Leptin调控食源性肥胖大鼠组织自噬影响其能量代谢的机制研究
发布时间：2018-08-12 16:37
【摘要】：目的:1、通过脑室内给药的方法给予食源性肥胖大鼠leptin短期和长期干预,探讨中枢给予leptin干预对食源性肥胖大鼠组织自噬的作用及其能量代谢平衡的影响。2、通过体外培养3T3-L1前脂肪细胞后给予leptin和自噬工具药物干预,观察leptin对于3T3-L1前脂肪细胞分化及表型的影响;探究leptin对于3T3-L1前脂肪细胞自噬的作用及机制。方法:1、选取SD大鼠通过给予高脂饲料喂养建立食源性肥胖(diet induced obesity,DIO)动物模型。在喂养过程中监测大鼠体重增长情况,在高脂饲料喂养16周后,将高脂组(high fat diet,HFD)大鼠根据体重进一步分为食源性肥胖组和肥胖抵抗组(diet resistant,DR);普通饲料喂养的大鼠作为对照组(chow feed,CF)。建模期间监测大鼠体重和进食量;使用EILSA试剂盒检测不同组别中SD大鼠的血清leptin水平。2、对SD大鼠通过第三脑室内注射leptin进行短期干预。分别将DIO、DR和CF组大鼠随机分为三组,即:注射leptin的大鼠分为高浓度组(High,H)和低浓度组(Low,L),设立对照组(Control,C)给予人工脑脊液注射。脑室内注射leptin后24 h监测大鼠体重和进食量;使用EILSA试剂盒检测不同组别SD大鼠的血清leptin水平;通过HE染色观察干预后肝脏、脂肪组织的形态变化;通过Western Blot方法检测大鼠中枢(下丘脑)和外周(肝脏组织、脂肪组织)内自噬相关因子LC3B、Beclin-1和P62蛋白水平的表达,观察中枢给予leptin短期干预后大鼠中枢和外周组织的自噬变化;探讨中枢给予leptin短期干预对DIO大鼠中枢及外周组织的自噬及能量代谢的影响。3、对SD大鼠进行侧脑室置管后连续注射leptin一周进行长期干预。分别将CF和DIO和DR组大鼠随机分为两组,即:注射高浓度leptin的大鼠为干预组(High,H),设立对照组(Control,C)给予人工脑脊液注射。在置管成功后,给予各组大鼠连续注射leptin或人工脑脊液干预一周。在干预过程中,每天定时监测大鼠体重和进食量;使用EILSA试剂盒检测不同组别中SD大鼠的血清leptin水平;通过油红O染色和HE染色观察干预后大鼠肝脏、脂肪组织的形态变化;通过Western Blot检测大鼠中枢(下丘脑)和外周(脂肪组织、肝脏组织)内自噬相关因子LC3B、Beclin-1和P62蛋白水平的表达,观察中枢给予leptin蛋白长期干预后大鼠中枢和外周组织的自噬变化。探讨中枢给予leptin长期干预对DIO大鼠中枢及外周组织的自噬及能量代谢的影响。4、体外培养3T3-L1前脂肪细胞并诱导其分化为成熟脂肪细胞,在诱导分化过程中加入leptin干预,通过油红O染色观察leptin对于3T3-L1前脂肪细胞分化及细胞表型的影响;通过RT-PCR检测脂肪细胞分化、甘油三酯合成、分解,脂肪酸β氧化及棕色脂肪特征性基因的mRNA表达水平来探讨leptin对脂肪细胞分化及成脂的影响;在细胞诱导分化的不同阶段给予leptin及自噬工具药(Rapamyin和Bafilomycin A1)干预,检测自噬相关因子LC3B蛋白水平的表达并在免疫荧光显微镜下观察P62的变化,分析leptin干预在3T3-L1前脂肪细胞诱导分化的不同阶段对其自噬的作用。检测leptin干预后3T3-L1前脂肪细胞自噬及mTOR信号通路相关因子LC3B、P62、Beclin-1、4E-BP1、p-4E-BP1的蛋白水平表达。探讨leptin对脂肪细胞分化及自噬的影响是否通过mTOR依赖的信号通路实现。结果:1、造模成功后,DIO组大鼠的体重和能量摄入均高于CF和DR组大鼠。DIO大鼠血清leptin水平高于CF和DR组,存在leptin抵抗。2、中枢给予leptin短期干预后,CF组大鼠的体重下降,但能量摄入较干预前增多;DIO组体重下降,leptin高浓度组大鼠能量摄入较干预前减少,leptin低浓度组大鼠能量摄入与干预前比较差异无统计学意义。DR组大鼠体重下降,但能量摄入与干预前比较无明显变化。Leptin干预后CF、DIO和DR组大鼠血清leptin水平均下降,但差异无统计学意义。高脂饲料喂养的DIO和DR大鼠存在肝脂肪变性,且DIO大鼠的脂肪细胞体积明显增大,给予leptin干预24h后,肝细胞和脂肪细胞形态无改变。给予高脂饲料喂养的DIO和DR大鼠与普通饲料喂养的CF组大鼠比较,其肝脏组织自噬下调,脂肪组织和下丘脑组织的自噬上调。给予leptin干预后,DIO和DR大鼠的肝脏组织自噬上调,脂肪组织和下丘脑组织自噬下调;CF组大鼠的肝脏组织的自噬下调,脂肪组织和下丘脑组织自噬上调。3、中枢给予leptin长期干预后,CF组大鼠的体重无明显改变,但能量摄入较干预前增多;DIO组大鼠体重下降,其能量摄入波动下降;DR大鼠体重下降,同时能量摄入也减少。Leptin长期干预后,CF、DIO和DR组大鼠血清leptin水平较干预前有所增加,但差异无统计学意义;给予leptin干预一周后,DIO和DR组大鼠肝脏组织脂肪变性得到一定程度改善,DIO组大鼠脂肪细胞体积减小,但CF和DR组脂肪细胞体积没有明显改变。给予leptin干预后,DIO和DR大鼠的肝脏组织自噬上调,脂肪组织和下丘脑组织自噬下调;CF组大鼠的肝脏组织的自噬下调,脂肪组织和下丘脑组织自噬下调。4、Leptin干预后3T3-L1前脂肪细胞形态无改变。在诱导分化过程中给予leptin持续干预,3T3-L1前脂肪细胞成脂率降低。在给予3T3-L1前脂肪细胞诱导分化早期给予leptin干预后PPARγ、HSL mRNA表达量较对照组增高,在细胞分化的晚期干预组的aP2、DGAT1、DGAT2 mRNA表达量降低。给予leptin干预后,UCP1和PGC-1αmRNA增高。5、在3T3-L1前脂肪细胞诱导分化早期给予leptin干预促进自噬上调,在3T3-L1前脂肪细胞诱导分化晚期给予leptin干预后细胞自噬下调。在3T3-L1前脂肪细胞分化的早期leptin能增加自噬体的合成,促进自噬体降解,加快3T3-L1前脂肪细胞自噬进程,促进自噬;但在3T3-L1前脂肪细胞分化的中、晚期leptin增加自噬体的合成,但抑制自噬体降解,抑制自噬。Leptin对于3T3-L1前脂肪细胞自噬的调控是通过mTOR依赖的信号通路实现的。结论:1、中枢给予leptin干预后,可以促进DIO、DR大鼠体重下降,能量消耗增加;CF组大鼠能量消耗增加。同时调节大鼠肝脏、脂肪和下丘脑组织自噬,维持能量代谢平衡。2、3T3-L1前脂肪细胞诱导分化时给予leptin干预,细胞形态未改变,但其成脂分化率降低;在细胞诱导分化早期leptin促进3T3-L1前脂肪细胞自噬上调,促进细胞分化;在细胞诱导分化晚期,leptin抑制3T3-L1前脂肪细胞自噬,促进甘油三酯分解,减少脂滴聚集。且leptin对3T3-L1前脂肪细胞自噬的调节通过mTOR依赖的信号通路实现。
