| BackgroundIt is well known that obesity can induce a series of metabolic syndrome,such as insulin resistance,hyperglycemia,hypertension,hyperlipidemia.Obesity can lead to cardiovascular diseases such as diabetes.atherosclerosis,even and cancer.thus threatening human health.Excess energy is stored as fat when energy intake is greater than its consumption.Adipose tissues can be divided into white adipose tissues and brown adipose tissues according to their functions.White adipose tissues store energy and alleviate mechanical damage,while brown adipose tissues consume energy to regulate body temperature.In response to cold and high-fat diet,hypothalamus regulate the activation of the to release norepinephrine,and norepinephrine can work on noradrenergic receptor in brown adipose tissues,which activating cAMP-PKA-CREB pathway,that consuming energy through uncoupled respiratory processes mediated uncoupling protein-1(UCP-1).In addition,muscle tissue is also regulated by hypothalamus-sympathetic nerve system.which mediates metabolic adaptation and is involved in energy metabolism.Spermidine is a kind of polyamine,which is presented in all organisms.Emerging evidence has suggested that exogenous polyamines can promote longevity in mice.In addition,spermidine supplementation exerts cardioprotective effects in animal models.And spermidine supplementation can reduce body weight and alleviate hepatic steatosis in high-fat diet induced obese mice by inducing autophagy of white adipose tissue,resulting in promoting glucose tolerance and insulin sensitivity.However,the roles of oral spermidine in diet-obesity had not been reported.and influences of oral spermidine on other metabolic tissues remain largely unclear.In this study,spermidine was orally administrated into diet-induced obese mice.We showed that oral spermidine promoted the expression of tyrosine hydroxylase in the hypothalamus,induced the activation of brown adipose tissues and metabolic adaptation of skeletal muscle,thus reducing obesity and metabolic disturbance in HFD-fed mice.So,this study can provide a novel idea and a potential therapy for diet-induced obesity.Methods1.Establishment of diet-induced obesity model and intervention of oral spermidine in mice8 weeks age C57BL/6 male mice were fed on high-fat diet(HFD)and normal chow diet(ND)for 28-32 weeks.Diet-induced obesity model was established.Oral spermidine intervention was fed in mice of 12-16 weeks on a high-fat diet2.Effects of oral spermidine on obesity in HFD-fed miceDuring high-fat diet feeding,body weight was evaluated during the intervention of oral spermidine.After the intervention,the mice were sacrificed under anesthesia.White adipose tissues were collected from epididymal and subcutaneous areas for observation and weighing,and the percentage of adipose tissues weight to body weight were calculated for statistical analysis.3.Effect of oral spermidine on insulin sensitivity and glucose tolerance in HFD-fed miceIn the process of oral spermidine treatment,glucose tolerance test(GTT)and insulin tolerance test(ITT)were performed.For GTT.the mice were intraperitoneally injected with glucose and the blood glucose levels were measured at different time points.For ITT,the mice were intraperitoneally injected with insulin and the blood glucose levels were measured and analyzed at different time points.4.Effect of oral spermidine on white adipose tissues inflammation in HFD-fed miceAfter the intervention of spermidine,epididymal and subcutaneous white adipose tissues of mice were collected.White adipose tissues embedded in paraffin and sectioned.HE staining was used to observe adipocyte hypertrophy.Immunohistochemistry was used to observe infiltration of macrophages in white adipose tissues.5.Effect of oral spermidine on hepatic steatosis in HFD-fed miceAfter the intervention of oral spermidine,the liver tissues of the mice were collected,the weight of the liver was recorded,and the percentage of the weight of the liver in the body weight was calculated.HE staining was used to observe hepatic steatosis.ELISA was used to detect the levels of triglyceride in liver6.Effect of oral spermidine on activation of brown adipose tissues in HFD-fed miceAfter oral spermidine intervention,the brown adipose tissues of mice were