Resveratrol Ameliorates Lipid Droplets Accumulation By SIRT1/ATF6-Dependent Mechanism

BackgroundMetabolic syndrome?MetS?is a multi-metabolic abnormal disease characterized by central obesity,nonalcoholic fatty liver disease?NAFLD?,hypertension,glucose metabolism and dyslipidemia.The mortality of MetS continue to rise,and it has become a major nutritional-related disease that seriously jeopardizes the health of our residents and hinders social development.Many studies showed that obesity,especially central obesity,which performs as the accumulation of excess fat in metabolic target organs?such as liver and adipose tissue?,is the common pathological basis of MetS.Lipid droplets?LDs?are highly dynamic organelles produced from the endoplasmic reticulum and their main physiological function is to store neutral lipids.LDs is almost abundant in liver and adipose tissue,and the formation,fusion and accumulation of LDs is an important part in the development of MetS.The unfolded protein response?UPR?induced by endoplasmic reticulum stress?ERS?not only promotes the formation of LDs,but also affects the fusion and accumulation of LDs in liver and adipose tissue,which has become an important intervention target for regulating LDs accumulation.A large amount of evidence indicates that dietary structural changes and consequently nutritional imbalances and metabolic abnormalities are the key causes of the high incidence of MetS.Nutrition intervention has become the simplest and most practical way to prevent and control metabolic syndrome.Human studies have found that plant food intake is negatively correlated with the occurrence of a variety of chronic metabolic diseases,and the efficacy components of its health effects are mainly phytochemicals.Our previous clinical trials and experimental studies found that RSV can significantly improve the lipids and glucose metabolism,but the underlying mechanism is not clear.Based on our previous studies and other studies carried out by other people,the current study propose the following hypothesis:RSV may improve the progress of MetS by inhibiting lipid droplets accumulation in liver and fat tissue.The current study will conduct in cells and mouse fed by high-fat diet to assess the influence of in RSV on the accumulation of LDs in liver?liver cells?and adipose tissue?fat cells?,and underlying mechanism including the influence of RSV on the regulation ofsilent mating-type information regulation 2 homolog 1?SIRT1?and ATF6?a key gene in UPR?,and LDs accumulation related gene?Fsp27 and CREBH,etc.?,and the final effect on the accumulation of LDs in liver?liver cells?and adipose tissue?fat cells?.This study will provide a new experimental evidence for the prevention of MetS by RSV and new intervention targets for the prevention of MetS.ObjectiveTo investigate the effect and mechanism of RSV on ameliorating lipid droplets accumulation through SIRT1/ATF6-mediated LD-related proteins in hepatocytes and adipocytes.Methods1.The SIRT1 knockdown?SIRT1 KD?mice and wild-type?WT?mice fed with high-fat diet?HFD?was administered intragastric RSV of 400 mg/kg/day for 30 days.Body weight and food intake of mouse were recorded.The liver and adipose tissue lipid accumulation degree and the pathological changes were assayed by HE staining and immunofluorescence cytochemistry method,magnetic resonance?MRI?methods.Serum glucose and lipids metabolism index were measured by kits.Expression levels of LDs related gene?Fsp27 PLIN1CREBH?,ERS related gene?GRP78?and UPR related gene?PERK,IRE-1,ATF6?in the liver and fat tissue were tested by qRT PCR and western blot method.All above tests were to assess the effect of RSV on the accumulation of LDs in liver and adipose tissue of HFD fed mice.2.Primary hepatocytes isolated from WT mice and human hepatoma cell line HepG2 cells were exposed to PA to mimic hepatic steatosis in vivo.Differentiated 3T3-L1 cells as adipocytes in vitro experiments.Liver cells and adipocytes were subjected to RSV intervention at different concentrations,cell viability was detected by CCK-8,and LDs accumulation was detected by immunofluorescence cytochemistry and MRI.The cells were transfected with siRNA or SIRT1 overexpression plasmid of SIRT1 and ATF6,respectively,and the expression levels of LDs accumulation related genes?Fsp27,PLIN1 and CREBH?were detected to elucidate the effect of RSV on the accumulation of LDs in hepatocytes and adipocytes3.To explore the effect of RSV on the expression level of ERS related genes?GRP78?and UPR related genes?PERK,IRE-1 and ATF6?in hepatocytes and adipocytes by interfering siRNA and overexpression plasmid.The effect of RSV on the combination of SIRT1 and ATF6was further detected by co-immunoprecipitation experiment?co-IP?and ortho-junction experiment?PLA?.The effect