二甲双胍促肝脏瘦素受体基因表达上调血浆可溶性瘦素受体的初步研究
发布时间：2018-08-05 13:37
【摘要】：研究背景:2型糖尿病(T2DM)是一种常见的内分泌代谢疾病,占糖尿病(DM)总发病率的90%~95%,是遗传和环境因素共同作用导致的复杂疾病。近年来,因环境及生活方式的改变,T2DM在我国呈爆发式增长,目前我国已成为糖尿病第一大国[1]。胰岛素抵抗是T2DM发病的重要机制也是其重要特征。大量研究证实,肥胖与T2DM紧密相关[2,3,4,5],肥胖尤其是中心性肥胖是T2DM的独立重要危险因素[6,7,8]。随着体脂的增加,肥胖患者通常表现为血浆瘦素水平升高[9],而血浆s LR(soluble leptin receptor,可溶性瘦素受体)水平显著降低的特点[10,11]。瘦素由脂肪组织分泌,具有调节摄食、糖脂代谢及免疫炎症等诸多功能[12]。s LR是人体血浆中结合瘦素的最主要血浆蛋白[13],调节瘦素的生物功能。多项研究显示,血浆sLR水平降低与肥胖T2DM患者血糖、低密度脂蛋白胆固醇、血压等代谢综合征指标升高呈负相关[14],经过手术、控制饮食、体育锻炼等措施降低体重后,血浆s LR水平显著升高[15,16,17],提示血浆sLR水平可能作为肥胖T2DM患者代谢状态改善的生化指征。我们前期采用二甲双胍干预高脂喂养的胰岛抵抗小鼠及初诊T2DM病人,均发现二甲双胍能显著上调血浆s LR的水平[18],但其机制尚未阐明。已知,人体血浆s LR来源于膜型LR(leptin receptor,瘦素受体)胞外域水解脱落[19],人体中存在四种LR亚型,即LR-a、LR-b、LR-c及LR-d,它们均由同一基因(LR)编码,转录后经RNA差异剪接后生成[20]。其中,LRb是传递瘦素信号的唯一全长型瘦素受体[21]。小鼠体内还存在其他亚型的瘦素受体,如膜型LR-f及分泌型LR-e[22]。已有报道,肝脏是瘦素受体表达量最高的外周组织器官,在调节血浆s LR水平中具有重要作用[23,24]。此外,肾脏组织中也存在丰富的瘦素受体[23]。对于水解LR的酶总体上了解较少。近期报道,ADAM10(A Disintegrin And Metalloproteinase 10,解聚素样金属蛋白酶10)、ADAM17参与了LR的水解脱落过程,其中ADAM10起主要作用[25]。但二甲双胍是否通过上调瘦素受体及其水解酶的基因表达上调s LR的水平,目前尚未见报道。为此,本研究通过观察二甲双胍对小鼠肝脏、肾脏组织瘦素受体总量(lrt)及四种主要膜型受体(lra,lrb,lrc,lrd)的基因表达,同时观察该药物对解聚素样金属蛋白酶adam10及adam17的基因表达以及其蛋白水平的改变,拟初步阐明二甲双胍上调血浆可溶性瘦素受体的可能机制。材料和方法:42只c57bl/6小鼠(6~8周龄,体重17.02±1.91g)用随机数字表法随机分为2组,分别给予正常饮食及高脂饮食喂养5月后,各组再以随机数字表法分为3亚组,分别给予蒸馏水及低剂量(50mg/kg.d)、高剂量(200mg/kg.d)二甲双胍灌胃(1次/天,共计15d),处死后取各组小鼠肝脏、肾脏组织存于-80℃冰箱[18]。剔除喂养过程中死亡小鼠,根据饮食和蒸馏水、二甲双胍剂量的不同处理共为6亚组,如下所示:正常饮食—蒸馏水组(cd-w,n=7);正常饮食—低剂量二甲双胍组(cd-lm,n=7);正常饮食—高剂量二甲双胍组(cd-hm,n=7);高脂饮食—蒸馏水组(hf-w,n=5);高脂饮食—低剂量二甲双胍组(hf-lm,n=6),高脂饮食—高剂量二甲双胍组(hf-hm,n=7)。q-pcr检测各小组肝脏、肾脏组织瘦素受体(lrt,lra,lrb,lrc,lrd)及(adam10、adam17)的基因表达水平,western-blot检测各组肝脏、肾脏组织adam10、adam17蛋白表达水平。结果:1.二甲双胍能剂量依赖性上调肝脏组织瘦素受体基因的表达,而对肾脏组织瘦素受体基因的表达无显著影响。肝脏组织瘦素受体总量lrtmrna的相对表达量分别为:cd组【cd-wvscd-lmvscd-hm,1.04±0.34vs1.63±0.18vs5.26±0.89(p0.01)】;hf组【hf-wvshf-lmvshf-hm,2.25±0.10vs4.96±0.88(p0.05)vs9.11±1.33(p0.01)】。2.肝脏中受二甲双胍上调的膜型瘦素受体亚型主要是三种,即lra{cd组【1.02±0.24vs1.42±0.47vs2.92±0.68(p0.05)】;hf组【1.13±0.62vs2.80±0.18(p0.05)vs6.04±1.00(p0.01)】}、lrc{cd组【1.00±0.06vs1.02±0.13vs1.97±0.25(p0.01)】;hf组【0.97±0.12vs2.60±0.18(p0.01)vs4.58±0.11(p0.01)】}、lrd{cd组【1.02±0.25vs0.69±0.27vs2.48±0.15(p0.01)】;hf组【1.22±0.23vs2.55±0.16(p0.01)vs4.82±0.03(p0.01)】}。3.肝脏长型跨膜瘦素受体lrbmrna在二甲双胍处理前后的cd组小鼠中并无显著变化(1.02±0.30 vs 0.69±0.16 vs 0.32±0.09),有趣的是,高剂量处理的HF组小鼠肝脏中,LRb m RNA明显上调【0.69±0.26 vs 0.77±0.15 vs 2.39±0.59(P0.01)】。4.二甲双胍对肾脏组织中瘦素受体的mRNA表达无论在正常饮食组或高脂组均无显著影响LRt【CD组(1.06±0.43 vs 1.03±0.32 vs 0.85±0.08);HF组(0.37±0.13 vs0.36±0.17 vs 0.74±0.27)】,LRa【CD组(1.05±0.45 vs 0.87±0.22 vs 0.36±0.07);HF组(0.56±0.21 vs 0.57±0.23 vs 0.57±0.08)】,LRb{CD组【1.00±0.15 vs 0.58±0.16(P0.01)vs 0.12±0.02(P0.01)】;HF组(0.14±0.04 vs 0.08±0.05 vs 0.08±0.03)},LRc【CD组(1.03±0.32 vs 1.05±0.16 vs 0.40±0.06);HF组(0.69±0.25 vs 0.67±0.21 vs 0.48±0.14)】,LRd{CD组【1.11±0.52 vs 0.79±0.18 vs 0.44±0.14(P0.05)】;HF组(0.28±0.07 vs0.21±0.04 vs 0.67±0.13)}。5.二甲双胍对肝脏及肾脏组织中ADAM10的mRNA【肝脏:CD组(1.02±0.22vs 1.06±0.14 vs 0.76±0.10);HF组(0.79±0.14 vs 0.82±0.07 vs 0.88±0.05)】{肾脏:CD组【1.01±0.17 vs 0.95±0.16 vs 0.56±0.16(P0.05)】;HF组(0.38±0.19 vs 0.23±0.04vs 0.33±0.09)}、ADAM17【肝脏:CD组(1.00±0.01 vs 1.07±0.07 vs 0.85±0.09);HF组(0.81±0.02 vs 0.82±0.06 vs 0.84±0.09)】{肾脏:CD组【1.02±0.29 vs 1.00±0.08vs 0.42±0.07(P0.05)】;HF组(0.40±0.18 vs 0.21±0.06 vs 0.40±0.16)}及蛋白表达水平均无显著影响。结论:1、二甲双胍对肝脏组织的LR基因表达有上调作用,但对肾脏组织无此作用。2、二甲双胍上调血浆sLR是通过上调肝脏组织LR基因表达实现的,但对产生sLR的水解酶ADAM10、ADAM17基因及蛋白的表达均无上调作用。3、二甲双胍上调血浆sLR可能是通过促进小鼠肝脏组织短型跨膜瘦素受体,主要是LRa、LRc、LRd基因表达实现的。LRb对sLR不起主要作用,但LRb上调能调节瘦素功能。
