胰岛素样生长因子-Ⅰ在肥胖大鼠行Roux-en-Y胃旁路术后脂肪组织中的表达变化及其作用
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摘要
目的检测胰岛素样生长因子-Ⅰ(insulin-like growth factor-Ⅰ,IGF-Ⅰ)蛋白及其m RNA在肥胖大鼠行Roux-en-Y胃旁路手术(RYGB)前后的表达水平变化,并探讨IGF-Ⅰ表达和脂肪细胞增殖、凋亡的关系。方法1选取SPF级SD雄性大鼠70只,随机分为空白对照组(NC组)10只和高脂饮食组60只。高脂饮食组大鼠给予特定配方高脂饲料,NC组大鼠给予特定配方维持饲料,6周后测量高脂饮食组大鼠的体质量,选择体质量增加在前20位的肥胖大鼠,随机分为胃旁路术组(GB组)10只和假手术组(SO组)10只。GB组大鼠行RYGB,SO组大鼠行假手术,NC组大鼠不接受任何手术。GB组和SO组大鼠于术中、3组大鼠于处理后12周取腹股沟脂肪组织〔代表皮下脂肪组织(subcutaneous adipose tissue,SAT)〕和附睾脂肪组织〔代表内脏脂肪组织(visceral adipose tissue,VAT)〕各0.5 g,行免疫印迹法和实时荧光定量PCR法分别检测脂肪组织中IGF-Ⅰ蛋白及其m RNA的表达。2转染实验。将SAT细胞分为空白对照组(BC组,未行转染)、IGF-Ⅰ(+)组(即基因过表达组)、IGF-Ⅰ(+)空载体组、IGF-Ⅰ(-)组(即基因沉默组)及IGF-Ⅰ(-)空载体组,转染相应载体,每组设3个复孔。转染48 h后进行细胞增殖与凋亡实验,并采用免疫印迹法检测蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)及磷脂酰肌醇3-激酶(PI3K)的表达水平。3 Wortmannin实验。将SAT细胞分为Wortmannin(+)IGF-Ⅰ(+)组、Wortmannin(+)IGF-Ⅰ(-)、Wortmannin(-)IGF-Ⅰ(+)及Wortmannin(-)IGF-Ⅰ(-)组,转染相应载体类型24 h后,加入0.1 mmol/L Wortmannin,24 h后采用免疫印迹法检测AKT、p-AKT、p-PI3K、PI3K及GAPDH的表达水平。结果 1 PCR结果表明,在SAT组织中,同GB术前组比较,GB术后组的IGF-Ⅰ蛋白及其m RNA的表达水平均降低(P<0.01),而SO术前组和SO术后组的IGF-Ⅰ蛋白及其m RNA的表达水平比较差异均无统计学意义(P>0.05);VAT中,5组大鼠的IGF-Ⅰ蛋白及其m RNA的表达水平比较差异均无统计学意义(P>0.05)。2四甲基偶氮唑盐(MTT)比色法结果表明,同对应的空载体组比较,IGF-Ⅰ(+)组的增殖能力升高(P=0.04),IGF-Ⅰ(-)组的细胞增殖缓慢(P=0.04);流式细胞仪检测结果表明,同对应的空载体组比较,IGF-Ⅰ(+)组的细胞凋亡率较低(P=0.04),IGF-Ⅰ(-)组的细胞凋亡率较高(P=0.04)。3与IGF-Ⅰ(+)空载体组比较,IGF-Ⅰ(+)组的p-PI3K/PI3K(P=0.03)和p-AKT/AKT(P=0.04)比值均升高;与IGF-Ⅰ(-)空载体组相比,IGF-Ⅰ(-)组的p-PI3K/PI3K(P=0.04)和p-AKT/AKT(P=0.04)比值均降低。与Wortmannin(+)IGF-Ⅰ(+)组比较,Wortmannin(-)IGF-Ⅰ(+)组的p-AKT/AKT比值较高(P<0.05);与Wortmannin(-)IGF-Ⅰ(+)组比较,Wortmannin(-)IGF-Ⅰ(-)组的p-AKT/AKT比值较低(P<0.05)。结论 IGF-Ⅰ参与大鼠皮下脂肪富集,RYGB能明显降低大鼠皮下脂肪中IGF-Ⅰ蛋白及其m RNA的表达水平,从而达到减重效果。
Objective To verify the expression change of insulin-like growth factor-I(IGF-I) protein and its mRNA before and after Roux-en-Y gastric bypass surgery(RYGB) in obese rats,and to investigate the relationship between the expression of IGF-I and proliferation/apoptosis of adipose cells.Methods ① Seventy male SD rats were raised at the SPF level circumstance and were randomly divided into control group(NC group,10 rats) and high fat diet group(60 rats).Rats of high fat diet group were given specific high fat formula diet,rats of NC group were given particular formula diet.After 6 weeks,the body weights of the rats in high fat diet group were measured,and the 20 rats of top weight were selected.The 20 obese rats were randomly divided into 2 groups:gastric bypass(GB) group(n=10) and sham-operation group(SO group,n=10).RYGB were administered to the rats of GB group,and for rats of SO group,sham operations were performed.Rats of NC group did not receive any surgery.Inguinal adipose tissues[represented the subcutaneous adipose tissue(SAT)) and epididymal adipose tissues[on behalf of visceral adipose tissue(VAT)) were taken during operation in rats of GB group and SO group respectively(0.5 g),and 12 weeks after operation in all rats of three groups.The expressions of IGF-I protein and its mRNA in adipose tissue were detected by Western blot and real-time fluorescence quantitative PCR.