YBX3通过调节UCP1的表达调控棕色脂肪产热和能量消耗

YBX3 regulates thermogenesis and energy expenditure by regulating UCP1 in brown adipose tissue

目的探讨棕色脂肪组织(brown adipose tissue,BAT)中Y盒蛋白3(Y-box binding protein 3,YBX3)水平对小鼠产热和能量消耗的影响作用机制。方法将小鼠置于4℃环境作为冷刺激方法。用慢病毒侵染法构建过表达YBX3或者抑制YBX3表达的人血管基质组分细胞系。测量细胞的耗氧率用于评估细胞的线粒体功能。向小鼠BAT注射YBX3的小干扰RNA(small interfering RNA,siRNA)构建抑制小鼠BAT中YBX3表达的模型。检测小鼠的O_(2)消耗速率、CO_(2)释放速率和能量消耗速率用于评估小鼠能量代谢情况。采用qRT-PCR、Western blotting、免疫荧光染色法检测过氧化物酶体增殖物激活受体γ共激活因子1α(peroxisome proliferator-activated receptor gamma coactivator 1α,PGC-1α)、解偶联蛋白1(uncoupling protein 1,UCP1)和YBX3基因的表达。采用苏木精-伊红法染色检测BAT中脂肪细胞脂滴大小。采用RNA结合蛋白免疫沉淀提取与YBX3特异性结合的mRNA。采用二代测序方法鉴定mRNA。采用双荧光素酶报告基因系统检测YBX3与目的序列结合的情况。结果在寒冷刺激下野生型小鼠BAT中YBX3的表达显著增加(P<0.05)。过表达YBX3的人血管基质组分细胞系诱导为成熟棕色脂肪细胞后可以促进产热基因PGC-1α和UCP1的表达(P<0.05)。抑制YBX3表达的人血管基质组分细胞系诱导为成熟棕色脂肪细胞后可以抑制PGC-1α和UCP1的表达(P<0.05)、抑制线粒体氧化磷酸化(P<0.05)。与对照组相比,小鼠注射si-Ybx3之后能量消耗显著降低(P<0.05),在寒冷环境难以维持体温(P<0.05),产热基因表达被显著抑制(P<0.01),BAT中脂肪细胞脂滴大小显著增加(P<0.05)。与在25℃相比,4℃环境刺激下BAT中YBX3在细胞核中的表达显著增加(P<0.05)。YBX3能够特异性地与PGC-1αmRNA 3′非翻译区特定位点相结合(P<0.05)。YBX3能够特异性地与PGC-1α和UCP1启动子区域相结合(P<0.05)。结论BAT中YBX3能够通过调控PGC-1α和UCP1的表达影响产热和能量代谢。

Objective To observe the effects and mechanisms of Y-box protein 3(YBX3)deletion in brown adipose tissue(BAT)on thermogenesis and energy consumption in mice.Methods The mice were placed in a 4℃environment as a cold stimulation method.Lentiviral infection assay was used to construct a human stromal vascular fraction cell line that overexpresses YBX3 or inhibits YBX3 expression.Oxygen consumption rate was used to assess mitochondrial function.Mouse BAT was injected with YBX3 siRNA(si-Ybx3)to inhibit YBX3 expression in BAT.The oxygen consumption rate,CO_(2) release rate and energy consumption rate of mice were detected to evaluate the energy metabolism of mice.qRT-PCR,Western blotting,and immunofluorescence staining assays were used to detect peroxisome proliferator-activated receptor gamma coactivator 1α(PGC-1α),uncoupling protein 1(UCP1)and YBX3 expression.HE staining was used to detect the size of lipid droplets of adipocytes in BAT.RNA-binding protein immunoprecipitation was used to extract the mRNA bindsing specifically to YBX3.Next-generation sequencing assay was used to identify mRNA.Dual-luciferase reporter gene systems were used to detect the binding of YBX3 to the target sequence.Results YBX3 expression in BAT of wild-type mice was induced by cold stimulation(P<0.05).Overexpressing YBX3 promoted the expression of the thermogenic gene PGC-1αand UCP1 after human stromal vascular fraction cell line cells being induced into mature brown adipocytes(P<0.05).Silencing YBX3 in human stromal vascular fraction cell line cells inhibited the expression of PGC-1αand UCP1(P<0.05)and mitochondrial oxidative phosphorylation after being induced into mature brown adipocytes(P<0.05).Compared with the control group,mice injected with si-Ybx3 had significantly lower energy consumption(P<0.05),lower body temperature in a cold environment(P<0.05),and lower expression levels of thermogenic genes(P<0.05).The size of lipid droplets in adipocytes increased significantly(P<0.05).Compared with at 25℃,the expression of YBX3 in the nucleus of brown adipocytes significantly increased from mice kept at 4℃(P<0.05).YBX3 specifically bound to the target sequence in the 3'untranslated region of PGC-1αmRNA(P<0.05).YBX3 specifically bound to PGC-1αand UCP1 promoter regions(P<0.05).Conclusion YBX3 in BAT can affect thermogenesis and energy metabolism by regulating the expressions of PGC-1αand UCP1.