亮氨酸干预对胰岛素抵抗大鼠胆汁酸代谢的影响
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摘要
目的观察亮氨酸对大鼠胰岛素抵抗、血清胆汁酸水平、胆汁酸代谢相关基因等指标的影响,进一步揭示亮氨酸调节胰岛素抵抗大鼠糖脂代谢的机制。方法雄性6周龄SD大鼠24只随机分为3组,分别喂饲普通饲料、高脂饲料,高脂饲料添加1.5%亮氨酸。喂养32周,进行葡萄糖耐量实验(GTT)和胰岛素耐量实验(ITT),计算葡萄糖曲线下面积(AUC)。实验结束后,收集大鼠血清和肝脏组织,测定血清胰岛素、葡萄糖水平,计算胰岛素抵抗指数(HOMA-IR),评价胰岛素敏感性。检测血清及肝脏中总胆固醇(TC)和三酰甘油(TG)水平,实时荧光定量逆转录-聚合酶链反应(Real-time PCR)测定大鼠肝脏中胆汁酸合成相关基因肝X受体(LXRα)、法尼酯X受体(FXR)、胆固醇7α羟化酶(CYP7A1)、胆固醇27α羟化酶(CYP27A1)mRNA的表达,超高效液相色谱-串联质谱法(UPLC-MS/MS)测定大鼠血清中17种胆汁酸的水平。结果亮氨酸干预能够显著降低高脂喂养大鼠血清TC、TG和肝脏TG水平,改善脂代谢。亮氨酸对血糖和血胰岛素水平没有显著影响,但是能够有效降低HOMA-IR和葡萄糖耐量实验AUC水平,改善胰岛素抵抗。亮氨酸能够显著升高高脂喂养大鼠血清胆汁酸水平和LXRα、FXR、CYP27A1mRNA水平,但对CYP7A1mRNA的表达没有影响。结论亮氨酸干预能够显著增加胰岛素抵抗大鼠的胆汁酸合成,减少血清中脂肪的堆积,改善糖脂代谢,增强其胰岛素敏感性,胆汁酸合成相关基因表达的改变可能是其重要机制。
Objective To observe the effects of leucine on insulin resistance,serum bile acid levels,and expression of genes related to bile acid metabolism,and to reveal the mechanism of leucine intervention on glucose and lipid metabolism in insulin resistant rats.Methods Twenty-four male Sprague-Dawley rats aged 6 weeks were randomly divided into three groups and fed on normal diet,high fat diet,and high fat diet supplemented with 1.5%leucine respectively.At the end of 32 weeks,glucose tolerance test(GTT)and insulin tolerance test(ITT)were performed,and the area under the curve of glucose(AUC)was calculated.Then the rats were sacrificed,the serum and tissues samples were collected.Serum insulin and glucose levels were measured,insulin resistance index(HOMA-IR)calculated,and insulin sensitivity evaluated.The levels of total cholesterol(TC)and triglycerides(TG)in the serum and liver were detected.The expressions of bile acid synthesis related genes liver X recepter(LXRα),farnesoid X receptor(FXR),cholesterol 7α-hydroxylase(CYP7 A1)and cholesterol 27-hydroxylase(CYP27 A1)mRNA in the liver were determined by real-time reverse transcription PCR(RT-PCR),and the levels of 17 bile acids in the serum by ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).Results Leucine intervention were determined significantly reduced serum TC,TG and liver TG levels and improved lipid metabolism in rats on high-fat diet.Leucine had no significant effect on glucose and insulin levels,but could effectively reduce the HOMA-IR and AUC of glucose tolerance test and decrease insulin resistance.Leucine significantly increased serum bile acid levels and LXRα,FXR,CYP27 A1 mRNA levels but had no effect on CYP7 A1 mRNA expression.Conclusion Leucine intervention significantly increased bile acid synthesis in insulin resistant rats,thereby reducing the accumulation of serum lipids,improving glucose and lipid metabolism and insulin sensitivity.
Objective To observe the effects of leucine on insulin resistance,serum bile acid levels,and expression of genes related to bile acid metabolism,and to reveal the mechanism of leucine intervention on glucose and lipid metabolism in insulin resistant rats.Methods Twenty-four male Sprague-Dawley rats aged 6 weeks were randomly divided into three groups and fed on normal diet,high fat diet,and high fat diet supplemented with 1.5%leucine respectively.At the end of 32 weeks,glucose tolerance test(GTT)and insulin tolerance test(ITT)were performed,and the area under the curve of glucose(AUC)was calculated.Then the rats were sacrificed,the serum and tissues samples were collected.Serum insulin and glucose levels were measured,insulin resistance index(HOMA-IR)calculated,and insulin sensitivity evaluated.The levels of total cholesterol(TC)and triglycerides(TG)in the serum and liver were detected.The expressions of bile acid synthesis related genes liver X recepter(LXRα),farnesoid X receptor(FXR),cholesterol 7α-hydroxylase(CYP7 A1)and cholesterol 27-hydroxylase(CYP27 A1)mRNA in the liver were determined by real-time reverse transcription PCR(RT-PCR),and the levels of 17 bile acids in the serum by ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).Results Leucine intervention were determined significantly reduced serum TC,TG and liver TG levels and improved lipid metabolism in rats on high-fat diet.Leucine had no significant effect on glucose and insulin levels,but could effectively reduce the HOMA-IR and AUC of glucose tolerance test and decrease insulin resistance.Leucine significantly increased serum bile acid levels and LXRα,FXR,CYP27 A1 mRNA levels but had no effect on CYP7 A1 mRNA expression.Conclusion Leucine intervention significantly increased bile acid synthesis in insulin resistant rats,thereby reducing the accumulation of serum lipids,improving glucose and lipid metabolism and insulin sensitivity.
