EphA3 deficiency in the hypothalamus promotes high-fat diet-induced obesity in mice

Erythropoietin-producing hepatocellular carcinoma A3(EphA3)is a member of the largest subfamily of tyrosine kinase receptors-Eph receptors.Previous studies have shown that EphA3 is associated with tissue development.Recently,we have found that the expression of EphA3 is elevated in the hypothalamus of mice with diet-induced obesity(DIO).However,the role of EphA3 in hypothalamic-controlled energy metabolism remains unclear.In the current study,we demonstrated that the deletion of EphA3 in the hypothalamus by CRISPR/Cas9-mediated gene editing promotes obesity in male mice with high-fat diet feeding rather than those with normal chow diet feeding.Moreover,the deletion of hypothalamic EphA3 promotes high-fat DIO by increasing food intake and reducing energy expenditure.Knockdown of EphA3 leads to smaller intracellular vesicles in GT1-7 cells.The current study reveals that hypothalamic EphA3 plays important roles in promoting DIO.

Insulin:A connection between pancreaticβcells and the hypothalamus

Insulin is a hormone secreted by pancreaticβcells.The concentration of glucose in circulation is proportional to the secretion of insulin by these cells.In target cells,insulin binds to its receptors and activates phosphatidylinositol-3-kinase/protein kinase B,inducing different mechanisms depending on the cell type.In the liver it activates the synthesis of glycogen,in adipose tissue and muscle it allows the capture of glucose,and in the hypothalamus,it regulates thermogenesis and appetite.Defects in insulin function[insulin resistance(IR)]are related to the development of neurodegenerative diseases in obese people.Furthermore,in obesity and diabetes,its role as an anorexigenic hormone in the hypothalamus is diminished during IR.Therefore,hyperphagia prevails,which aggravates hyperglycemia and IR further,becoming a vicious circle in which the patient cannot regulate their need to eat.Uncontrolled calorie intake induces an increase in reactive oxygen species,overcoming cellular antioxidant defenses(oxidative stress).Reactive oxygen species activate stress-sensitive kinases,such as c-Jun Nterminal kinase and p38 mitogen-activated protein kinase,that induce phosphorylation in serine residues in the insulin receptor,which blocks the insulin signaling pathway,continuing the mechanism of IR.The brain and pancreas are organs mainly affected by oxidative stress.The use of drugs that regulate food intake and improve glucose metabolism is the conventional therapy to improve the quality of life of these patients.Currently,the use of antioxidants that regulate oxidative stress has given good results because they reduce oxidative stress and inflammatory processes,and they also have fewer side effects than synthetic drugs.

Cold exposure alters proteomic profiles of the hypothalamus and pituitary in female rats

Objective:Studies have shown that both short-term and long-term cold exposures disturb the biological process.The aim of the present study is to investigate the effects of intermittent cold exposure on proteomic profiles in the hypothalamus and pituitary of female Sprague-Dawley(SD)rats.Materials and methods:The rats were exposed to-10°C in a cabin for 4 h per day,and the treatment lasted for 14 days.The comparative label-free LC-MS/MS analysis was performed to investigate the changes of proteomic profiles in the hypothalamus and pituitary.ELISA analysis was used to validate the expression of differential proteins.Results:22 differential proteins in the hypothalamus and 75 differential proteins in the pituitary were identified by the label-free proteomic analysis.Gene ontology annotation and enrichment analysis indicated that cold exposure disrupted protein phosphorylation,filopodium assembly,intracellular protein transport,peripheral nervous system neuron axonogenesis,spinal cord development,Golgi organization,positive regulation of pseudopodium assembly,and cell-cell adhesion.Three proteins(Cdc42,Ptprs,and Setd7)were down-regulated in the cold exposure group.Conclusion:The results indicate that intermittent cold exposure alters the proteomic profiles of hypothalamus and pituitary in female rats.

Neuropeptide Y and leptin receptor expression in the hypothalamus of rats with chronic immobilization stress

In this study, Sprague-Dawley rats were immobilized to a frame for 3 hours a day for 21 days to establish a model of chronic immobilization stress. The body weight and food intake of rats subjected to chronic immobilization stress were significantly decreased compared with the control group. Dual-labeling immunofluorescence revealed that the expression of leptin receptor and the co-localization coeffient in these leptic receptor neurons in the arcuate nucleus of the hypothalamus were both upregulated, while the number of neuropeptide Y neurons was decreased. Chronic immobilization stress induced high expression of leptin receptor in the arcuate nucleus and suppressed the synthesis and secretion of neuropeptide Y, thereby disrupting the pathways in the arcuate nucleus that regulate feeding behavior, resulting in diminished food intake and reduced body weight.

