack of leptin and enhancement of endocannabinoid tone in the hypothalamus of ob/ob mice. We found that acetate reduces food-intake in concomitance with a reduction of orexin neuronal activity in ob/ob mice. This was demonstrated by evaluating food-intake behaviour and orexin-A/c-FOS immunoreactivity coupled with patch-clamp recordings in Hcrt-eGFP neurons, quantification of prepro-orexin mRNA, and immunolabeling of GPR-43, the main acetate receptor. Our data provide new insights into the mechanisms of the effects of chronic dietary supplementation with acetate, or complex carbohydrates, on energy intake and body weight, which may be partly mediated by inhibition of orexinergic neuron activity.

Transcriptomic analysis of sheep hypothalamus discloses regulatory genes potentially involved in sex-dependent differences in body weight of progeny born to dams supplemented with omega-3 fatty acids or methionine during late-gestation.

Fetal programming research conducted in sheep has reported sexually dimorphic responses on growth of the progeny born to in-utero methionine or omega-3 fatty acids supplementation. However, the biological mechanism behind the nutrient by sex interaction as a source of variation in offspring body weight is still unknown. A high-throughput RNA sequencing data of hypothalamus samples from 17 lambs were used in the current study to identify differentially expressed genes (DEGs) between males and females born to dams supplemented with different nutrients during late-gestation. Ewes received a basal diet without omega-3 fatty acids or methionine supplementation as the control (CONT); omega-3 fatty acids supplementation (FAS), or methionine supplementation (METS). A list of regulated genes was generated. Data were compared as CONT vs. FAS and CONT vs. METS. For CONT vs. METS, a treatment by sex interaction was found (adjusted P-value < 0.05) on 121 DEGs (112 upregulated and 9 downregulated) on female lambs born to METS compared with METS males. Importantly, with the sex interaction term, more than 100 genes were upregulated in female lamb's hypothalamuses born to METS. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were performed using the DEGs from female lambs. Terms under biological process (related to morphogenesis, organism, and tissue development), cellular component (related to chromatin, extracellular components), and molecular function (involved in chromatin structure and transcription and factors linked to binding DNA) were presented (adjusted P-value < 0.05) for GO. For the IPA, the top-scoring network was developmental disorder, endocrine system development and function, and organ morphology. Only a few differences were observed in the comparison between the interaction of sex and treatment for the CONT vs. FAS comparison. The markedly increased number of DEGs substantially involved in developmental and growth processes indicates the extent to which maternal methionine supplementation causes the sexually dimorphic effects observed in the offspring.

Feeding dams during gestation affects the development of the offspring for their entire life. The objective of the current experiment was to evaluate the changes of the transcriptome in the hypothalamus of the offspring lambs born from dams supplemented with (i) a control diet (without lipids or methionine supplementation), (ii) an omega-3 fatty acid supplementation, or (iii) a methionine supplementation. The supplementation took place in the last third of gestation and the hypothalamus of male and female offspring was collected after being on a fattening diet for 54 d. Hypothalamus samples were used to extract RNA and analyzed using RNA sequencing. There was an interaction due to sex and methionine supplementation. The pathways that were modified were chromatin structure, developmental processes, and organ morphology. The modification observed on these pathways could explain the sex by treatment interaction differences previously observed in growth. There were few sex by omega-3 fatty acid interactions on the hypothalamus transcriptome. Therefore, the sexual dimorphism observed by methionine supplementation may be regulated by the hypothalamus.