二甲雙胍促肝臟瘦素受體基因表達上調(diào)血漿可溶性瘦素受體的初步研究
發(fā)布時間：2018-08-05 13:37
【摘要】：研究背景:2型糖尿病(T2DM)是一種常見的內(nèi)分泌代謝疾病,占糖尿病(DM)總發(fā)病率的90%~95%,是遺傳和環(huán)境因素共同作用導致的復雜疾病。近年來,因環(huán)境及生活方式的改變,T2DM在我國呈爆發(fā)式增長,目前我國已成為糖尿病第一大國[1]。胰島素抵抗是T2DM發(fā)病的重要機制也是其重要特征。大量研究證實,肥胖與T2DM緊密相關[2,3,4,5],肥胖尤其是中心性肥胖是T2DM的獨立重要危險因素[6,7,8]。隨著體脂的增加,肥胖患者通常表現(xiàn)為血漿瘦素水平升高[9],而血漿s LR(soluble leptin receptor,可溶性瘦素受體)水平顯著降低的特點[10,11]。瘦素由脂肪組織分泌,具有調(diào)節(jié)攝食、糖脂代謝及免疫炎癥等諸多功能[12]。s LR是人體血漿中結合瘦素的最主要血漿蛋白[13],調(diào)節(jié)瘦素的生物功能。多項研究顯示,血漿sLR水平降低與肥胖T2DM患者血糖、低密度脂蛋白膽固醇、血壓等代謝綜合征指標升高呈負相關[14],經(jīng)過手術、控制飲食、體育鍛煉等措施降低體重后,血漿s LR水平顯著升高[15,16,17],提示血漿sLR水平可能作為肥胖T2DM患者代謝狀態(tài)改善的生化指征。我們前期采用二甲雙胍干預高脂喂養(yǎng)的胰島抵抗小鼠及初診T2DM病人,均發(fā)現(xiàn)二甲雙胍能顯著上調(diào)血漿s LR的水平[18],但其機制尚未闡明。已知,人體血漿s LR來源于膜型LR(leptin receptor,瘦素受體)胞外域水解脫落[19],人體中存在四種LR亞型,即LR-a、LR-b、LR-c及LR-d,它們均由同一基因(LR)編碼,轉錄后經(jīng)RNA差異剪接后生成[20]。其中,LRb是傳遞瘦素信號的唯一全長型瘦素受體[21]。小鼠體內(nèi)還存在其他亞型的瘦素受體,如膜型LR-f及分泌型LR-e[22]。已有報道,肝臟是瘦素受體表達量最高的外周組織器官,在調(diào)節(jié)血漿s LR水平中具有重要作用[23,24]。此外,腎臟組織中也存在豐富的瘦素受體[23]。對于水解LR的酶總體上了解較少。近期報道,ADAM10(A Disintegrin And Metalloproteinase 10,解聚素樣金屬蛋白酶10)、ADAM17參與了LR的水解脫落過程,其中ADAM10起主要作用[25]。但二甲雙胍是否通過上調(diào)瘦素受體及其水解酶的基因表達上調(diào)s LR的水平,目前尚未見報道。為此,本研究通過觀察二甲雙胍對小鼠肝臟、腎臟組織瘦素受體總量(lrt)及四種主要膜型受體(lra,lrb,lrc,lrd)的基因表達,同時觀察該藥物對解聚素樣金屬蛋白酶adam10及adam17的基因表達以及其蛋白水平的改變,擬初步闡明二甲雙胍上調(diào)血漿可溶性瘦素受體的可能機制。材料和方法:42只c57bl/6小鼠(6~8周齡,體重17.02±1.91g)用隨機數(shù)字表法隨機分為2組,分別給予正常飲食及高脂飲食喂養(yǎng)5月后,各組再以隨機數(shù)字表法分為3亞組,分別給予蒸餾水及低劑量(50mg/kg.d)、高劑量(200mg/kg.d)二甲雙胍灌胃(1次/天,共計15d),處死后取各組小鼠肝臟、腎臟組織存于-80℃冰箱[18]。剔除喂養(yǎng)過程中死亡小鼠,根據(jù)飲食和蒸餾水、二甲雙胍劑量的不同處理共為6亞組,如下所示:正常飲食—蒸餾水組(cd-w,n=7);正常飲食—低劑量二甲雙胍組(cd-lm,n=7);正常飲食—高劑量二甲雙胍組(cd-hm,n=7);高脂飲食—蒸餾水組(hf-w,n=5);高脂飲食—低劑量二甲雙胍組(hf-lm,n=6),高脂飲食—高劑量二甲雙胍組(hf-hm,n=7)。q-pcr檢測各小組肝臟、腎臟組織瘦素受體(lrt,lra,lrb,lrc,lrd)及(adam10、adam17)的基因表達水平,western-blot檢測各組肝臟、腎臟組織adam10、adam17蛋白表達水平。結果:1.二甲雙胍能劑量依賴性上調(diào)肝臟組織瘦素受體基因的表達,而對腎臟組織瘦素受體基因的表達無顯著影響。肝臟組織瘦素受體總量lrtmrna的相對表達量分別為:cd組【cd-wvscd-lmvscd-hm,1.04±0.34vs1.63±0.18vs5.26±0.89(p0.01)】;hf組【hf-wvshf-lmvshf-hm,2.25±0.10vs4.96±0.88(p0.05)vs9.11±1.33(p0.01)】。