| BackgroundWith the improvement of people's living standard and the aging of the population,the incidence of arteriosclerotic cardiovascular disease?ASCVD?has increased year by year.However,the certain pathogenesis of ASCVD is unclear.There are a variety of risk factors for the development of ASCVD,including age,gender,dyslipidemia,hypertension,diabetes,obesity,smoking,and family history.The 2013 American Heart Association/American College of Cardiology?AHA/ACC?Guidelines for Adult Overweight and Obesity Management states that the obesity is the key point of lipid metabolism disorders and an important risk factor for ASCVD.The incidence of obesity involves the pathological process of excessive food intake and/or reduced energy consumption.It is worth noting that Neuropeptide Y?NPY?,as a regulator of energy metabolism,participating in the occurrence and development of obesity.NPY is a polypeptide composed of 36 amino acids,distributed in the central nervous system,peripheral nervous system and surrounding tissues.In the central nervous system,NPY is expressed in the whole brain,but the expression level in the hypothalamus arcuate nucleus is the highest.NPY plays a key role in regulating many physiological processes,including promoting food intake,increasing energy intake and reducing energy consumption.NPY/agouti gene-related protein?AgRP?and opioid-melanocyte procortin?POMC?neurons are the most widely distributed neurons in the arcuate nucleus.NPY/AgRP neurons play an appetite-stimulating role and can also inhibit the anorexia effect of POMC neurons through NPY-Y1 receptor or gamma-aminobutyric acid?GABA?.Therefore,the hypothalamic arcuate nucleus-related neurons are closely associated with obesity.Recent studies have suggested that cold stimulation can be used as an intervention to prevent and control obesity,mainly based on the discovery that cold stimulation can lead to the activation of brown adipose tissue?BAT?and beige adipose tissue?BeAT?in mice and humans,leading to increased energy consumption.Cold stimulation is a"stress"to the body,which can activate the sympathetic nervous system and is involved in the diet-related mechanisms,especially the mechanism that promotes weight loss,including lipolysis mediated by beta-adrenergic receptors,inhibition of adipocyte proliferation in white adipose tissue?WAT?,and BAT.However,what confuses the researchers is that there appears to be an increasd sympathetic activity in obese people when compared with skinny people,suggesting that beta-adrenergic activity may promote weight gain in certain conditions.In order to elucidate the comprehensive effects of chronic cold stimulation on energy balance and glucose homeostasis,and the central mechanism leading to these changes,the first part of this study explored whether the cold stimulation could play a role in weight loss under the condition of high-fat diet and investigate the regulation mechanism of NPY in the central nervous system in this process by constructing a cold stimulation mouse model.There are mutiple interventions for obesity treatment,mainly through adjusting diet structure,strengthening exercise and inhibiting appetite.Recent studies have demonstrated that there is a certain relationship between the Receptor Activator for Nuclear Factor-kappa B Ligand?RANKL?/Receptor Activator for Nuclear Factor-kappa B?RANK?and energy homeostasis in the central nervous system of the brain.RANKL is a 317 amino acid polypeptide belonging to the tumor necrosis factor?TNF?family.The expressions of RANKL/RANK protein and mRNA are expressed in bone,bone marrow,lymphoid tissue,hypothalamus and septum of brain.The elevated levels of RANKL have been observed in the blood of patients with anorexia nervosa,which are consistent with the severity of anorexia nervosa.In that case,we speculate that RANKL/RANK signalling path way may be related to reduce appetite.At the same time,other researches have found that the RANKL/RANK signaling is regulated by Cocaine-Amphetamine-Regulated transcript?CART?and NPY.Therefore,we investigated whether the presence of RANKL/RANK in the hypothalamic region can affect appetite by acting on NPY neurons distributed in the same region.In order to verify the above assumption,the second part of this study investigates whether RANKL/RANK can reduce food intake and lead to weight loss by down-regulating hypothalamic NPY neurons,and its possible specific mechanisms.Obesity and abnormal blood glucose and lipid often exist at the same time,which all belong to the category of metabolic syndrome?MetS?.There are many interrelated mechanisms among them,especially in the central nervous system.In clinical practice,we observed that obese patients with cardiovascular diseases often have abnormal blood