[Abstract]:AIM: 1. To investigate the effect of leptin on autophagy and energy metabolism balance in rats with food-borne obesity by intraventricular administration of leptin. 2. To observe the effect of leptin on autophagy and energy metabolism balance in rats with food-borne obesity. To investigate the effect of leptin on the autophagy of 3T3-L1 preadipocytes and its mechanism. Methods: 1. SD rats were fed with high fat diet to establish an animal model of diet-induced obesity (DIO). Sixteen weeks later, the rats in the high fat diet (HFD) group were further divided into the diet resistant (DR) group and the diet resistant (DR) group according to their body weight; the rats fed with ordinary diet were used as the control group (CF). The body weight and food intake of the rats were monitored during the modeling period; the serum leptin levels of SD rats in different groups were detected by EILSA kit. 2. Short-term intervention was carried out by injecting leptin into the third ventricle of SD rats. The rats in DIO, DR and C F groups were randomly divided into three groups. The rats injected with leptin were divided into high concentration group (High, H) and low concentration group (Low, L), and the control group (Control, C) was injected with artificial cerebrospinal fluid. Levels of serum leptin in SD rats of different groups were detected by EILSA kit, morphological changes of liver and adipose tissue were observed by HE staining, and the expression of autophagy-related factors LC3B, Beclin-1 and P62 protein in central (hypothalamus) and peripheral (liver, adipose tissue) were detected by Western Blot method. The changes of autophagy in central and peripheral tissues of SD rats after short-term intervention with leptin were investigated. The effects of short-term intervention with leptin on autophagy and energy metabolism in central and peripheral tissues of DIO rats were investigated. 3. SD rats were injected with leptin for a week after catheterization in lateral ventricle for long-term intervention. The rats in each group were injected with leptin or artificial cerebrospinal fluid continuously for one week after successful catheterization. During the intervention, the rats'body weight and food intake were monitored regularly every day, and different groups were detected by EILSA kit. Levels of serum leptin in SD rats were observed by oil red O staining and HE staining, and the morphological changes of liver and adipose tissue were observed after intervention. To investigate the effects of long-term intervention with leptin on autophagy and energy metabolism in central and peripheral tissues of DIO rats. 4. Preadipocytes 3T3-L1 were cultured in vitro and induced to differentiate into mature adipocytes. Leptin was added into the process of differentiation and observed by oil red O staining. To investigate the effect of leptin on the differentiation and cell phenotype of 3T3-L1 preadipocytes, to investigate the effect of leptin on adipocyte differentiation and adipogenesis by RT-PCR detection of adipocyte differentiation, triglyceride synthesis, decomposition, fatty acid beta oxidation and mRNA expression of brown fat characteristic genes. The expression of autophagy-related factor LC3B protein was detected by immunofluorescence microscopy, and the effect of leptin on autophagy in different stages of 3T3-L1 preadipocyte differentiation was analyzed. The protein levels of LC3B, P62, Beclin-1, 4E-BP1 and p-4E-BP1 were detected. The effects of leptin on adipocyte differentiation and autophagy were investigated by mTOR-dependent signaling pathway. Results: 1. After successful modeling, the body weight and energy intake of DIO rats were higher than those of CF and DR rats. The serum leptin levels of DIO rats were higher than those of CF and DR rats. The body weight of CF rats decreased, but the energy intake increased after short-term intervention with leptin. The body weight of DIO rats decreased, the