[Abstract]:Background: type 2 diabetes (T2DM) is a common endocrine and metabolic disease, accounting for the 90%~95% of the total incidence of diabetes (DM). It is a complex disease caused by the combination of genetic and environmental factors. In recent years, because of the change of environment and lifestyle, T2DM has increased in China. At present, China has become the first country of diabetes, [1]. islet. Vegetal resistance is an important mechanism for the pathogenesis of T2DM. A large number of studies have confirmed that obesity is closely related to T2DM and is closely related to [2,3,4,5]. Obesity, especially central obesity, is an independent and important risk factor for T2DM, [6,7,8]., with the increase of body fat, obese patients usually show a level of [9] in plasma leptin, and the plasma s LR (soluble leptin receptor). A significant reduction in the level of soluble leptin receptor, [10,11]. leptin is secreted by adipose tissue, and has many functions such as regulating feeding, glycolipid metabolism and immune inflammation. [12].s LR is the most important plasma protein [13] associated with leptin in human plasma and regulates the biological function of leptin. A number of studies show that the level of sLR in plasma is lower than that of obese T2DM. Blood glucose, low density lipoprotein cholesterol, blood pressure and other metabolic syndrome were negatively correlated [14]. After surgery, diet control, physical exercise and other measures to reduce weight, the plasma s LR level increased significantly [15,16,17], suggesting that plasma sLR level may be a biochemical indication of the improvement of metabolic status of obese T2DM patients. We used two in the early period. Metformin interfered with high fat fed islet resistant mice and first diagnosed T2DM patients. It was found that metformin can significantly increase the level of plasma s LR level [18], but its mechanism has not been elucidated. It is known that the human plasma s LR is derived from the extracellular domain of LR (leptin receptor, leptin receptor) from the extracellular domain of [19], and there are four LR subtypes in the human body. LR-d, which are encoded by the same gene (LR), are transcribed and transcribed after RNA differential splicing to generate [20].. LRb is the only full-length leptin receptor that transacts leptin signal in [21]. mice and there are other subtypes of leptin receptors in the body, such as membrane LR-f and secretory LR-e[22]., and the liver is the highest expression of leptin receptor in the peripheral tissue. It has an important role in regulating the level of plasma s LR, [23,24]., in