Soy Isoflavone and Its Effect to Regulate Hypothalamus and Peripheral Orexigenic Gene Expression in Ovariectomized Rats Fed on A High-Fat Diet

Objective To explore the effect of soy isoflavone on obesity in the light of hypothalamus and peripheral orexigenic gene regulation. Methods Fifty-four female rats were randomly assigned to 6 groups： one sham-operated group （SHAM）, one ovariectomized （OVX） control group, three OVX groups fed with 400 ppm （L-SI）, 1 200 ppm （M-SI） and 3 600 ppm （H-SI） isoflavone respectively, and one OVX group receiving 0.45 ppm diethylstilbestrol （EC）. All rats were allowed to take high-fat diet for 4 weeks. Some neuropeptides were measured by RT-PCR. These neuropeptides included NPY, pro-opiomelanocortin （POMC）, cocaine and amphetamine regulated transcript （CART）, orexin, melanin-concentrating hormone （MCH）, ting hormone precursor （P-MCH）, ghrelin, and leptin. Results Compared with the OVX control group, the body weight and food intake in the H-S1 group were reduced significantly and there was a significant dose-dependent manner in the 3 isoflavone groups. The results of RT-PCR showed that the NPY level in the 3 isoflavone groups was significantly increased and the POMC/CART gene expression decreased significantly in rats＇ hypothalamus compared with that in the OVX control group. However, the expression of orexin, MCH and P-MCH had no change. The peripheral grelin mRNA expression was higher in the 3 isoflavone groups, while leptin gene expression in the fat was not consistent. Conclusions This research showed that isoflavone could prevent obesity induced by high-fat diet and ovariectomy through regulating hypothalamus and peripheral orexigenic gene expressions associated with food intake.

Transcriptome responses to heat stress in hypothalamus of a meat-type chicken

Background： Heat stress has resulted in great losses in poultry production. To address this issue, we systematically analyzed chicken hypothalamus transcriptome responses to thermal stress using a 44 k chicken Agilent microarray, Methods： Hypothalamus samples were collected from a control group reared at 25℃, a heat-stress group treated at 34℃ for 24 h, and a temperature-recovery group reared at 25℃ for 24 h following a heat-stress treatment. We compared the expression profiles between each pair of the three groups using microarray data. Results： A total of 1,967 probe sets were found to be differentially expressed in the three comparisons with P 〈 0.05 and a fold change （FC） higher than 1.5, and the genes were mainly involved in self-regulation and compensation required to maintain homeostasis. Consistent expression results were found for 11 selected genes by quantitative real-time PCR. Thirty-eight interesting differential expression genes were found from GO term annotation and those genes were related to meat quality, growth, and crucial enzymes. Using these genes for genetic network analysis, we obtained three genetic networks. Moreover, the transcripts of heat-shock protein, including Hsp 40 and Hsp 90, were significantly altered in response to thermal stress. Conclusions： This study provides a broader understanding of molecular mechanisms underlying stress response in chickens and discovery of novel genes that are regulated in a specific thermal-stress manner.

Metformin inhibits food intake and neuropeptide Y gene expression in the hypothalamus

Metformin may reduce food intake and body weight, but the anorexigenic effects of metformin are still poorly understood. In this study, Sprague-Dawley rats were administered a single intracere- broventricular dose of metformin and compound C, in a broader attempt to investigate the regula- tory effects of metformin on food intake and to explore the possible mechanism. Results showed that central administration of metformin significantly reduced food intake and body weight gain, par- ticularly after 4 hours. A reduction of neuropeptide Y expression and induction of AMP-activated protein kinase phosphorylation in the hypothalamus were also observed 4 hours after metformin administration, which could be reversed by compound C, a commonly-used antagonist of AMP-activated protein kinase. Furthermore, metformin also improved lipid metabolism by reducing plasma low-density lipoprotein. Our findings suggest that under normal physiological conditions, central regulation of appetite by metformin is related to a decrease in neuropeptide Y gene expres- sion, and that the activation of AMP-activated protein kinase may simply be a response to the anorexigenic effect of metformin.