② Transfection experiment.SAT cells were divided into blank control group(BC group,without transfection),IGF-I(+)group(gene overexpression group),IGF-I(+) empty vector group,IGF-I(-) group(gene silencing group),and IGF-I(-) empty vector group.Cells were transfected with corresponding vectors with 3 duplicated holes of each group.Cell viability and apoptosis assays were carried out in 48 hours after transfection.Expressions of protein kinase B(AKT),phosphorylated protein kinase B(p-AKT),phosphoinositide 3-kinase(PI3K),and phosphorylated phosphoinositide 3-kinase(p-PI3K) were detected by Western blot meanwhile.③ Wortmannin experiment.SAT cells were divided into Wortmannin(+)IGF-I(+) group,Wortmannin(+)IGF-I(-)group,Wortmannin(-)IGF-I(+) group,and Wortmannin(-) IGF-I(-) group,which were transfected with corresponding vectors for24 hours,then adding Wortmannin(0.1 mmol/L).After 24 hours,the expression levels of AKT,p-AKT,p-PI3 K,PI3K,and GAPDH were detected by Western blot.Results ① PCR results showed that,in SAT,compared with preoperative GB group,the expression levels of IGF-I mRNA and its protein in postoperative GB group were both lower(P<0.01).However,the expression levels of IGF-I mRNA and its protein between preoperative SO group and postoperative SO group showed no significant difference(P>0.05).In VAT,the expression levels of IGF-I mRNA and its protein in 5 groups showed no significant difference(P>0.05).② The MTT results showed that,IGF-I(+) group harbored stronger proliferation abilities compared with its negative control group(P=0.04),whereas IGF-I(-) group had lower abilities compared with its negative control group(P=0.04).The results of flow cytometry assay showed that,the apoptosis rate of IGF-I(+) group was lower(P=0.04) than that of the corresponding negative control group,and it was higher in IGF-I(-) group than that of the corresponding negative control group(P=0.04).③ Compared with IGF-I(+) empty vector group,P-PI3K/PI3 K ratio(P=0.03) and p-AKT/AKT(P=0.04) ratio of IGF-I(+) group were increased;compared with IGF-I(-) empty vector group,p-PI3K/PI3 K ratio(P=0.04) and p-AKT/AKT ratio(P=0.04) of IGF-I(-) group were decreased.The p-AKT/AKT ratio of Wortmannin(-) IGF-I(+) group was higher(P<0.05) than that of Wortmannin(+) IGF-I(+) group;the p-AKT/AKT ratio of Wortmannin(-) IGF-I(-) group was lower than that of Wortmannin(-) IGF-I(+) group(P<0.05).Conclusions IGF-I is involved in the accumulation of subcutaneous fat in rats.RYGB can significantly reduce the expression levels of IGF-I mRNA and its protein in subcutaneous fat of rats,so as to achieve the effect of weight loss.