2.肝臟中受二甲雙胍上調(diào)的膜型瘦素受體亞型主要是三種,即lra{cd組【1.02±0.24vs1.42±0.47vs2.92±0.68(p0.05)】;hf組【1.13±0.62vs2.80±0.18(p0.05)vs6.04±1.00(p0.01)】}、lrc{cd組【1.00±0.06vs1.02±0.13vs1.97±0.25(p0.01)】;hf組【0.97±0.12vs2.60±0.18(p0.01)vs4.58±0.11(p0.01)】}、lrd{cd組【1.02±0.25vs0.69±0.27vs2.48±0.15(p0.01)】;hf組【1.22±0.23vs2.55±0.16(p0.01)vs4.82±0.03(p0.01)】}。3.肝臟長型跨膜瘦素受體lrbmrna在二甲雙胍處理前后的cd組小鼠中并無顯著變化(1.02±0.30 vs 0.69±0.16 vs 0.32±0.09),有趣的是,高劑量處理的HF組小鼠肝臟中,LRb m RNA明顯上調(diào)【0.69±0.26 vs 0.77±0.15 vs 2.39±0.59(P0.01)】。4.二甲雙胍對腎臟組織中瘦素受體的mRNA表達無論在正常飲食組或高脂組均無顯著影響LRt【CD組(1.06±0.43 vs 1.03±0.32 vs 0.85±0.08);HF組(0.37±0.13 vs0.36±0.17 vs 0.74±0.27)】,LRa【CD組(1.05±0.45 vs 0.87±0.22 vs 0.36±0.07);HF組(0.56±0.21 vs 0.57±0.23 vs 0.57±0.08)】,LRb{CD組【1.00±0.15 vs 0.58±0.16(P0.01)vs 0.12±0.02(P0.01)】;HF組(0.14±0.04 vs 0.08±0.05 vs 0.08±0.03)},LRc【CD組(1.03±0.32 vs 1.05±0.16 vs 0.40±0.06);HF組(0.69±0.25 vs 0.67±0.21 vs 0.48±0.14)】,LRd{CD組【1.11±0.52 vs 0.79±0.18 vs 0.44±0.14(P0.05)】;HF組(0.28±0.07 vs0.21±0.04 vs 0.67±0.13)}。5.二甲雙胍對肝臟及腎臟組織中ADAM10的mRNA【肝臟:CD組(1.02±0.22vs 1.06±0.14 vs 0.76±0.10);HF組(0.79±0.14 vs 0.82±0.07 vs 0.88±0.05)】{腎臟:CD組【1.01±0.17 vs 0.95±0.16 vs 0.56±0.16(P0.05)】;HF組(0.38±0.19 vs 0.23±0.04vs 0.33±0.09)}、ADAM17【肝臟:CD組(1.00±0.01 vs 1.07±0.07 vs 0.85±0.09);HF組(0.81±0.02 vs 0.82±0.06 vs 0.84±0.09)】{腎臟:CD組【1.02±0.29 vs 1.00±0.08vs 0.42±0.07(P0.05)】;HF組(0.40±0.18 vs 0.21±0.06 vs 0.40±0.16)}及蛋白表達水平均無顯著影響。結論:1、二甲雙胍對肝臟組織的LR基因表達有上調(diào)作用,但對腎臟組織無此作用。2、二甲雙胍上調(diào)血漿sLR是通過上調(diào)肝臟組織LR基因表達實現(xiàn)的,但對產(chǎn)生sLR的水解酶ADAM10、ADAM17基因及蛋白的表達均無上調(diào)作用。3、二甲雙胍上調(diào)血漿sLR可能是通過促進小鼠肝臟組織短型跨膜瘦素受體,主要是LRa、LRc、LRd基因表達實現(xiàn)的。LRb對sLR不起主要作用,但LRb上調(diào)能調(diào)節(jié)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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R587.1
本文編號：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R587.1
【參考文獻】
相關期刊論文 前1條
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，本文編號：2165958
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