Sexual maturation in hens is not associated with increases in serum leptin and the expression of leptin receptor mRNA in hypothalamus

Background： In mammals, leptin is an attractive candidate for mediating the metabolic signal and the reproductive function via the specific receptor in hypothalamus. However, till now, the role of leptin on reproduction in birds is less well established. This experiment was conducted to elucidate the role of leptin on the onset of reproduction in bird, as a first step, to detect the changes of peripheral leptin and leptin receptor mRNA expression in hypothalamus between mature and immature hens at the same age. 120 ISA brown pullets at D60 were allocated randomly into two groups, long light （LL） group being raised under artificial light regimes with incrementally increased light phase （from 8 L：]6D to 14 L：]2D） and short light （SL） group raised on consistent light （8 L：16D） for 12 wk. Results： The results showed that pullets in LL group reached sexual maturation 15 d earlier than those in SL group. Serum E2 showed a significant increase with age, but no difference was observed between two groups. Serum leptin concentration decreased significantly from D112 to D136 in LL, and was markedly higher in LL group than that in SL at D112, while there was no significant difference between two groups at D136. Leptin receptor and GnRH-I mRNA expression in hypothalamus were significantly increased with age, yet there was no significant difference between SL and LL chickens at the same age. The expression of FSH-13 and LH-13 mRNA in pituitary was increased with age but did not show significant difference between LL and gland, and decreased from D112 to D136 in LL but not groups at the same age. SL group. GnfiH-I mRNA expression was very rich in pinea n SL group, and there was no difference between two Conclusions： These results indicate that the earlier sexual maturation in hens induced by long-light regime is not accompanied with an increase in serum leptin or leptin receptor gene expression in hypothalamus, or genes expression in HPG axis.

Changes of Neuronal Activities after Gut Electrical Stimulation with Different Parameters and Locations in Lateral Hypothalamus Area of Obese Rats

This study tested the effects of the gastrointestinal pulse train electrical stimulation with different parameters and at different locations on the neuronal activities of the lateral hypothalamus area（LHA） in obese rats in order to find the optimal stimulation parameter and location. Eight gastric electrical stimulations（GES） with different parameters were performed and the neuronal activities of gastric-distension responsive（GD-R） neurons in LHA were observed. The effects of stimulations with 8 parameters were compared to find the optimal parameter. Then the optimal parameter was used to perform electrical stimulation at duodenum and ileum, and the effects of the duodenal and ileac stimulation on the GD-R neurons in LHA were compared with the gastric stimulation of optimal parameter. The results showed that GES with the lowest energy parameter（0.3 ms, 3 mA, 20 Hz, 2 s on, 3 s off） activated the least neurons. The effects of GES with other parameters whose pulse width was 0.3 ms were not significantly different from those of the lowest energy parameter. Most gastric stimulations whose pulse width was 3 ms activated more LHA neurons than the smallest energy parameter stimulation, and the effects of those 3 ms gastric stimulations were similar. Accordingly, the lowest energy parameter was recognized as the optimal parameter. The effects of stimulations with the optimal parameter at stomach, duodenum and ileum on the LHA neuronal activities were not different. Collectively, gastrointestinal electrical stimulation（GIES） with relatively large pulse width might have stronger effects to the neuronal activities of GD-R neurons in LHA of obese rats. The effects of the GIES at different locations（stomach, duodenum and ileum） on those neurons are similar, and GES is preferential because of its easy clinical performance and safety.

THE EFFECT OF DECOCTION REHMANNIA ON THE CYTOCHEMICAL COMPONENTS OF THE LOCAL CEREBRUM, HYPOTHALAMUS AND ADRENAL GLAND OF EXPERIMENTAL CEREBRAL EMBOLISM

There have been fairly good effects ofDecoction Rehmannia(地黄饮子)in treat-ing stroke syndromes.The model of theexperimental cerebral embolism has been es-tablished and the morphological andcytochemical changes of the effect of

Chronic corticosterone disrupts the circadian rhythm of CRH expression and m^(6)A RNA methylation in the chicken hypothalamus

Background:Corticotropin-releasing hormone(CRH),the major secretagogue of the hypothalamic-pituitary-adrenal(HPA)axis,is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions.N6-methyladenosine(m^(6)A)RNA methylation is involved in the regulation of circadian rhythm,yet it remains unknown whether CRH expression and m^(6)A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress.Results:Chronic exposure to corticosterone(CORT)eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken.The circadian rhythms of clock genes in hippocampus,hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens.The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened,together with mRNA of other feeding-related neuropeptides.Interestingly,hypothalamic m^(6)A level oscillates in an opposite pattern to CRH mRNA,with lowestm^(6)A level after midnight(ZT18)corresponding to the peak of CRH mRNA before dawn(ZT22).CORT diminished the circadian rhythm of m^(6)A methylation with significantly increased level at night.Further site-specific m^(6)A analysis on 3’UTR of CRH mRNA indicates that higher m^(6)A on 3’UTR of CRH mRNA coincides with lower CRH mRNA at night(ZT18 and ZT22).Conclusions:Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus,leading to dysfunction of HPA axis in the chicken.RNA m^(6)A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.