collected Paraffin-embedded tissue sections and HE staining were used to observe adipocyte sizes.Immunohistochemistry staining of UCP-1 was observed in brown adipose tissues.In addition.Ucp-1,Pgc-1α,Pgc-1β,Cpt-1α,Cpt-1β and Cidea were detected by real-time quantitative PCR.And the expression of CREB.p-CREB(Ser133).PGC-1α and UCP-1 at protein levels were detected by western-blot7.Effect of oral spermidine on muscle metabolism in HFD-fed miceAfter oral spermidine intervention,the soleus tissues were collected.The expression of Ucp-1 and Glut-4 were detected by real-time quantitative PCR.the expression of UCP-1.PGC-1α,CREB.p-CREB(Ser133),AKT,p-AKT(Ser473)at protein levels were detected by western-blot.8.Effect of oral spermidine on the expression of tyrosine hydroxylase in hypothalamus and brown adipose tissues of HFD-fed miceAfter oral spermidine intervention,the expression of tyrosine hydroxylase(TH)at mRNA levels in hypothalamus and brown adipose tissues was detected by real-time quantitative PCR.The expression of TH at protein levels was detected by western-blot9.Effect of spermidine on the expression of tyrosine hydroxylase in SH-SY5Y cells and the norepinephrine content in supernatantsAfter 16 hours of spermidine intervention,the expression of TH and SIRT1 in SH-SY5Y cells were detected by real-time quantitative PCR.And the expression of CREB,p-CREB(Ser133)and TH at protein levels were measured by western-blot after spermidine intervention for 24 hours.The supernatant from cells stimulated by spermidine for 24 hours was collected.Norepinephrine in the supernatant was detected by ELISA10.Direct and indirect effects of spermidine on HIB-1B brown adipocytes and C2C12 myotubesHIB-1B preadipocytes,HIB-1B brown adipocytes and C2C12 myotubes were stimulated by spermidine,and the expression of CREB,p-CREB(Ser133),UCP-1 and PGC-1α at the protein levels were detected by western-blot.The supernatants from SH-SY5Y cells stimulated with spermidine were collected.HIB-1B preadipocytes,HIB-1B brown adipocytes and C2C12 myotubes were stimulated by the supernatants.The expression of Ucp-1 and Pgc-1α mRNA were detected by real-time quantitative PCR.The expression of UCP-1,PGC-1α,p-CREB(Ser133)and CREB in protein levels were detected by western-blot.Results1.Oral spermidine attenuates diet-induced obesityObesity model mice were divided into spermidine intervention group(HFD+SPD)and normal drinking water group(HFD).Mice fed with normal chow diet and normal drinking water were used as control group(ND).Compared with ND group,the body weight of HFD-fed mice were increased significantly.And oral spermidine intervention reduced the mice weight compared with HFD group2.Oral spermidine improves insulin sensitivity in HFD-fed miceITT results showed that insulin sensitivity in mice fed on HFD plus spermidine were significantly improved comparing with HFD-fed mice.And the blood glucose levels of mice fed on HFD plus spermidine were significantly lower than that of HFD-fed mice.GTT results showed no significant difference between mice fed on HFD plus spermidine and HFD-fed mice3.Oral spermidine attenuates white adipose tissues expansion and inflammation in HFD-fed miceCompared with ND group,the white adipose tissues weight of HFD-fed mice were increased significantly.And oral spermidine intervention reduced the weight of white adipose tissues in mice compared with HFD group.Compared with HFD-fed mice,adipocyte sizes in inguinal and epididymal white adipose tissues of mice fed on HFD plus spermidine were significantly decreased.Immunohistochemistry staining showed that oral spermidine intervention reduced the infiltration of macrophages in white adipose tissues4.Oral spermidine reduces hepatic steatosis in HFD-fed miceThe results showed that the weight of liver in mice fed on HFD plus spermidine was significantly reduced compared with that of HFD-fed mice.HE staining results showed that the hepatic steatosis in mice fed on HFD plus spermidine were attenuated significantly.The level of hepatic triglyceride of mice fed on HFD plus spermidine were significantly lower than that of HFD-fed mice5.Oral spermidine promotes the activation of brown adipose tissues in HFD-fed miceThe real-time quantitative PCR results showed that the mRNA levels expression of