addition, there are also abundant leptin receptor [23]. in renal tissues that have less understanding of the hydrolysis of LR enzymes. In the near future, ADAM10 (A Disintegrin And Metalloproteinase 10, depolymer like metal protease 10), ADAM17 participated in the process of hydrolysis and abscission of LR. The main role of [25]. but whether metformin up regulation of the level of s LR by up regulation of leptin receptor and its hydrolase gene expression has not yet been reported. To this end, the present study observed the gene expression of the total amount of leptin receptor (LRT) and four major membrane receptors (LRA, LRB, LRC, LRD) in the liver of mice by metformin, and to observe the gene expression of the four major membrane receptors (LRB, LRC, LRD). The possible mechanism of upregulation of soluble leptin receptor in plasma by metformin was preliminarily elucidated. Materials and methods: 42 c57bl/6 mice (6~8 weeks, weight 17.02 + 1.91g) were randomly divided into 2 groups by random digital table method, which were given to normal, respectively. After diet and high fat diet feeding in May, each group was divided into 3 subgroups by random digital table method, which were treated with distilled water and low dose (50mg/kg.d), high dose (200mg/kg.d) metformin gavage (1 times / day, total 15d). After death, the mice liver was taken from each group. The kidney tissues were stored in the [18]. culling of -80 C fridge in the [18]. culling and feeding process. The dosage of distilled water and metformin was treated in a total of 6 subgroups, as follows: normal diet - cd-w (n=7); normal diet - low dose metformin group (cd-lm, n=7); normal diet - high dose metformin group (cd-hm, n=7); high fat diet distilled water group (Hf-W, n=5); high fat diet - low dose metformin group (hf-lm, n=6), High fat diet (hf-hm, n=7).Q-pcr was used to detect the gene expression levels of leptin receptor (LRT, LRA, LRB, LRC, LRD) and (ADAM10, ADAM17) in all groups of liver, kidney tissue, kidney tissue ADAM10, and egg white expression level. Results: 1. metformin can increase the liver tissue in a dose dependent manner. The expression of the hormone receptor gene had no significant influence on the expression of the renal tissue leptin receptor gene. The relative expression of the total amount of leptin receptor lrtmrna in the liver tissue was: Group CD [cd-wvscd-lmvscd-hm, 1.04 + 0.34vs1.63 + 0.18vs5.26 + 0.89 (P0.01)], and HF group [hf-wvshf-lmvshf-hm, 2.25 + 0.10vs4.96 + 0.88 (P0.05) vs9.11 + 1.33 (P0.01)) The membrane type leptin receptor subtypes in the liver of.2. were mainly three, that is, group lra{cd [1.02 + 0.24vs1.42 + 0.47vs2.92 + 0.68 (P0.05)], HF group [1.13 + 0.62vs2.80 + 0.18 (P0.05) vs6.04 + 1 (P0.01)]}, lrc{cd Group [1 + 1 + 0.25]. 