Objective To verify the expression change of insulin-like growth factor-I(IGF-I) protein and its mRNA before and after Roux-en-Y gastric bypass surgery(RYGB) in obese rats,and to investigate the relationship between the expression of IGF-I and proliferation/apoptosis of adipose cells.Methods ① Seventy male SD rats were raised at the SPF level circumstance and were randomly divided into control group(NC group,10 rats) and high fat diet group(60 rats).Rats of high fat diet group were given specific high fat formula diet,rats of NC group were given particular formula diet.After 6 weeks,the body weights of the rats in high fat diet group were measured,and the 20 rats of top weight were selected.The 20 obese rats were randomly divided into 2 groups:gastric bypass(GB) group(n=10) and sham-operation group(SO group,n=10).RYGB were administered to the rats of GB group,and for rats of SO group,sham operations were performed.Rats of NC group did not receive any surgery.Inguinal adipose tissues[represented the subcutaneous adipose tissue(SAT)) and epididymal adipose tissues[on behalf of visceral adipose tissue(VAT)) were taken during operation in rats of GB group and SO group respectively(0.5 g),and 12 weeks after operation in all rats of three groups.The expressions of IGF-I protein and its mRNA in adipose tissue were detected by Western blot and real-time fluorescence quantitative PCR.② Transfection experiment.SAT cells were divided into blank control group(BC group,without transfection),IGF-I(+)group(gene overexpression group),IGF-I(+) empty vector group,IGF-I(-) group(gene silencing group),and IGF-I(-) empty vector group.Cells were transfected with corresponding vectors with 3 duplicated holes of each group.Cell viability and apoptosis assays were carried out in 48 hours after transfection.Expressions of protein kinase B(AKT),phosphorylated protein kinase B(p-AKT),phosphoinositide 3-kinase(PI3K),and phosphorylated phosphoinositide 3-kinase(p-PI3K) were detected by Western blot meanwhile.③ Wortmannin experiment.SAT cells were divided into Wortmannin(+)IGF-I(+) group,Wortmannin(+)IGF-I(-)group,Wortmannin(-)IGF-I(+) group,and Wortmannin(-) IGF-I(-) group,which were transfected with corresponding vectors for24 hours,then adding Wortmannin(0.1 mmol/L).After 24 hours,the expression levels of AKT,p-AKT,p-PI3 K,PI3K,and GAPDH were detected by Western blot.Results ① PCR results showed that,in SAT,compared with preoperative GB group,the expression levels of IGF-I mRNA and its protein in postoperative GB group were both lower(P<0.01).However,the expression levels of IGF-I mRNA and its protein between preoperative SO group and postoperative SO group showed no significant difference(P>0.05).In VAT,the expression levels of IGF-I mRNA and its protein in 5 groups showed no significant difference(P>0.05).② The MTT results showed that,IGF-I(+) group harbored stronger proliferation abilities compared with its negative control group(P=0.04),whereas IGF-I(-) group had lower abilities compared with its negative control group(P=0.04).The results of flow cytometry assay showed that,the apoptosis rate of IGF-I(+) group was lower(P=0.04) than that of the corresponding negative control group,and it was higher in IGF-I(-) group than that of the corresponding negative control group(P=0.04).③ Compared with IGF-I(+) empty vector group,P-PI3K/PI3 K ratio(P=0.03) and p-AKT/AKT(P=0.04) ratio of IGF-I(+) group were increased;compared with IGF-I(-) empty vector group,p-PI3K/PI3 K ratio(P=0.04) and p-AKT/AKT ratio(P=0.04) of IGF-I(-) group were decreased.The p-AKT/AKT ratio of Wortmannin(-) IGF-I(+) group was higher(P<0.05) than that of Wortmannin(+) IGF-I(+) group;the p-AKT/AKT ratio of Wortmannin(-) IGF-I(-) group was lower than that of Wortmannin(-) IGF-I(+) group(P<0.05).Conclusions IGF-I is involved in the accumulation of subcutaneous fat in rats.RYGB can significantly reduce the expression levels of IGF-I mRNA and its protein in subcutaneous fat of rats,so as to achieve the effect of weight loss.