Vcp-1,Pgc-1α,Pgc-1β,Cpt-1α,Cpt-1β and Cidea genes in brown adipose tissues of mice fed on HFD plus spermidine were higher than that in HFD-fed mice.Western-blot results showed that,compared with HFD-fed mice,the expression of CREB.p-CREB(Ser133),PGC-1α and UCP-1 protein in brown adipose tissues of mice fed on HFD plus spermidine was significantly higher than that in HFD-fed mice.The adipocyte sizes in brown adipocytes of mice fed on HFD plus spermidine were decreased compared with HFD-fed mice.Immunohistochemistry showed that the expression of UCP-1 in the brown adipose tissues of mice fed on HFD plus spermidine was higher than that in HFD-fed mice6.Oral spermidine improves metabolic adaptation of skeletal muscle in HFD-fed miceThe real-time quantitative PCR results showed that the mRNA level expression of Ucp-1 in soleus tissues of mice fed on HFD plus spermidine was significantly higher than that in HFD-fed mice,and the expression of Glut-4 was also increased.Western-blot showed that,compared with HFD-fed mice,the protein levels expression of PGC-1α,UCP-1 were increased.And oral spermidine induced the expression and phosphorylation of CREB in soleus tissues of HFD-fed mice.In addition,oral spermidine also induced AKT activation in soleus tissues of HFD-fed mice7.Oral spermidine promotes tyrosine hydroxylase expression in hypothalamus of HFD-fed miceThe results showed that Th expression in hypothalamus of mice fed on HFD plus spermidine was significantly higher than that of HFD-fed mice.Western-blot results showed that TH expression in hypothalamus of mice fed on HFD plus spermidine was significantly higher than that of HFD-fed mice at protein level.But the mRNA and protein levels expression of TH in brown adipose tissues of mice fed on HFD plus spermidine were not significantly different from that in HFD-fed mice8.Spermidine increased norepinephrine by promoting tyrosine hydroxylase expression in hypothalamusSH-SY5Y cells were stimulated by spermidine in vitro.The qPCR results showed that spermidine promoted TH and SIRT1 expression in SH-SY5Y cells compared with control group.The western-blot results showed that,compared with control group,spermidine promoted p-CREB(Ser133).CREB and TH expression in SH-SY5Y cells.In addition,concentration of norepinephrine in supernatants of SH-SY5Y cells stimulated by spermidine was higher than those stimulated by PBS9.Spermidine acts on brown adipocytes and skeletal muscle cells through neural cellsThe supernatants from SH-SY5Y cells stimulated by spermidine was used to stimulate the HIB-1B preadipocytes,HIB-1B brown adipocytes and C2C12 myotubes.The results showed that the expression of UCP-1,PGC-1α,CREB and p-CREB(Ser133)were higher than control group at protein levels in HIB-1B brown adipocytes and C2C12 myotubes stimulated by the supernatants.In addition,the supernatants promoted the expression of UCP-1 and PGC-1α at protein levels in HIB-1B preadipocytes,compared with control group And the expression of Ucp-1 and Pgc-1α were higher than control group at mRNA levels in C2C12 myotubes.10.Spermidine directly acts on HIB-1B brown adipocytes and C2C12 myotubesSpermidine was used to stimulate the HIB-1B preadipocytes,HIB-1B brown adipocytes and C2C12 myotubes.The results showed that the expression of UCP-1 and PGC-1α were higher than control groups at protein levels in HIB-1B preadipocytes stimulated by spermidine.And the expression of UCP-1,PGC-1α,p-CREB(Ser133)were higher at protein levels in HIB-1B brown adipocytes stimulated by spermidine.In addition,the results showed that the expression of UCP-1,PGC-1α,CREB and p-CREB(Ser133)were higher than control groups at protein levels in C2C12 myotubes stimulated by spermidine.Conclusions1.Oral spermidine attenuates diet-induced obesity and metabolic disorders in HFD-fed mice2.Oral spermidine activates brown adipose tissues in HFD-fed mice3.Oral spermidine improves metabolic adaptation of muscle tissues in HFD-fed mice4.Oral spermidine promotes tyrosine hydroxylase expression in hypothalamus,thus inducing the activation of brown adipocytes and metabolic adaption of skeletal muscle cells in HFD-fed mice5.Spermidine also directly acts on brown adipocytes and skeletal muscle cells to improve metabolism... |
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