Effects Of High Fat And Baicalin On The Hypothalamic FNDC5/Autophagy Pathway And Its Relationship With Alterations In Food Intake And Body Weight

IntroductionWith economic development and continuous improvement of living standards,obesity is becoming a global epidemic disease.As a potential hazardous factor for human health,obesity can cause various complications(including diabetes,cardiovascular disease cancer,and etc)which seriously affect life quality and span,and bring a huge burden to the country and society.The major factor for obesity is that the energy intake is higher than the energy consumption.The hypothalamus,a critical brain region for the control of energy metabolism and feeding behavior,can sense metabolic signals such as nutrients and hormones and is involved in the regulation of energy balance.It is demonstrated that cellular autophagy is essential for the functions of hypothalamic neurons which control food intake and energy homeostasis.Overconsumption of saturated fatty acid can cause defective hypothalamic autophagy,which leads to an imbalance in energy metabolism,increase in food intake and body weight.However,the detailed molecular mechanisms underlying the high-fat-induced defective hypothalamic autophagy and increase in food intake remain to further investigated.FNDC5 is a novel myokine,which can regulate the browning of white fat cells and increase energy consumption.FNDC5 has been identified to reduce glucose and lipid metabolism disorders,insulin resistance,oxidative stress and cellular inflammation that induced by high-fat diet.It can also alleviate tissue autophagy damage by participating in autophagy regulation pathways in liver and myocardial tissue.The role of FNDC5 in the regulation of autophagy in the hypothalamus is not reported.Baicalin,a major active component from the Scutellaria baicalensis,plays a neuroprotective role by freely crossing the blood-brain barrier.Baicalin has been identified to play variable pharmacological functions,such as anti-oxidation,anti-inflammatory,anti-dyslipidemia and anti-tumor.Recent studies have shown that baicalin could prevent obesity induced by high-fat diet,but the role of baicalin in the hypothalamus referring to eating behavior and body weight is not known.ObjectiveThis study intends to determine the effects of high fatty acids and baicalin on the hypothalamic autophagy pathway and its relationship with food intake and body weight,which will reveal the molecular mechanism of high-fat-induced hypothalamic defective autophagy referring to increases in food intake and body weight,and the role of baicalin in reversing the effects of high fat.Methods1.High-fat-diet mice:C57BL/6 mice were respectively fed with high-fat diet(HFD)and regular chow diet(SD)for 7 days.The body weight and food intake were assessed once per day.After 7-day treatment,total RNA and protein of the hypothalamus were extracted.The expression levels of Fndc5 mRNAs of the two groups were detected by qRT-PCR,and the protein expression levels of hypothalamic FNDC5,p62 and LC3-II of the two groups were detected by Western blot.2.Palmitic-acid-treated mice:palmitic acid(PAL)and saline(SAL)were intraperitoneally injected into the mice for 7 days,respectively.The body weight and food intake of the two groups were assessed once per day.After 7-day treatment,total RNA and protein of the hypothalamus were extracted.The Fndc5 mRNA levels were detected by qRT-PCR,and the protein levels of hypothalamic FNDC5,p62 and LC3-II were detected by Western blot.3.PAL-treated GT1-7 cells with:GT1-7 cells were treated with saline(SAL)and 50μM palmitic acid(PAL)for 48 hr.The total proteins of the treated cells were extracted.The protein levels of FNDC5,p62 and LC3-Ⅱ were detected by Western blot.4.Overexpression of FNDC5 in GT1-7 cells:After 48-hr treatment with FNDC5 overexpression,total proteins wre extracted.The protein levels of FNDC5,p62 and LC3-Ⅱ were detected by Western blot.5.GT1-7 cells treated with different concentrations of baicalin:GT1-7 cells were treated with 50 μM palmitic acid and baicalin titrated from 0,25 μM,50 μM,100 μM,and 200 μM.After 48-hr treatment,the expression of FNDC5 protein was detected by Western blot to determine whether baicalin could reverse the effects of PA on the expression of FNDC5.6.The optimal concentration of baicalin to treat GT1-7 cells:the cells were divided into three groups,the