引文
1 Sjoholm A,Nystrom T.Endothelial inflammation in insulin resistance.Lancet,2005,365(9459):610-612.
2 Hotamisligil GS.Inflammation and metabolic disorders.Nature,2006,444(7121):860-867.
3 Cohen DH,Leroith D.Obesity,type 2 diabetes,and cancer:the insulin and IGF connection.Endocr Relat Cancer,2012,19(5):F27-F45.
4 Mah AT,Van Landeghem L,Gavin HE,et al.Impact of diet-induced obesity on intestinal stem cells:hyperproliferation but impaired intrinsic function that requires insulin/IGF-I.Endocrinology,2014,155(9):3302-3314.
5 Pozdniakov AL,Pogozheva AV,Gadzhieva ZM,et al.Experimental model of the early stages of alimentary obesity in the rat.Biull Eksp BiolMed,1982,94(11):116-119.
6 Kindel TL,Yoder SM,D'Alessio DA,et al.The effect of duodenaljejunal bypass on glucose-dependent insulinotropic polypeptide secretion in Wistar rats.Obes Surg,2010,20(6):768-775.
7 Okada T,Kawano Y,Sakakibara T,et al.Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes.Studies with a selective inhibitor wortmannin.J Biol Chem,1994,269(5):3568-3573.
8 Walser M,Sama MT,Wickelgren R,et al.Local overexpression of GH and GH/IGF-I effects in the adult mouse hippocampus.J Endocrinol,2012,215(2):257-268.
9 DeMaria EJ.Bariatric surgery for morbid obesity.N Engl J Med,2007,356(21):2176-2183.
10 Giles ED,Wellberg EA,Astling DP,et al.Obesity and overfeeding affecting both tumor and systemic metabolism activates the progesterone receptor to contribute to postmenopausal breast cancer.Cancer Res,2012,72(24):6490-6501.
11 Frumovitz M,Jhingran A,Soliman PT,et al.Morbid obesity as an independent risk factor for disease-specific mortality in women with cervical cancer.Obstet Gynecol,2014,124(6):1098-1104.
12 Sahin IH.Obesity,bisphosphonates,and colorectal cancer.J Clinl Oncol,2013,31(20):2639.
13 Whitson BA,Leslie DB,Kellogg TA,et al.Adipokine response in diabetics and nondiabetics following the Roux-en-Y gastric bypass:a preliminary study.J Surg Res,2007,142(2):295-300.
14 Vetter ML,Cardillo S,Rickels MR,et al.Narrative review:effect of bariatric surgery on type 2 diabetes mellitus.Ann Intern Med,2009,150(2):94-103.
15 Vidal J,Jimenez A,De Hollanda A,et al.Metabolic surgery in type 2 diabetes:Roux-en-Y gastric bypass or sleeve gastrectomy as procedure of choice?Curr Atheroscler Rep,2015,17(10):58.
16 Talar T,Candice J,Erik D.Bariatric surgery and type 2 diabetes mellitus:surgically induced remission.Diabetes Sci Technol,2008,2(4):685-691.