energy intake of high leptin concentration group decreased, and the energy intake of low leptin concentration group was not significantly different from that before intervention. The serum leptin levels of CF, DIO and DR rats decreased after Leptin intervention, but there was no significant difference between the two groups. Compared with CF group, DIO and DR rats fed with high fat diet had lower autophagy in liver tissue and higher autophagy in adipose tissue and hypothalamus tissue. Tissue autophagy was down-regulated, adipose tissue and hypothalamic tissue autophagy was up-regulated. 3. After long-term intervention with leptin, the body weight of CF rats did not change significantly, but energy intake increased. The body weight of DIO rats decreased and energy intake fluctuated. The body weight of DR rats decreased and energy intake decreased. After long-term intervention with Leptin, CF and DIO rats lost weight and energy intake. The serum leptin level of rats in DIO and DR groups was higher than that before the intervention, but there was no significant difference between the two groups.One week after the intervention with leptin, the fatty degeneration of liver tissue in DIO and DR groups was improved to a certain extent, the volume of adipocytes in DIO group decreased, but the volume of adipocytes in CF and DR groups did not change significantly.After the intervention with leptin, DIO and DR increased. Autophagy of liver tissue was up-regulated, autophagy of adipose tissue and hypothalamus tissue was down-regulated; autophagy of liver tissue was down-regulated, autophagy of adipose tissue and hypothalamus tissue was down-regulated in CF group. The morphology of 3T3-L1 preadipocytes did not change after Leptin intervention. The expression of PPAR-gamma and HSL-mRNA increased after leptin intervention in the early stage of 3T3-L1 preadipocyte differentiation, but decreased in the late stage of cell differentiation. After leptin intervention, the expression of UCP1 and PCG-1alpha mRNA increased. 5. Leptin intervention was given in the early stage of 3T3-L1 preadipocyte differentiation. In the early stage of 3T3-L1 preadipocyte differentiation, leptin can increase autophagy synthesis, promote autophagy degradation, accelerate the autophagy process of 3T3-L1 preadipocyte, and promote autophagy; but in the late stage of 3T3-L1 preadipocyte differentiation, leptin increases. Leptin regulates autophagy of 3T3-L1 preadipocytes through a mTOR-dependent signaling pathway. Conclusion: 1. Leptin can promote the weight loss and energy consumption of DIO, DR rats, and CF rats. 2,3T3-L1 preadipocytes were treated with leptin when they were induced to differentiate, but the adipogenic differentiation rate was decreased; leptin promoted autophagy of 3T3-L1 preadipocytes in the early stage of cell differentiation, and promoted cell differentiation; leptin inhibited 3T3-L1 preadipocytes in the late stage of cell differentiation. L1 preadipocyte autophagy promotes triglyceride decomposition and decreases lipid droplet aggregation, and leptin modulates 3T3-L1 preadipocyte autophagy via a mTOR-dependent signaling pathway.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R589.2
本文编号：2179640