lipids levels such as high low-density lipoprotein?LDL?,low high-density lipoprotein?HDL?and high triglyceride.More and more studies have proved that LDL/LDLR?low-density lipoprotein receptor?plays an important role in the development of obesity.Moreover,other studies have found that low-density lipoprotein receptor?LDLR?deficiency reduces fat synthesis and increases calorie production,leading to weight loss.Therefore,we further speculate that some specific blood lipids may activate hypothalamic NPY neurons and these blood lipids may include LDL/LDLR.In the third part of this study,we preliminarily explored whether LDL/LDLR could participate in the regulation of body fat and energy metabolism through the NPY pathway of the central nervous system.Methods1.The effect of cold stimulation on the metabolism of mice fed with high fat diet?HFD?and the role of NPY in the relevant central mechanisms1.1 Selection and grouping of experimental animals56 wild-type C57BL mice and 28 C57BL mice with NPY-GFP?expressing green fluorescent protein GFP in brain under the control of NPY promoter?were divided into three groups for study.Among them,28 wild-type C57BL mice were used to study energy intake,including meaurement of body weight,adipose tissue weight,glucose and insulin tolerance test,and in situ hybridization of hypothalamic target proteins.Anothe r 28wild-type C57BL mice were used to study c-fos expression in the hypothalamic amygdala region.In addition,28 NPY-GFP mice were used to determine whether NPY neurons in NPY-GFP mice were activated by cold stimulation and HFD.1.2 Experimental animal feeding methodsWhen the mice were 10 weeks of age,each group was fed with two diets:one half were fed with standard chow diet and the other half were fed HFD.1.3 Cold-stimulation intervention in experimental animalsAfter 10 weeks of age,14 mice in each group?7 standard chow diets,7 high-fat diets?were simultaneously subjected to cold stimulation intervention.Method:Mice were placed in an ice-water mixture?0°C?with a depth of 0.5 cm,1 hour per day for 7 weeks.Non-cold-stimulated mice were placed in room temperature water?23°C?at the same time.1.4 Body weight measurement and food intake assessment of experimental animals28 wild-type C57BL mice used for energy intake studies were weighed once a week at the same time.When the mice were at the age of 10,12,14 and 17 weeks,the food intake was measured within 24 hours and was calculated as energy intake?kJ/day?.1.5 Determination of glucose metabolism in experimental animalsIn the energy intake study,28 wild-type C57BL mice at the age of 16 weeks?6 weeks after initiation of HFD and cold stimulation?were used to glucose and insulin tolerance tests.During experiments,the insulin tolerance test was conducted after 2 days of the glucose test.1.6 Tissues sample collectionThe tissues of experimental animals were taken at the age of 17 weeks?after 7 weeks of HFD and cold stimulation intervention?.After anaesthetized with 100 mg/kg ketamine and 20 mg/kg xylazine,mice of three groups sacrificed.28 wild-type C57BL mice used in the energy intake study were dissected.We collected and weighed the BAT between the scapula and the groin,epididymis,mesenteric and retroperitoneal WAT.As for the brain tissue collection methods for the three groups,mice were all perfused with saline,fixed with 4%paraformaldehyde,then dehydrated with 30%sucrose solution,and finally stored at-70°C.1.7 Immunohistochemical detection of C-fosAfter 7 weeks of HFD and cold stimulation intervention,the brain tissues of 28wild-type C57BL mice used for C-fos expression studies were collected.The brain tissues were used to detect C-of immunoreactive in the amygdala region by immunohistochemistry.The results were quantified by Zeiss Axioplan optical microscope?Carl Zeiss Imaging Solutions GmbH,Munich,Germany?.1.8 Immunohistochemical detection of C-fos and NPY in NPY-GFP mice groupAfter 7 weeks of HFD and cold stimulation intervention in 28 transgenic mice,the brain tissues were collected to detect red C-fos active neurons and green GFP-positive NPY neurons by immunohistochemistry.The results were quantified by Zeiss Axioplan optical microscope?Carl Zeiss Imaging Solutions GmbH,Munich,Germany?.1.9 In situ hybridization of brain tissueIn situ hybridization experiments were performed using brain tissues of 28 wild-type C57BL mice used for energy intake studies.We detected the mRNA expression of brain-derived neurotrophic factor?BDNF?and growth hormone releasing hormone?GHRH?in ventromedial hypohalamus?VMH?and paravenricular hypohalamus?PVH?.1.10 Data StatisticsAll statistical analyses were performed using GraphPad Prism Version 6.0?GraphPad Software,Inc?.2.The effect of RANKL on food intake and body weight in mice and the