17 Salameh BS,Khoukaz MT,Bell RL.Metabolic and nutritional changes after bariatric surgery.Expert Rev Gastroenterol Hepatol,2010,4(2):217-223.
18 Kashyap SR,Daud S,Kelly KR,et al.Acute effects of gastric bypass versus gastric restrictive surgery on beta-cell function and insulinotropic hormones in severely obese patients with type 2diabetes.Int J Obes(Lond),2010,34(3):462-471.
19 Heindel JJ,Newbold R,Schug TT.Endocrine disruptors and obesity.Nat Rev Endocrinol,2015,11(11):653-661.
20 Clemmons DR.Modifying IGF-I activity:an approach to treat endocrine disorders,atherosclerosis and cancer.Nat Rev Drug Discov,2007,6(10):821-833.
21 Chesnokova V,Zhou CQ,Ben-Shlomo A,et al.Growth hormone is a cellular senescence target in pituitary and nonpituitary cells.Proc Natl Acad Sci U S A,2013,110(35):E3331-E3339.
22 Yakar S,Liu JL,Le Roith D.The growth hormone/insulin-like growth factor-I system:implications for organ growth and development.Pediatr Nephrol,2000,14(7):544-549.
23 Eggert ML,Wallaschofski H,Grotevendt A,et al.Cross-sectional and longitudinal relation of IGF-I and IGF-binding protein 3 with lipid metabolism.Eur J Endocrinol,2014,171(1):9-19.
24 Hu L,Yang G,Hagg D,et al.IGF-I promotes adipogenesis by a lineage bias of endogenous adipose stem/progenitor cells.Stem Cells,2015,33(8):2483-2495.
25 Tang QQ,Lane MD.Adipogenesis:from stem cell to adipocyte.Annu Rev Biochem,2012,81:715-736.
26 Chang HR,Kim HJ,Xu X,et al.Macrophage and adipocyte IGF-I maintain adipose tissue homeostasis during metabolic stresses.Obesity(Silver Spring),2016,24(1):172-183.
27 Fowke J,Matthews C,Yu H,et al.Racial differences in the association between body mass index and serum IGF-I,IGF2,and IGFBP3.Endocr Relat Cancer,2010,17(1):51-60.
28 Hegedus B,Yeh TH,Lee DY,et al.Neurofibromin regulates somatic growth through the hypothalamic-pituitary axis.Hum Mol Genet,2008,17(19):2956-2966.
2 Hotamisligil GS.Inflammation and metabolic disorders.Nature,2006,444(7121):860-867.
3 Cohen DH,Leroith D.Obesity,type 2 diabetes,and cancer:the insulin and IGF connection.Endocr Relat Cancer,2012,19(5):F27-F45.
4 Mah AT,Van Landeghem L,Gavin HE,et al.Impact of diet-induced obesity on intestinal stem cells:hyperproliferation but impaired intrinsic function that requires insulin/IGF-I.Endocrinology,2014,155(9):3302-3314.
5 Pozdniakov AL,Pogozheva AV,Gadzhieva ZM,et al.Experimental model of the early stages of alimentary obesity in the rat.Biull Eksp BiolMed,1982,94(11):116-119.
6 Kindel TL,Yoder SM,D'Alessio DA,et al.The effect of duodenaljejunal bypass on glucose-dependent insulinotropic polypeptide secretion in Wistar rats.Obes Surg,2010,20(6):768-775.
7 Okada T,Kawano Y,Sakakibara T,et al.Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes.Studies with a selective inhibitor wortmannin.J Biol Chem,1994,269(5):3568-3573.
8 Walser M,Sama MT,Wickelgren R,et al.Local overexpression of GH and GH/IGF-I effects in the adult mouse hippocampus.J Endocrinol,2012,215(2):257-268.