negative control group was treated with saline,the positive control group was treated with 50 μM palmitic acid,and the experimental group was treated with both 50 μM palmitic acid and 50 μM baicalin.After 48-hr treatment,the total RNA and total protein of each group of cells were extracted.The expression level of Agrp mRNA in each group was detected by qRT-PCR.The protein expression levels of FNDC5,p62,LC3-Ⅱ and AGRP in each group were detected by Western blot.7.Baicalin-treated mice:the mice were divided into three groups and treated for 7 days respectively.The negative control group and the positive control group were intraperitoneally injected with saline and palmitic acid,respectively.The experimental mice were intraperitoneally injected with palmitic acid and baicalin(400 mg/kg/day)by oral gavage.The body weight and food intake of these mice were assessed according to above descriptions.After 7-day treatment,total RNA and total protein of the hypothalamus of the mice were extracted.The expression of Fndc5 and Agrp mRNAs in each group of mice were detected by qRT-PCR.The protein levels of hypothalamic FNDC5,p62,LC3-Ⅱ and AGRP were detected by Western blot.Results1.Compared with the regular chow diet(SD)mice,the food intake decreased and the body weight increased in the high-fat-diet(HFD)mice.Futhermore,compared with saline(control)mice,no significant difference in food intake but increase in body weight were observsed in the palmitic-acid treated(PAL)mice.2.Compared with mice in SD group and SAL group,the hypothalamic Fndc5 mRNA and protein levels of HFD group and PAL group significantly increased,suggesting that high fat promotes Fndc5 expression in the hypothalamus.3.Compared with SD group and SAL group,the hypothalamic LC3-Ⅱ levels of HFD and PAL group were significantly down-regulated,and the p62 levels were significantly up-regulated,indicating that high fat caused defective autophagy in the hypothalamus.4.Compared with the control GT1-7 cells,the levels of FNDC5 and p62 in the PAL-treated cells increased,and the LC3-Ⅱ levels decreased.Further analyses shown that overexpression of FNDC5 in the GT1-7 cells could lead to increase of the p62 level and decrease of LC3-Ⅱ level.These data suggest FNDC5 is involved in the high-fat-induced defective autophagy.5.Compared with PAL-treated GT1-7 cells,the expression levels of FNDC5 and p62 in GT1-7 cells treated with 50 μM palmitic acid and 50 μM baicalin were down-regulated,and the expression levels of LC3-Ⅱ was up-regulated.Compared with the PAL-treated mice,the expression levels of FNDC5 and p62 in the hypothalamus of mice that treated with palmitic acid and baicalin were down-regulated,and the expression levels of LC3-Ⅱwere up-regulated.These data suggest that baicalin reversed the effects of PAL-induced defective autophagy through down-regulating FNDC5 expression.6.Compared with the control cells,there was no significant change in Agrp mRNA and protein levels in cells treated with palmitic acid alone.Compared with PAL-treated cells,the Agrp mRNA and protein levels were down-regulated in the cells treated with PAL and baicalin.Furthermore,compared with the control mice,no significant change was observed in hypothalamic Agrp mRNA and protein levels in the PAL-treated mice.Compared with the PAL-treated mice,the levels of Agrp mRNA and protein were down-regulated in the mice treated with PAL and baicalin.Our data showed that PAL did not affect the expression of AgRP,while baicalin could down-regulate the expression of AgRP.7.Compared with the control mice,no alteration of the food intake and the increase in body weight were observed in the PAL-treated mice.Compared with the PAL-treated mice,both food intake and body weight were reduced in the mice treated with PAL and baicalin.ConclusionOur data suggest that FNDC5 is involved in the regulation of autophagy pathway in the hypothalamus,and high fat leads to increases in body weight through the defective FNDC5/autophagy pathway;while baicalin could alleviate the defective effect of FNDC5/autophagy induced by high fat.Baicalin also reduces the expression of hypothalamic AgRP,and thus leads to decrease in food intake and body weight.This study reveals the mechanism of FNDC5/autophagy pathway in high-fat-and baicalin-induced changes of feeding and body weight,and provides potential targets for further development of obesity interventions.