引文
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[18]Watanabe M,Houten S M,Wang L,et al.Bile acids lower triglyceride levels via a pathway involving FXR,SHP,and SREBP-1c[J].J Clin Invest,2004,113(10):1408-1418.
[19]Zhang Y,Lee F Y,Barrera G,et al.Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in diabetic mice[J].Proc Natl Acad Sci U S A,2006,103(4):1006-1011.
[20]黄凤杰.复杂生物样本中胆汁酸谱检测方法研究及老鸭嘴化学成分研究[D].上海:上海交通大学,2013.
[21]胡军,白秋方,杨少娟,等.肥胖对胰岛β细胞凋亡的影响[J].华中科技大学学报:医学版,2017,46(6):652-655.
[22]Pedersen H K,Gudmundsdottir V,Nielsen H B,et al.Human gut microbes impact host serum metabolome and insulin sensitivity[J].Nature,2016,535(7612):376-381.
[2]Binder E,Bermudez-Silva F J,Andre C,et al.Leucine supplementation protects from insulin resistance by regulating adiposity levels[J].PLoS One,2013,8(9):e74705.
[3]Sener A,Malaisse W.L-leucine and a nonmetabolized analogue activate pancreatic islet glutamate dehydrogenase[J].Nature,1980,288(5787):187-189.
[4]Arakawa M,Masaki T,Nishimura J,et al.The effects of branched-chain amino acid granules on the accumulation of tissue triglycerides and uncoupling proteins in diet-induced obese mice[J].Endocr J,2011,58(3):161-170.
[5]Nie C,He T,Zhang W,et al.Branched chain amino acid:beyond nutrition metabolism[J].Int J Mol Sci,2018,19(4):954-969.
[6]Zhao Y,Dai X Y,Zhou Z,et al.Leucine supplementation via drinking water reduces atherosclerotic lesions in apoE null mice[J].Acta Pharmacol Sin,2016,37(2):196-203.
[7]Higuchi N,Kato M,Miyazaki M,et al.Potential role of branched-chain amino acids in glucose metabolism through the accelerated induction of the glucose-sensing apparatus in the liver[J].J Cell Biochem,2011,112(1):30-38.
[8]Russell D W.The enzymes,regulation,and genetics of bile acid synthesis[J].Annu Rev Biochem,2003,72(1):137-174.
[9]Li T,Chiang J Y L.Bile acid signaling in metabolic disease and drug therapy[J].Pharmacol Rev,2014,66(4):948-983.
[10]Zhang Z,Li D,Blanchard D E,et al.Key regulatory oxysterols in liver:analysis as delta4-3-ketone derivatives by HPLC and response to physiological perturbations[J].J Lipid Res,2001,42(4):649-658.
[11]邱冬妮,孙旭,钟良,等.高胆固醇摄入对新西兰兔胆汁酸代谢的影响[J].复旦学报:医学版,2009,36(5):601-604.
[12]Dardevet D,Sornet C,Bayle G,et al.Postprandial stimulation of muscle protein synthesis in old rats can be restored by a leucine-supplemented meal[J].J Nutr,2002,132(1):95-100.
[13]Pedroso J A B,Nishimura L S,de Matos-Neto E M,et al.L-eucine improves protein nutritional status and regulates hepatic lipid metabolism in calorie-restricted rats[J].Cell Biochem Funct,2014,32(4):326-332.
[14]Li H,Xu M,Lee J,et al.Leucine supplementation increases SIRT1expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice[J].Am J Physiol Endocrinol Metab,2012,303(10):1234-1244.
[15]Binder E,Bermudez-Silva F J,Elie M,et al.Leucine supplementation modulates fuel substrates utilization and glucose metabolism in previously obese mice[J].Obesity,2014,22(3):713-720.
[16]Cariou B,van Harmelen K,Duran-Sandoval D,et al.The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice[J].J Biol Chem,2006,281(16):11039-11049.
[17]Ma K,Saha P K,Chan L,et al.Farnesoid X receptor is essential for normal glucose homeostasis[J].J Clin Invest,2006,116(4):1102-1109.
[18]Watanabe M,Houten S M,Wang L,et al.Bile acids lower triglyceride levels via a pathway involving FXR,SHP,and SREBP-1c[J].J Clin Invest,2004,113(10):1408-1418.
[19]Zhang Y,Lee F Y,Barrera G,et al.Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in diabetic mice[J].Proc Natl Acad Sci U S A,2006,103(4):1006-1011.
[20]黄凤杰.复杂生物样本中胆汁酸谱检测方法研究及老鸭嘴化学成分研究[D].上海:上海交通大学,2013.
[21]胡军,白秋方,杨少娟,等.肥胖对胰岛β细胞凋亡的影响[J].华中科技大学学报:医学版,2017,46(6):652-655.
[22]Pedersen H K,Gudmundsdottir V,Nielsen H B,et al.Human gut microbes impact host serum metabolome and insulin sensitivity[J].Nature,2016,535(7612):376-381.