regulation mechanism of central NPY2.1 Selection and grouping of experimental animals30 wild-type C57BL6 male mice and 8 transgenic NPY-GFP mice were divided into 4groups for the study.Among them,12 wild-type C57BL6 male mice were implanted with brain micropump to observe the relationship between RANKL injection and body weight and food intake;10 wild-type C57BL6 male mice were used to detect C-fos expression in the thalamus region after 30 minutes of RANKL injection;8 transgenic NPY-GFP mice were used to detect co-expression of C-fos and NPY in the hypothalamic region after 30minutes of RANKL injection;8 wild-type C57BL6 male mice were used to detect C-fos and CART mRNA co-expression in the hypothalamic region after 30 minutes of RANKL injection2.2 Intervention of experimental animalsThere are two ways to give RANKL in mice.Micro-infiltration syringe was implanted in the brain of the mice used for measuring body weight and food intake after RANKL injection,keeping sustained and stable administration within one week.The other three groups of mice were administered in a single dose via the tail vein injection.The mice in each group were divided into the intervention group and the control group.The intervention group was injected with 10?g of RANKL/1ml in saline per day,and the control group was injected with 1 ml of saline.2.3 Body weight measurement and food intake evaluation of experimental animalsThe mice used for measuring body weight and food intake were fed with normal diet,and injected with 10?g of RANKL/1 ml in saline or 1 ml saline into the brain every day.Body weight was measured every day.1 mouse died in the saline injection group.On the8th day,cumulative food intake of 24 hours was measured for the intervention group and the control?from 10:00 on the 7th day to 10:00?.2.4 In situ hybridization of mice brain tissues after 30 minutes of interventionThree groups of mice used for observing immediate effect after RANKL injection were injected with 10?g RANKL/1 ml in saline or 1 ml saline via the tail vein injection.30minutes after the injection,the mice anesthetized with 100 mg/kg ketamine and 20 mg/kg xylazine were sacrificed.After perfused with saline and fixed with 4%paraformaldehyde,the brain tissue was removed and dehydrated with a 30%sucrose solution overnight.The brain tissue was cut into 30?m thick coronal sections.The in situ hybridization method was used to detect the staining of c-fos,double-labeled c-fos and NPY,and double-labeled c-fos and CART mRNA in the hypothalamic region.2.5 In situ hybridization of mice brain tissue after 1 week of interventionThe brain tissue samples of mice used for measuring body weight and food intake were collected?method described in 2.4?on the 8th day.In situ hybridization were performed to dectect NPY,CART,POMC,AgRP mRNA levels in Arc,as well as CART mRNA levels in DMH.2.6 Data Statistics?described in 1.10.?3.The effect of LDL on the body weight of mice and the regulation mechanism of central NPY3.1 Selection and grouping of experimental animals12 wild-type C57BL6 male mice,18 LDLR-/-mice,and 6 transgenic NPY-GFP mice were divided into 3 groups for the study.Among them,12 mice of LDLR-/-were used to detect changes in body weight;12 wild-type C57BL6 male mice and 6 LDLR-/-mice were used to determine immediate c-fos expression;6 transgenic NPY-GFP mice were used to detect co-expression of C-fos and NPY.3.2 Feeding methods and measurement of body weight12 LDLR-/-mice were divided into two groups:HFD and standard chow diet.The body weight of the mice was measured at the same time of every week for 10 weeks.3.3 Determination of C-fos in mice brain tissue12 wild-type C57BL6 male mice and 6 LDLR-/-mice were divided into control group?C57BL mice intravenously injected with saline?,injection group?C57BL mice intravenously injected with LDL?,knockout injection group(LDLR-/-mice intravenously injected with LDL).After 30 minutes of the tail vein injection,the mice anesthetized with100 mg/kg ketamine and 20 mg/kg xylazine were sacrificed.Mice were perfused with saline and fixed with 4%paraformaldehyde.The brain tissue was removed and dehydrated with 30%sucrose solution overnight.The brain tissue was cut into 30?m thick coronal sections,and the expression of C-fos in the hypothalamic region was determined by in situ hybridization.3.4 Co-expression of c-fos and NPY in mice brain tissueAfter 30 minutes of 6 transgenic NPY-GFP mice injected with LDL,the co-expression of C-fos and NPY in the hypothalamic arcuate nucleus was detected by immunohistochemistry described in 3.3.3.5 Data statistics?described in 1.10.?Result1 The effect of cold stimulation on the metabolism of mice with HFD and the regulation mechanism of central NPY1.1 Synergistic effects of cold