8 + 0.11 (P0.01)}, group lrd{cd [1.02 + 0.25vs0.69 + 0.27vs2.48 + 0.15 (P0.01)], HF group [1.22 + 0.23vs2.55 + 0.16 (P0.01) vs4.82 + 0.03 (P0.01)],}.3. liver long type transmembrane leptin receptor was not significantly changed (1.02 + 0.30 0.69 + 0.69 + 0.32 + 0.09) before and after metformin treatment (1.02 + 0.30 0.69 + 0.32 + 0.09), interesting, high dose In the liver of the treated HF mice, the LRb m RNA was obviously up-regulated [0.69 + 0.26 vs, 0.77 + 0.15 vs 2.39 + 0.59 (P0.01)]. The mRNA expression of the.4. dimethyl metformin on the renal tissue was not significantly affected in the normal diet group or the high fat group (CD group (1.06 + 0.43 vs 1.03 + 0.32 0.85 + 0.08). Vs 0.74 + 0.27), LRa [group CD (1.05 + 0.45 vs 0.87 + 0.22 vs 0.36 + 0.07), HF group (0.56 + 0.21 vs 0.57 + 0.23 vs 0.57 + 0.08). 25 vs 0.67 + 0.21 vs 0.48 + 0.14), group LRd{CD [1.11 + 0.52 vs 0.79 + 0.18 vs 0.44 + 0.14 (P0.05)], HF group (0.28 + 0.07 vs0.21 +)}.5. metformin to the liver and kidney tissue ADAM10 mRNA [liver:] Kidneys: group CD [1.01 + 0.17 vs 0.95 + 0.16 vs 0.56 + 0.16 (P0.05)]; HF group (0.38 + 0.19 vs 0.23 + 0.04vs 0.33 + 0.09)}, ADAM17 18 vs 0.21 + 0.06 vs 0.40 + 0.16)} and protein expression level had no significant effect. Conclusion: 1, metformin has an up-regulated effect on LR gene expression in liver tissue, but it has no effect on renal tissue.2, and metformin up regulation of plasma sLR is realized by up regulation of LR gene expression in liver tissue, but sLR hydrolase ADAM10, ADAM17 gene is produced. There is no up regulation of.3, and the up regulation of sLR by metformin may be by promoting the short transmembrane leptin receptor in the liver tissue of mice, and.LRb, which is mainly expressed in LRa, LRc, and LRd gene, does not play a major role in sLR, but the up regulation of LRb can regulate the function of leptin.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1
本文编号：2165958
[Abstract]:Background: type 2 diabetes (T2DM) is a common endocrine and metabolic disease, accounting for the 90%~95% of the total incidence of diabetes (DM). It is a complex disease caused by the combination of genetic and environmental factors. In recent years, because of the change of environment and lifestyle, T2DM has increased in China. At present, China has become the first country of diabetes, [1]. islet. Vegetal resistance is an important mechanism for the pathogenesis of T2DM. A large number of studies have confirmed that obesity is closely related to T2DM and is closely related to [2,3,4,5]. Obesity, especially central obesity, is an independent and important risk factor for T2DM, [6,7,8]., with the increase of body fat, obese patients usually show a level of [9] in plasma leptin, and the plasma s LR (soluble leptin receptor). A significant reduction in the level of soluble leptin receptor, [10,11]. leptin is secreted by adipose tissue, and has many functions such as regulating feeding, glycolipid metabolism and immune inflammation. [12].s LR is the most important plasma protein [13] associated with leptin in human plasma and regulates the biological function of leptin. A number of studies show that the level of sLR in plasma is lower than that of obese T2DM. Blood glucose, low density lipoprotein cholesterol, blood pressure and other metabolic