Inhibition Of Mitochondrial Calcium Overload By SIRT3 Prevents Whitening Of Brown Adipose Tissue

Background and Purpose:With irregular lifestyles and rapidly-changing dietary habits,energy intake is far greater than energy consumption that greatly increase the risks of obesity.Obesity and associated metabolic disorders,often lead to the occurrence of numerous metabolic diseases,which further affect people’s quality of life and physical health.Therefore,it is of great significance to prevent obesity and associated metabolic disorders.It is well acknowledged that not only white adipose tissue（WAT）,but also brown adipose tissue（BAT）play pivotal roles in the pathophysiology of obesity.BAT is a highly vascularized organ and rich in mitochondria,and thus contributes significantly to systemic metabolism.The contend and function of BAT decline with occurance of obesity or aging,which is also called BAT whitening that are characterized with decreased UCP-1 expression,accumulated lipid droplets,and mitochondrial dysfunction.Of note,mitochondrial dysfunction is the main culprit of BAT whitening.However,there are multiple factors,including oxidative stress,impaired respiratory function and calcium overload,that could exacerbate the mitochondrial function.Therefore,it is of great significance to explore the mechanism of regulating mitochondrial function and energy metabolism in order to prevent obesity.Previous studies have shown that capsaicin could inhibit high-fat diet（HFD）induced obesity through activating the transient receptor potential channel vanillic acid subtype V1（TRPV1）.In addition,the effect of capsaicin in improving obesity might be related with SIRT3,which belongs to is a NAD+-dependent deacetylase family and is mainly located in mitochondria that regulate the deacetylation of numerous proteins or enzymes.Researches have found that SIRT3 knockout mice had increased reactive oxygen species（ROS）generation that resulted in mitochondrial dysfunction.Calcium homeostasis is the premise of cell physiological activities and mitochondrial calcium overload is the culprit of the ROS overproduction and mitochondrial dysfunction.Ca²⁺uptake is mainly regulated by mitochondrial calcium uniporter（MCU）,which distributes in inner mitochondrial membrane and is is a low-affinity and low-conductance channel.However,whether or not SIRT3 plays important role in obesity-or aging-related mitochondrial calcium homeostasis and associated dysfunction,and whether or not SIRT3mediates the effect of capsaicin in improving obesity are still unknown.Therefore,we aimed to explore the role of SIRT3 in obesity and aging,in hoping of providing more information about obesity management.Materials and Methods:There were two parts,including animal experiments and in vitro experiments,in our study.WT,SIRT3 KO and TRPV1 KO mice and were randomly divided into groups that were feed with normal diet（ND）,high-fat diet（HFD）or high-fat+0.01%capsaicin diet（HFCD）,respectively.There were 10 mice in each group and dietary intervention duration were 32 weeks.The effect of capsaicin or SIRT3 on body weight and energy metabolism were explored.The culture of primary brown adipose cells from WT and SIRT3 KO mice were considered as the in vitro experiments.Methods:1.The body weight,food intake,intraperitoneal glucose tolerance test（IPGTT）,weight of BAT and WAT were determined.H&E staining was used to assess fat cell area.2.The energy expenditure（EE）and respiratory exchange ratio（RER）were measured by Comprehensive Laboratory Animal Monitoring System（CLAMS）,and DHE and Mito SOX red were used to determine the reactive oxygen species（ROS）.3.High resolution respirometry system was used to determine mitochondrial respiratory function.4.Intracellular and mitochondrial Ca²⁺uptake was measured using Fluorescence Master System and Multiskan Spectrum.5.Realtime quantitative PCR and Western Blot were used to detect the expression and distribution of TRPV1,SIRT3,MCU,MICU1,AMPK,UCP1,PRDM16 and PGC-1α.Result:1.Compared with WT-ND group,the body weight of WT-HFD and SIRT3 KO groups increased significantly.Similar with TRPV1 KO,SIRT3 KO abolished the effect of capsaicin in improving HFD-induced obesity.Nevertheless,there were no significant difference of food intake among groups.HFD resulted in significant glucose intolerance,however,the protective effects of capsaicin against HFD induced glucose intolerance disappeared in TRPV1 KO and SIRT3 KO group.Although BAT weight was almost equal in all groups,H&E staining showed that capsaicin could significantly inhibit the whitening of BAT that were induced by HFD,which were blocked in SIRT3 group.2.HFD decreased the expression of UCP-1,PRDM-16,and PGC-1α,accompanied with decreased EE,O₂consumption and CO₂ production and increased cytoplasmic and mitochondrial ROS generation.Capsaicin increased BAT activity and aerobic respiratory function;however,all of these effects disappeared in SIRT3-KO mice.3.HFD increased the mitochondrial Ca²⁺uptake of primary brown adipocyte.Capsaicin inhibited the HFD induced Ca²⁺overload,however,the effect was blocked in SIRT3 KO mice.In vitro experiment showed that capsaicin could improve palmitic acid induced mitochondrial Ca²⁺overload,which could be blocked by si-Sirt3 treatment and be promoted by SIRT3 overexpression.4.Capsaicin could inhibit HFD induced expression of MCU and MICU,which were disappeared in SIRT3 KO mice.In vitro experiments showed that mitochondrialdysfunction and Ca²⁺overload could be exacerbated by MCU overexpression on thepremise of SIRT3 overexpression and be reversed by si-Mcu on the premise of si-Sirt3 or SIRT3 KO.5.According to Chromatin Immunoprecipitation（Ch IP）,capsaicin reduced MCU expression and H3K27ac level on its promoter,of which the latter one could be increased by si-Sirt3.In vitro experiments showed that inhibition of H3K27ac could reduce MCU expression and mitigate PA induced mitochondrial Ca²⁺uptake.In addition,H3K27ac level and MCU expression could be increased by AMPK inhibition or si-AMPKαand bedecreased by AMPK activation.6.Capsaicin blocked HFD induced p-AMPK,PGC-1αand SIRT3 down-regulation,which were consistent with in vitro experiments.SIRT3 could be promoted by AMPK activation or PGC-1αoverexpression and be reduced by AMPK inhibition or si-Pgc-1α.ChIP indicated that there existed an obvious positive binding signal of PGC-1a on thepromoter of the Sirt3 gene,which could be reduced by PA and enhanced by capsaicin or AMPK activation.Conclusion:1.The expression of SIRT3 in BAT were significantly reduced in HFD-induced obesity.Capsaicin inhibited BAT whitening and thus prevented obesity through TRPV1activation,however,all the effects were disappeared in SIRT3 KO mice,which suggested the significant role of SIRT3 in BAT whitening and thus could be an effective target of capsaicin in preventing obesity.2.Capsaicin improved HFD induced mitochondrial dysfunction,however,the effects were blocked by SIRT3 KO,which suggested that SIRT3 mediated beneficial roles of capsaicin in alleviating oxidative stress and mitochondrial dysfunction through TRPV1activation.Therefore,SIRT3 plays significant role in preventing BAT whitening.3.AMPK induced SIRT3 activation alleviated mitochondrial Ca²⁺overload through MCU down-regulation via decreasing H3K27ac level on its promoter.Capsaicin restored SIRT3 expression and alleviated mitochondrial Ca²⁺overload that were induced by AMPK inactivation during obesity,which suggested that AMPK/SIRT3 play crucial roles in capsaicin mediated mitochondrial Ca²⁺homeostasis and BAT whitening inhibition.Thus,SIRT3 could be a promising target in preventing obesity.