of ATF6 on transcription regulation of SIRT1 after RSV intervention was detected by chromatin immunoprecipitation?ChIP?and biluciferase reporter genes.The effect of RSV on the acetylation modification of ATF6 protein through SIRT1 was detected by acetylation antibody.Results1.RSV can effectively improve lipid accumulation in liver and adipose tissue of mice induced by HFD diet and regulate LDs accumulation related gene expression,while this effect is eliminated after SIRT1 knockdown.RSV intervention significantly inhibited HFD-induced weight and liver weight and lipid level in WT mouse.RSV intervention significantly improved hepatic steatosis,reduced TG content and lipid accumulation in liver and fat of HFD-induced WT mouse.GRP78?ERS marker protein?expression was increased in liver and adipose tissue of WT mice after HFD treatment,UPR^ER was activated,and expressions of PERK,IRE-1,ATF6 and other related proteins were increased,while SIRT1 expression was decreased.RSV intervention effectively reduced ERS level and GRP78 and ATF6 expressions,and up-regulated SIRT1 expression,but had no significant effect on PERK and IRE-1 expressions.After depletion of SIRT1 in vivo,the inhibition of RSV on LDs accumulation and GRP78 and ATF6 expression was weakened.2.RSV significantly inhibited PA-induced lipid accumulation in liver cells and adipogenic differentiation of 3T3-l1 adipocytes,enhanced the expression of SIRT1,and reduced the expressions of ERS marker protein GRP78 and UPR-related gene ATF6.After SIRT1 siRNA or ATF6 overexpression plasmid was transfected,the inhibition of RSV on LDs accumulation was weakened,and the expressions of GRP78 and ATF6 was inhibited.After SIRT1 overexpression plasmid or ATF6 siRNA transfection,the accumulation of RSV on LDs and the expression of GRP78 and ATF6 were significantly enhanced.It was found that PA can induce the accumulation of LDs in hepatocytes,while the adipogenic differentiation of 3T3-l1 adipocytes also increases the intracellular LDs,while ERS level increases and UPR is activated.After treatment of hepatocytes and adipocytes by RSV,PA-induced reduction of cellular activity and intracellular LDs accumulation were inhibited,and the expression levels of ERS and UPR^ER as well as Fsp27,CREBH,PLIN1 and ATF6proteins were reduced,but the expression levels of PERK and IRE-1 remained unchanged.After SIRT1 siRNA transfection of hepatocytes and adipocytes,the accumulation of RSV on LDs was weakened,while overexpression of SIRT1 significantly enhanced the effect of RSV.After further use of ATF6 siRNA,the inhibitory effect of RSV on the accumulation of TG and LDs in hepatocytes and adipocytes was more significant,while the effect was reversed after transfection of ATF6 overexpressed plasmid.3.RSV inhibited the binding of ATF6 to SIRT1,promoted the deacetylation modification of SIRT1 to ATF6,and thereby weakened the transcriptional inhibition of SIRT1 by ATF6,and increased the expression of SIRT1 through the positive feedback regulation mechanism,consequently regulated the expression of LDs related proteins and reduced the accumulation of LDs in liver and adipose tissue.ATF6 silencing led to significantly increased mRNA and protein expression of RSV on SIRT1,while its overexpression led to decreased SIRT1 expression in RSV treated cells,suggesting that ATF6 played a negative role in RSV regulation of SIRT1 expression in target cells.Through co-IP and PLA experiments,it was found that RSV inhibited the pa-induced binding between SIRT1 and ATF6 in hepatocytes.Through the dual-luciferase reporter gene detection,ATF6 silencing can significantly enhance the RSV-induced SIRT1 transcription activity,while the overexpression of ATF6 can inhibit SIRT1 transcription activity.Further ChIP detection showed that there were potential binding sites of ATF6 in the promoter region of SIRT1,suggesting that RSV transcriptional regulation of SIRT1 might be mediated by ATF6.Further use of anti-acetylation antibodies and immunoprecipitation detection revealed that the acetylation level of ATF6 after the treatment of cells with RSV,suggesting that RSV may promote the deacetylation of ATF6 through SIRT1.The above results suggested that RSV may weaken the transcriptional inhibition of SIRT1 by inhibiting the binding of ATF6 to SIRT1,and increase the transcriptional expression level of SIRT1.Meanwhile,it can also promote the deacetylation and inactivation of ATF6 through SIRT1,thereby affecting the expression of LDs accumulation related proteins and inhibiting LDs accumulation.ConclusionThe results of this study suggest that RSV can regulate LDs related protein expression through SIRT1/ATF6 signaling pathway in liver?hepatocytes?and adipose tissue?adipocytes?,inhibit the accumulation of LDs and delay the MetS process.