syndrome were negatively correlated [14]. After surgery, diet control, physical exercise and other measures to reduce weight, the plasma s LR level increased significantly [15,16,17], suggesting that plasma sLR level may be a biochemical indication of the improvement of metabolic status of obese T2DM patients. We used two in the early period. Metformin interfered with high fat fed islet resistant mice and first diagnosed T2DM patients. It was found that metformin can significantly increase the level of plasma s LR level [18], but its mechanism has not been elucidated. It is known that the human plasma s LR is derived from the extracellular domain of LR (leptin receptor, leptin receptor) from the extracellular domain of [19], and there are four LR subtypes in the human body. LR-d, which are encoded by the same gene (LR), are transcribed and transcribed after RNA differential splicing to generate [20].. LRb is the only full-length leptin receptor that transacts leptin signal in [21]. mice and there are other subtypes of leptin receptors in the body, such as membrane LR-f and secretory LR-e[22]., and the liver is the highest expression of leptin receptor in the peripheral tissue. It has an important role in regulating the level of plasma s LR, [23,24]., in addition, there are also abundant leptin receptor [23]. in renal tissues that have less understanding of the hydrolysis of LR enzymes. In the near future, ADAM10 (A Disintegrin And Metalloproteinase 10, depolymer like metal protease 10), ADAM17 participated in the process of hydrolysis and abscission of LR. The main role of [25]. but whether metformin up regulation of the level of s LR by up regulation of leptin receptor and its hydrolase gene expression has not yet been reported. To this end, the present study observed the gene expression of the total amount of leptin receptor (LRT) and four major membrane receptors (LRA, LRB, LRC, LRD) in the liver of mice by metformin, and to observe the gene expression of the four major membrane receptors (LRB, LRC, LRD). The possible mechanism of upregulation of soluble leptin receptor in plasma by metformin was preliminarily elucidated. Materials and methods: 42 c57bl/6 mice (6~8 weeks, weight 17.02 + 1.91g) were randomly divided into 2 groups by random digital table method, which were given to normal, respectively. After diet and high fat diet feeding in May, each group was divided into 3 subgroups by random digital table method, which were treated with distilled water and low dose (50mg/kg.d), high dose (200mg/kg.d) metformin gavage (1 times / day, total 15d). After death, the mice liver was taken from each group. The kidney tissues were stored in the [18]. culling of -80 C fridge in the [18]. culling and feeding process. The dosage of distilled water and metformin was treated in a total of 6 subgroups, as follows: normal diet - cd-w (n=7); normal diet - low dose metformin group (cd-lm, n=7); normal diet - high dose metformin group (cd-hm, n=7); high fat diet distilled water group (Hf-W, n=5); high fat