stimulation and HFD on body weight and food intakeIn the four groups,the mice in the cold-stimulation plus HFD group had the most weight gain,followed by the high-fat diet group,the third one is the cold-stimulated group and the last one is the standard chow diet group.At 2,4,and 7 weeks of intervention,food intake was measured,and the trend of the curve was consistent with the weight gain.1.2 Cold stimulation aggravated glucose metabolism in HFD-fed miceIn the glucose tolerance test,cold stimulation stimulation decreased glucose clearance in both standard diet and HFD-fed mice,showing poor glucose tolerance.However,cold stimulation did not change blood glucose levels in mice from different diet groups after insulin injection.1.3 Cold stimulation and HFD resulted in an increase in WAT weight in mice.The total WAT of the mice was the highest in the cold-stimulation plus HFD-fed group,followed by the HFD-fed group.There was no difference in the BAT weight among the four groups.1.4 Cold stimulation activated central amygdala neurons in mice.Cold-stimulation significantly increased C-fos-positive neurons in the central amygdala of mice in both standard chow diet and HFD-fed mice.At the same time,mice in the HFD group also showed more c-fos-positive neurons than the standard diet group.1.5 Cold stimulation activated the central amygdala NPY neuronsIn high-fat diet fed mice,cold stimulation stimulated more activated NPY neurons in the central amygdala of the NPY-GFP mouse,and the hypothalamic central amygdala sections showed that 61%of the activated neurons showed C-fos and NPY double staining.1.6 Cold stimulation inhibited the expression of BDNF mRNA and induces theexpression of GHRH mRNAIn both standard chow diet and HFD-fed mice,cold stimulation reduced the expression of BDNF mRNA in mouse VMH,and increased the expression of GHRH mRNA in PVN.There was a similar effect in HFD-fed mice compared to the standard diet.2 The effect of RANKL on food intake and body weight in mice and the regulation mechanism of central NPY2.1 Changes in body weight and food intake after RANKL administrationCompared to controls,the RANKL group showed a significant decrease in body weight and a lower food intake.2.2 Determination of C-fos immunoreactivity in hypothalamic neurons after RANKL administrationIn the arcuate nucleus?Arc?and dorsal medial nucleus?DMH?regions,C-fos-positive neurons in the RANKL-treated group were significantly more than controls group.2.3 Co-localization of NPY or CART mRNA and c-fos neurons in hypothalamic neurons after RANKL administrationIn Arc,there were 54%overlap of NPY-GFP neurons and C-fos in the RANKL group.Immunohistochemistry and in situ hybridization of C-fos showed that in the RANKL group,there were about 72%co-expression of the C-fos and CART mRNA in DMH.2.4 Expression of NPY,CART,POMC and AgRP mRNA in hypothalamic neurons after RANKL administrationCompared with the control group,in Arc,NPY mRNA expression was significantly decreased and CART mRNA expression was significantly increased in RANKL treated group.In DMH,CART mRNA expression was up-regulated in the RANKL group.3 The effect of LDL on body weight of mice and the regulation mechanism of central NPY3.1 LDL high-fat diet did not increase the weight of LDLR-/-mice.There was no significant difference in body weight between LDLR-/-mice in the high-fat diet group and the normal diet group.3.2 LDL activated downstream neurons through the hypothalamic arcuate nucleus LDLRAfter tail vein injection of LDL in C57BL6 mice,C-fos expression increased in hypothalamic arcuate nucleus,while there were no significant difference in LDLR-/-mice compared with the control group.Besides,the C-fos expression in LDLR-/-mice were lower than that in LDL treated C57BL6 mice.3.3 LDL increased NPY expression in hypothalamic arcuate nucleusAfter injection of LDL via the tail vein,the expression of NPY neurons in the arcuate nucleus of the NPY-GFP mice was significantly increased,and 81%of c-fos neurons were in the same region as NPY neurons..In conclusion1 Cold stimulation inhibited the expression of BDNF mRNA in VMH and induced GHRH mRNA expression in PVN by activating central amygdala NPY neurons,resulting in increased food intake,weight gain,increased WAT mass,and impaired glucose tolerance.In this process,HFD plays a synergistic role.2 RANKL decreased the expression of NPY mRNA in the hypothalamic Arc and the up-regulation of CART mRNA in Arc and DMH,which decreased the appetite of mice,thus reducing the effect of HFD on body weight.3 LDL activated NPY neurons through the LDLR of NPY neuron in hypothalamic Arc,resulting in peripheral lipid metabolism and energy metabolism disorders,leading to obesity. |
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