[Abstract]:AIM: 1. To investigate the effect of leptin on autophagy and energy metabolism balance in rats with food-borne obesity by intraventricular administration of leptin. 2. To observe the effect of leptin on autophagy and energy metabolism balance in rats with food-borne obesity. To investigate the effect of leptin on the autophagy of 3T3-L1 preadipocytes and its mechanism. Methods: 1. SD rats were fed with high fat diet to establish an animal model of diet-induced obesity (DIO). Sixteen weeks later, the rats in the high fat diet (HFD) group were further divided into the diet resistant (DR) group and the diet resistant (DR) group according to their body weight; the rats fed with ordinary diet were used as the control group (CF). The body weight and food intake of the rats were monitored during the modeling period; the serum leptin levels of SD rats in different groups were detected by EILSA kit. 2. Short-term intervention was carried out by injecting leptin into the third ventricle of SD rats. The rats in DIO, DR and C F groups were randomly divided into three groups. The rats injected with leptin were divided into high concentration group (High, H) and low concentration group (Low, L), and the control group (Control, C) was injected with artificial cerebrospinal fluid. Levels of serum leptin in SD rats of different groups were detected by EILSA kit, morphological changes of liver and adipose tissue were observed by HE staining, and the expression of autophagy-related factors LC3B, Beclin-1 and P62 protein in central (hypothalamus) and peripheral (liver, adipose tissue) were detected by Western Blot method. The changes of autophagy in central and peripheral tissues of SD rats after short-term intervention with leptin were investigated. The effects of short-term intervention with leptin on autophagy and energy metabolism in central and peripheral tissues of DIO rats were investigated. 3. SD rats were injected with leptin for a week after catheterization in lateral ventricle for long-term intervention. The rats in each group were injected with leptin or artificial cerebrospinal fluid continuously for one week after successful catheterization. During the intervention, the rats'body weight and food intake were monitored regularly every day, and different groups were detected by EILSA kit. Levels of serum leptin in SD rats were observed by oil red O staining and HE staining, and the morphological changes of liver and adipose tissue were observed after intervention. To investigate the effects of long-term intervention with leptin on autophagy and energy metabolism in central and peripheral tissues of DIO rats. 4. Preadipocytes 3T3-L1 were cultured in vitro and induced to differentiate into mature adipocytes. Leptin was added into the process of differentiation and observed by oil red O staining. To investigate the effect of leptin on the differentiation and cell phenotype of 3T3-L1 preadipocytes, to investigate the effect of leptin on adipocyte differentiation and adipogenesis by RT-PCR detection of adipocyte differentiation, triglyceride synthesis, decomposition, fatty acid beta oxidation and mRNA expression of brown fat characteristic genes. The expression of autophagy-related factor LC3B protein was detected by immunofluorescence microscopy, and the effect of leptin on autophagy in different stages of 3T3-L1 preadipocyte differentiation was analyzed. The protein levels of LC3B, P62, Beclin-1, 4E-BP1 and p-4E-BP1 were detected. The effects of leptin on adipocyte differentiation and autophagy were investigated by mTOR-dependent signaling pathway. Results: 1. After successful modeling, the body weight and energy intake of DIO rats were higher than those of CF and DR rats. The serum