Profiles of metabolic gene expression in the white adipose tissue, liver and hypothalamus in leptin knockout(Lep^(△I14/△I14)) rats

Leptin deficiency is principally linked to metabolic disorders. Leptin knockout（Lep△I14/△I14 Sprague Dawley rats created by CRISPR/Cas9 is a new model to study metabolic disorders. We used a whole rat genome oligonucleotide microarray to obtain tissue-specific gene expression profiles of the white adipose tissue, liver and hypothalamus in Lep△I14/△I14）and wild-type（WT） rats. We found 1,651 differentially expressed（enriched） genes in white adipose tissue,916 in the liver, and 306 in the hypothalamus in the Lep△I14/△I14 rats compared to WT. Gene ontology category and KEGG pathway analysis of the relationships among differentially expressed genes showed that these genes were represented in a variety of functional categories, including fatty acid metabolism, molecular transducers and cellular processes. The reliability of the data obtained from microarray was verified by quantitative real-time PCR on 14 representative genes. These data will contribute to a greater understanding of different metabolic disorders, such as obesity and diabetes.

The cerebellum, the hypothalamus and behavior

The cerebellum has been classically considered as the subcortical center for motor control. However, accumulating experimental evidence has revealed that it also plays an important role in cognition, for instance, in learning and memory, as well as in emotional behavior and nonsomatic activities, such as visceral and immunological responses.

Expression of mRNA-encoding subunits of N-methyl-D-aspartate receptor in the hypothalamus in sustained monaural block of auditory air-conduction model rats

A sustained monaural block of auditory air-conduction model was established in rats through subcutaneous suture in the right ear canal.The gene expression levels of hypothalamic N-methyl-D-aspartate receptor NR1,NR2A,NR2B and NR2C mRNA in the auditory central nervous system of Sprague-Dawley rats at postnatal 9,23,37 days were determined after an environmental change.Reverse transcription-PCR assay showed that the critical period for the development of NR1,NR2A,and NR2B subunits in the left hypothalamus and NR1-and NR2B-dependent auditory neurons in the right hypothalamus terminated 23 days after the suture in the right ear.The critical period for the development of NR2A subunit-dependent auditory neurons in the right hypothalamus was terminated by postnatal day 37.The results confirmed that N-methyl-D-aspartate receptor subunits in the hypothalamus may be regulated by the auditory environment.

Effects of Orexin A on mRNA Expression of Various Neuropeptides in the Hypothalamus and Pituitary, and on Serum LH Levels in Ovariectomized Gilts

Orexin has several biological functions, including the regulation of reproductive endocrine signaling, which has received much attention. However, little is known about the mechanism through which orexin regulates the levels of neuroendocrine hormones and peptides. We injected orexin A or physiological saline into the lateral ventricle of 10 ovariectomized （OVX） gilts, and determined the subsequent changes in serum luteinizing hormone （LH） concentration by using radioimmunoassay （RIA）. We also examined the expression of GnRH, NPY, and POMC mRNAs in the hypothalamus and that of LH, folliclestimulating hormone （FSH）, POMC, and ghrelin mRNAs in the pituitary by using semi-quantitative reverse transcription polymerase chain reaction. We found the following results： （1） Orexin A transiently promoted LH secretion; serum LH concentration started to increase at 10 min after the orexin injection, peaked at 30 min, and returned to its initial level at 1.5 h; （2） orexin A upregulated GnRH mRNA expression and downregulated NPY and POMC mRNAs expression in the hypothalamus; （3） orexin A upregulated LH and FSH mRNAs expression （FSH, P〉 0.05）, but downregulated ghrelin mRNA expression in the pituitary. No significant effects were observed on the pituitary expression of FSH and POMC mRNAs. Our data suggest that orexin A regulates reproductive function by stimulating GnRH and LH release directly and indirectly via its effects on NPY, POMC and ghrelin expression.

Altered Taurine Release in Hypothalamus of Aquaporin-4 Knockout Mice

Taurine is concentrated in glial cells in the hypothalamus and released in an osmo-dependent manner through volume-sensitive anion channels. Released tanrine acts on glycine receptors on vasopressin neurons to control vasopressin secretion. Water channel AQP4 is abundant in astrocytes in osmosensory areas such as the supraoptic nucleus of hypothalamus. An HPLC-based method was established to quantify taurine release from isolated hypothalamus tissues in wildtype and AQP4 knockout mice. Under the basal condition, there was no difference in taurine release from AQP4^＋/＋ and AQP4^-/- hypothalamuses. Taurine release from AQP4^-/- hypothalamus under hypoosmotic stimulation was significantly lower than that from AQP4^＋/＋ mice. AQP4 expression in the glial cells of the hypothalamus may play an important role in osmoregulation oftaurine release and subsequent vasopressin secretion.