9 DeMaria EJ.Bariatric surgery for morbid obesity.N Engl J Med,2007,356(21):2176-2183.
10 Giles ED,Wellberg EA,Astling DP,et al.Obesity and overfeeding affecting both tumor and systemic metabolism activates the progesterone receptor to contribute to postmenopausal breast cancer.Cancer Res,2012,72(24):6490-6501.
11 Frumovitz M,Jhingran A,Soliman PT,et al.Morbid obesity as an independent risk factor for disease-specific mortality in women with cervical cancer.Obstet Gynecol,2014,124(6):1098-1104.
12 Sahin IH.Obesity,bisphosphonates,and colorectal cancer.J Clinl Oncol,2013,31(20):2639.
13 Whitson BA,Leslie DB,Kellogg TA,et al.Adipokine response in diabetics and nondiabetics following the Roux-en-Y gastric bypass:a preliminary study.J Surg Res,2007,142(2):295-300.
14 Vetter ML,Cardillo S,Rickels MR,et al.Narrative review:effect of bariatric surgery on type 2 diabetes mellitus.Ann Intern Med,2009,150(2):94-103.
15 Vidal J,Jimenez A,De Hollanda A,et al.Metabolic surgery in type 2 diabetes:Roux-en-Y gastric bypass or sleeve gastrectomy as procedure of choice?Curr Atheroscler Rep,2015,17(10):58.
16 Talar T,Candice J,Erik D.Bariatric surgery and type 2 diabetes mellitus:surgically induced remission.Diabetes Sci Technol,2008,2(4):685-691.
17 Salameh BS,Khoukaz MT,Bell RL.Metabolic and nutritional changes after bariatric surgery.Expert Rev Gastroenterol Hepatol,2010,4(2):217-223.
18 Kashyap SR,Daud S,Kelly KR,et al.Acute effects of gastric bypass versus gastric restrictive surgery on beta-cell function and insulinotropic hormones in severely obese patients with type 2diabetes.Int J Obes(Lond),2010,34(3):462-471.
19 Heindel JJ,Newbold R,Schug TT.Endocrine disruptors and obesity.Nat Rev Endocrinol,2015,11(11):653-661.
20 Clemmons DR.Modifying IGF-I activity:an approach to treat endocrine disorders,atherosclerosis and cancer.Nat Rev Drug Discov,2007,6(10):821-833.
21 Chesnokova V,Zhou CQ,Ben-Shlomo A,et al.Growth hormone is a cellular senescence target in pituitary and nonpituitary cells.Proc Natl Acad Sci U S A,2013,110(35):E3331-E3339.
22 Yakar S,Liu JL,Le Roith D.The growth hormone/insulin-like growth factor-I system:implications for organ growth and development.Pediatr Nephrol,2000,14(7):544-549.
23 Eggert ML,Wallaschofski H,Grotevendt A,et al.Cross-sectional and longitudinal relation of IGF-I and IGF-binding protein 3 with lipid metabolism.Eur J Endocrinol,2014,171(1):9-19.
24 Hu L,Yang G,Hagg D,et al.IGF-I promotes adipogenesis by a lineage bias of endogenous adipose stem/progenitor cells.Stem Cells,2015,33(8):2483-2495.
25 Tang QQ,Lane MD.Adipogenesis:from stem cell to adipocyte.Annu Rev Biochem,2012,81:715-736.
26 Chang HR,Kim HJ,Xu X,et al.Macrophage and adipocyte IGF-I maintain adipose tissue homeostasis during metabolic stresses.Obesity(Silver Spring),2016,24(1):172-183.
27 Fowke J,Matthews C,Yu H,et al.Racial differences in the association between body mass index and serum IGF-I,IGF2,and IGFBP3.Endocr Relat Cancer,2010,17(1):51-60.
28 Hegedus B,Yeh TH,Lee DY,et al.Neurofibromin regulates somatic growth through the hypothalamic-pituitary axis.Hum Mol Genet,2008,17(19):2956-2966.