小檗碱介导BMAL1:CLOCK复合体调控糖脂代谢改善脂肪胰岛素抵抗的效应机制

Mechanism of berberine in improving adipocytic IR by mediating BMAL1:CLOCK complex and regulating glucose and lipid metabolism

探究小檗碱介导脑和肌肉芳香烃受体核转位样蛋白1(brain and muscle arnt-like 1,BMAL1):昼夜自发输出周期蛋白kaput(circadian locomotor output cycles kaput,CLOCK)复合体,调控糖脂代谢改善脂肪胰岛素抵抗(insulin resistance,IR)的作用机制。地塞米松诱导96 h建立IR-3T3-L1脂肪细胞模型,0.5、1、5、10、20μmol·L^(-1)小檗碱给药24 h。葡萄糖氧化酶法和细胞活性(cell counting kit-8,CCK-8)试剂盒分别检测胞外葡萄糖含量和细胞活力;酶比色法检测甘油三酯(triglyceride,TG)和甘油含量;油红O染色检测脂滴;荧光染色检测Ca2+、线粒体结构及活性氧(reactive oxygen species,ROS);蛋白免疫印迹(Western blot,WB)检测脂联素(adiponectin,ADPN)、BMAL1、CLOCK、激素敏感性脂肪酶(hormone-sensitive triglyceride lipase,HSL)、碳水化合物反应元件结合蛋白(carbohydrate-responsive element-binding protein,ChREBP)、固醇调节元件结合蛋白1C(sterol regulatory element-binding protein 1C,SREBP-1C)、过氧化物酶体增殖物激活受体γ共激活剂1α(peroxisome proliferator activated receptorγcoactivator 1α,PGC1α)、肉碱棕榈酰基转移酶1α(choline phosphotransferase 1α,CPT1α)、过氧化物酶体增殖物激活受体α(peroxisome proliferators-activated receptorsα,PPARα),免疫荧光检测BMAL1核定位。此外,添加20μmol·L^(-1)CLK8(CLOCK抑制剂)检测葡萄糖消耗量及BMAL1/ChREBP/PPARα蛋白。研究结果显示小檗碱增加IR-3T3-L1脂肪细胞葡萄糖消耗量,且不影响细胞活力;小檗碱降低IR-3T3-L1脂肪细胞胞内TG,5μmol·L^(-1)小檗碱升高甘油含量,减少脂滴积累,提示其加强脂解,而10μmol·L^(-1)小檗碱不影响甘油含量且脂滴更少,提示其可能同时加强脂解和甘油利用。此外,小檗碱降低IR-3T3-L1脂肪细胞胞内Ca^(2+)和ROS改善线粒体功能,上调PGC1α有助于维护线粒体结构。结果还显示小檗碱上调ADPN,提示其增加IR-3T3-L1脂肪细胞胰岛素敏感性,上调外周节律调控蛋白BMAL1和CLOCK并增加BMAL1核定位,上调脂解关键蛋白HSL和脂氧化限速酶CPT1α,下调TG合成关键蛋白SREBP-1C,小檗碱同时上调IR-3T3-L1脂肪细胞ChREBP和PPARα,以上结果共同提示其促糖转脂增强降糖效应。鉴于CLK8特异性抑制CLOCK酰基化修饰BMAL1形成复合体,结果显示CLK8添加小檗碱可降低葡萄糖消耗量,提示小檗碱上调BMAL1:CLOCK复合体改善糖代谢。CLK8添加小檗碱组上调BMAL1但下调ChREBP和PPARα,因而推测小檗碱介导BMAL1:CLOCK复合体调控细胞糖脂代谢减轻脂肪细胞IR。

To explore the action mechanism of berberine in improving adipocytic insulin resistance(IR)by mediating brain and muscle arnt-like 1(BMAL1):circadian locomotor output cycles kaput(CLOCK)complex and regulating glucose and lipid metabolism.After the IR-3T3-L1 adipocyte model was established by dexamethasone induction for 96 h,0.5,1,5,10,and 20μmol·L-1 berberine was administered for 24 h.The glucose oxidase method and cell counting kit-8(CCK-8)were used to detect extracellular glucose content and cell viability,respectively.The triglyceride(TG)and glycerol contents were detected by enzyme colorimetry.Oil red O staining was used to detect lipid droplets,and fluorescence staining was used to detect Ca2+,mitochondrial structure,and reactive oxygen species(ROS).Adiponectin(ADPN),BMAL1,CLOCK,hormone-sensitive triglyceride lipase(HSL),carbohydrate-response element-binding protein(ChREBP),sterol regulatory element-binding protein 1C(SREBP-1C),peroxisome proliferator-activated receptorγcoactivator 1α(PGC1α),carnitine palmitoyl transferase 1α(CPT1α),and peroxisome proliferator-activated receptorα(PPARα)were detected by Western blot(WB).Moreover,the nuclear localization of BMAL1 was detected by immunofluorescence.In addition,20μmol·L-1 CLK8 inhibitor was added to detect glucose consumption and BMAL1/ChREBP/PPARαprotein.The results showed that berberine increased glucose consumption in IR-3T3-L1 adipocytes without affecting cell viability and reduced TG content.In addition,5μmol·L-1 berberine increased glycerol content and reduced lipid droplet accumulation due to enhanced lipolysis,while 10μmol·L-1 berberine did not affect glycerol content,and fewer lipid droplets were observed due to enhanced lipolysis and glycerol utilization.Berberine improved mitochondrial function by reducing intracellular Ca2+and ROS in IR-3T3-L1 adipocytes and upregulated PGC1αto improve the mitochondrial structure.The results also showed that berberine elevated ADPN to increase the insulin sensitivity of IR-3T3-L1 adipocytes,upregulated peripheral rhythm-related proteins BMAL1 and CLOCK,and strengthened the nuclear localization of BMAL1.In addition,berberine increased key lipolysis protein and lipid oxidation rate-limiting enzyme CPT1αand downregulated the key protein of TG synthesis,SREBP-1C.Moreover,ChREBP and PPARαin IR-3T3-L1 adipocytes were upregulated.All the above results suggested that berberine may transform glucose into lipids to enhance the hypoglycemic effect.By considering that CLK8 specifically inhibited the CLOCK acylation to modify BMAL1 and form complex,the results showed that the addition of CLK8 to the berberine group reduced glucose consumption,which suggested that berberine upregulated the formation of BMAL1:CLOCK complex to improve glucose metabolism.The addition of CLK8 to the berberine group upregulated BMAL1 but downregulated ChREBP and PPARα,which suggested that berberine mediated BMAL1:CLOCK complex for the regulation of glucose and lipid metabolism to improve adipocytic IR.