Changes of neuroleukin mRNA levels in hypothalamus pituitary and splenic lymphocytes of rats during cold acclimation

Neuroleukin(NLK),a protein(Mr 56000),acts on both the nervous and the im-mune systems and its expression is regulated at mRNA levels.One hundred fifty Sprague-Daw-ley(SD)male rats were divided into 5 groups:control group(N)and groups of cold-exposurefor 1,2,3 and 4 weeks(ClW,C2W,C3W and C4W).With RNA dot blot experiments,changes of NLK mRNA levels in both neuroendocrine system(pituitary and hypothalamus)andimmune system(splenic lymphocytes,LC)of SD male rats during cold acclimation presentedsimilar responses.C/N ratios of scan gray intensity level in RNA dots are presented as follows:there was a rise in C/N ratio to 1.63～11.31 in CIW,a fall to 0.63 in C2W and a rise again to1.42～21.09 in C3W.It might thus be regarded as a general response to cold surroundings.Itwas reported that SDS-PAGE autoradiogram of synthetic proteins in LC and secretive proteinsin supernatant cultured in vitro showed that NLK content in LC was not parallel to its mRNAlevel because NLK was extremely meager over the 1-week cold exposure whereas it increasedobviously over the 3-and 4-week cold exposure.This is probably a major cause of why im-munoglobulin synthesis in cold-acclimated animals was greatly enlarged.

The Effect of Energy on Serotonin-Like Neurons in Duck Hypothalamus

In the present study, immunohistochemical method (SABC method) was used to examine the distribution of serotonin-like neurons in hypothalamus of Cherry Valley ducks bred with high energy diet and low energy diet respectively. All films were analysed by using a computer-assisted image analysis system. In high energy group, labelled neurons are widely distributed in paraventricular nucleus(PVN, 17.73±1.41 neurons/19.46×103 μm2) and ventromedial hypothalamus (VMH, 15.8±1.71 neurons/19.46×103 μm2); in low energy group, labelled cells are widely distributed in lateral hypothalamus (LH, 11.25±1.53 neurons/19.46×103 μm2), yet there is no positive neuron in PVN. These results indicate that serotonin-like neurons in hypothalamus are involved in the regulation of food intake and energy metabolism.

Changes of Serotonin content and Turnover Rate in Hypothalamus of Female Rat during Fluorosis

The animal models of subacute and chronic fluorosis were devel-oped with injection of large doses of NaF (IP)and drinking water containing100 ppm F^- in female rats respectively. Serotonin content and turnover rate inhypothalamus of rat were determined with spectrofluorometry combined degra-dation blockade. 5-HT turnover rate in hypothalamus decreased during bothsubacute and chronic fluorosis. 5-HT content in hypothalamus increased duringsubacute fluorosis, but decreased during chronic fluorosis. These results suggestthat the influences on 5-HT metabolism of two kinds of fluorosis are not com-pletely identical. The decrease of 5-HT turnover rate in hypothalamus may be one of thepossible mechanisms of deficiency of pituitary prolactin and milk secretion dur-ing fluorosis.

Effects of Sodium Diclofenac on the Distribution of Fos Protein in Central Amygdala and Lateral Hypothalamus during Experimental Tooth Movement in Rats

This study evaluated whether the administration of a NSAID, sodium diclofenac, can promote alterations in the expression of Fos protein in central amygdala (CEA) and the lateral hypothalamus (LH) after 6 h of experimental tooth movement with a controlled force of 70 g, applied to the superior central incisors of rats. Adult male rats were anesthetized and divided into four groups: Control, no orthodontic appliance (OA);OA activated with 70 g;OA activated with 70 g and pretreated with diclofenac sodium (5 mg/kg, intramuscular);and diclofenac sodium alone. Six hours after the onset of the experiment the rats were reanesthetized and perfused with 4% paraformaldehyde. The brains were removed and fixed, and sections containing the CEA and LH were processed for Fos protein immunohistochemistry. The results show that in the control group, intramuscular injection of a ketamine/xylazine mixture did not induce IR-Fos cells in the CEA or LH. However, in the 70 g group, IR-Fos was the strongest observed