diet - low dose metformin group (hf-lm, n=6), High fat diet (hf-hm, n=7).Q-pcr was used to detect the gene expression levels of leptin receptor (LRT, LRA, LRB, LRC, LRD) and (ADAM10, ADAM17) in all groups of liver, kidney tissue, kidney tissue ADAM10, and egg white expression level. Results: 1. metformin can increase the liver tissue in a dose dependent manner. The expression of the hormone receptor gene had no significant influence on the expression of the renal tissue leptin receptor gene. The relative expression of the total amount of leptin receptor lrtmrna in the liver tissue was: Group CD [cd-wvscd-lmvscd-hm, 1.04 + 0.34vs1.63 + 0.18vs5.26 + 0.89 (P0.01)], and HF group [hf-wvshf-lmvshf-hm, 2.25 + 0.10vs4.96 + 0.88 (P0.05) vs9.11 + 1.33 (P0.01)) The membrane type leptin receptor subtypes in the liver of.2. were mainly three, that is, group lra{cd [1.02 + 0.24vs1.42 + 0.47vs2.92 + 0.68 (P0.05)], HF group [1.13 + 0.62vs2.80 + 0.18 (P0.05) vs6.04 + 1 (P0.01)]}, lrc{cd Group [1 + 1 + 0.25]. 8 + 0.11 (P0.01)}, group lrd{cd [1.02 + 0.25vs0.69 + 0.27vs2.48 + 0.15 (P0.01)], HF group [1.22 + 0.23vs2.55 + 0.16 (P0.01) vs4.82 + 0.03 (P0.01)],}.3. liver long type transmembrane leptin receptor was not significantly changed (1.02 + 0.30 0.69 + 0.69 + 0.32 + 0.09) before and after metformin treatment (1.02 + 0.30 0.69 + 0.32 + 0.09), interesting, high dose In the liver of the treated HF mice, the LRb m RNA was obviously up-regulated [0.69 + 0.26 vs, 0.77 + 0.15 vs 2.39 + 0.59 (P0.01)]. The mRNA expression of the.4. dimethyl metformin on the renal tissue was not significantly affected in the normal diet group or the high fat group (CD group (1.06 + 0.43 vs 1.03 + 0.32 0.85 + 0.08). Vs 0.74 + 0.27), LRa [group CD (1.05 + 0.45 vs 0.87 + 0.22 vs 0.36 + 0.07), HF group (0.56 + 0.21 vs 0.57 + 0.23 vs 0.57 + 0.08). 25 vs 0.67 + 0.21 vs 0.48 + 0.14), group LRd{CD [1.11 + 0.52 vs 0.79 + 0.18 vs 0.44 + 0.14 (P0.05)], HF group (0.28 + 0.07 vs0.21 +)}.5. metformin to the liver and kidney tissue ADAM10 mRNA [liver:] Kidneys: group CD [1.01 + 0.17 vs 0.95 + 0.16 vs 0.56 + 0.16 (P0.05)]; HF group (0.38 + 0.19 vs 0.23 + 0.04vs 0.33 + 0.09)}, ADAM17 18 vs 0.21 + 0.06 vs 0.40 + 0.16)} and protein expression level had no significant effect. Conclusion: 1, metformin has an up-regulated effect on LR gene expression in liver tissue, but it has no effect on renal tissue.2, and metformin up regulation of plasma sLR is realized by up regulation of LR gene expression in liver tissue, but sLR hydrolase ADAM10, ADAM17 gene is produced. There is no up regulation of.3, and the up regulation of sLR by metformin may be by promoting the short transmembrane leptin receptor in the liver tissue of mice, and.LRb, which is mainly expressed in LRa, LRc, and LRd gene, does not play a major role in sLR, but the up regulation of LRb can regulate the function of leptin.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1
【参考文献】
相关期刊论文 前1条
1 王廷伟;陈秋;;胰岛素抵抗与胰岛素信号通路的相关关系研究进展[J];西南军医;2014年01期
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