leptin levels of DIO rats were higher than those of CF and DR rats. The body weight of CF rats decreased, but the energy intake increased after short-term intervention with leptin. The body weight of DIO rats decreased, the energy intake of high leptin concentration group decreased, and the energy intake of low leptin concentration group was not significantly different from that before intervention. The serum leptin levels of CF, DIO and DR rats decreased after Leptin intervention, but there was no significant difference between the two groups. Compared with CF group, DIO and DR rats fed with high fat diet had lower autophagy in liver tissue and higher autophagy in adipose tissue and hypothalamus tissue. Tissue autophagy was down-regulated, adipose tissue and hypothalamic tissue autophagy was up-regulated. 3. After long-term intervention with leptin, the body weight of CF rats did not change significantly, but energy intake increased. The body weight of DIO rats decreased and energy intake fluctuated. The body weight of DR rats decreased and energy intake decreased. After long-term intervention with Leptin, CF and DIO rats lost weight and energy intake. The serum leptin level of rats in DIO and DR groups was higher than that before the intervention, but there was no significant difference between the two groups.One week after the intervention with leptin, the fatty degeneration of liver tissue in DIO and DR groups was improved to a certain extent, the volume of adipocytes in DIO group decreased, but the volume of adipocytes in CF and DR groups did not change significantly.After the intervention with leptin, DIO and DR increased. Autophagy of liver tissue was up-regulated, autophagy of adipose tissue and hypothalamus tissue was down-regulated; autophagy of liver tissue was down-regulated, autophagy of adipose tissue and hypothalamus tissue was down-regulated in CF group. The morphology of 3T3-L1 preadipocytes did not change after Leptin intervention. The expression of PPAR-gamma and HSL-mRNA increased after leptin intervention in the early stage of 3T3-L1 preadipocyte differentiation, but decreased in the late stage of cell differentiation. After leptin intervention, the expression of UCP1 and PCG-1alpha mRNA increased. 5. Leptin intervention was given in the early stage of 3T3-L1 preadipocyte differentiation. In the early stage of 3T3-L1 preadipocyte differentiation, leptin can increase autophagy synthesis, promote autophagy degradation, accelerate the autophagy process of 3T3-L1 preadipocyte, and promote autophagy; but in the late stage of 3T3-L1 preadipocyte differentiation, leptin increases. Leptin regulates autophagy of 3T3-L1 preadipocytes through a mTOR-dependent signaling pathway. Conclusion: 1. Leptin can promote the weight loss and energy consumption of DIO, DR rats, and CF rats. 2,3T3-L1 preadipocytes were treated with leptin when they were induced to differentiate, but the adipogenic differentiation rate was decreased; leptin promoted autophagy of 3T3-L1 preadipocytes in the early stage of cell differentiation, and promoted cell differentiation; leptin inhibited 3T3-L1 preadipocytes in the late stage of cell differentiation. L1 preadipocyte autophagy promotes triglyceride decomposition and decreases lipid droplet aggregation, and leptin modulates 3T3-L1 preadipocyte autophagy via a mTOR-dependent signaling pathway.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R589.2
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