醛固酮参与代谢综合征的形成
摘要
目的
由于目前尚无特异有效的防治方法,胰岛素抵抗(insulin resistence,IR)和代谢综合征(metabolic syndrome,MS)的发病率一直居高不下。最近,肾素-血管紧张素系统(Renin-angiotensin system,RAS)引起关注,有证据提示RAS与IR/MS存在重要关联。既往集中于血管紧张素II(angiotensin II,Ang II)的研究,但近年来醛固酮(aldosterone,ALD)备受关注,为系统研究ALD与IR/MS的关系,本研究的目的在于:①建立以高盐高脂诱导的IR/MS动物模型;②以观察IR/MS中胰腺ALD的变化规律,并通过ALD拮抗剂干预观察动物病情和预后的改变;③在此基础上,进一步研究ALD拮抗剂螺内酯干预IR/MS细胞凋亡通路的机制。
研究内容和方法
第一部分: MS动物模型的评价
36只3周龄雄性Wistar大鼠随机分为3组(每组12只):(Ⅰ)正常盐组(0.5% NaCl, NS),(Ⅱ)高盐高脂组(49%fat+4% NaCl,FS), (Ⅲ)高盐高脂+螺内酯组(螺内酯80mg/kg/day, FS+S)。螺内酯溶于饮用水中,在观察12周后,测体重(WB),颈总动脉插管直接测血压,血浆测血脂指标(TC,TG,LDL-C,HDL-C),测血清胰岛素和血糖值,根据稳态胰岛素抵抗指数(HOMA-IR index:fasting insulin Xfasting glucose/22.5)[1])评价是否存在胰岛素抵抗,根据代谢综合征的诊断标准及统计学方法进行组间比较评价模型是否构建成功。
第二部分:在IR/MS模型中胰腺组织的ALD变化
建立以高盐高脂诱导的IR/MS动物模型,在观察12周后分离出胰腺组织,胰腺头部采用酶联免疫吸附试验(ELISA)方法测定ALD浓度的变化及Western blotting检测盐皮质激素受体(mineralocorticoid receptor,MR)的变化。
胰腺中部行病理检查观察胰岛β细胞变化,在此基础上,着重观察是否存在细胞凋亡。采用TUNEL检查凋亡细胞。及比较ALD拮抗剂作用后的效果。
第三部分:ALD拮抗剂干预IR/MS的可能机制
收集NS组、FS组和FS+S组的大鼠胰腺组织,分别提取胰腺尾部组织细胞提取总蛋白,采用Western blotting方法检测胰腺组织中丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)和磷脂酰肌醇3激酶/蛋白激酶B (PI3-K /Akt)信号通路的变化。
研究结果
第一部分:MS动物模型的评价
高盐高脂能够诱导大鼠在12周后出现典型的IR/MS改变:
①高血压:FS组较NS组大鼠血压明显增高(BP:189.43±12.83 vs 153.65±20.53,P<0.01);
②血脂异常:FS组较NS组大鼠血脂明显增高(TC:1.51±0.24 vs. 0.88±0.12, TG:0.97±0.54 vs. 0.31±0.17, LDL-C: 0.90±0.23 vs. 0.53±0.23,三者均P<0.05,但HDL-C:0.83±0.17 vs. 0.89±0.13,P>0.05);
③胰岛素抵抗:FS组大鼠胰岛素抵抗指数较NS组明显升高(HOMA-IR index:2.45±0.36 vs. 1.84±0.41,P<0.01);
④体重增加:FS组较NS组大鼠体重明显增加(BW:326.45±6.84 vs.303.3±9.13,P<0.01)。
该模型指标符合代谢综合征诊断标准,IR/MS模型构建成功。
第二部分:在IR/MS模型中胰腺组织的ALD变化
1、在IR/MS大鼠胰腺组织中ALD的含量较NS组明显增高(5722.49±1156.13 vs. 2337.22±1350.09,P<0.01),提示MS模型存在胰腺ALD浓度升高。Western blotting检测ALD结合的受体MR在FS组表达亦升高,提示ALD浓度升高同时也激活了其受体的表达。
2、免疫组化的结果显示,与NS组比较,IR/MS大鼠胰腺组织的胰岛素表达下降;而ALD拮抗剂螺内酯处理后胰岛素表达则明显增强。
3、TUNEL结果显示,与NS组比较,FS组细胞凋亡明显增加,提示可能是胰岛β细胞减少的原因。而ALD拮抗剂螺内酯处理后细胞凋亡明显减少,提示ALD拮抗剂螺内酯对胰腺组织损伤及凋亡有明显保护作用。
第三部分:ALD拮抗剂干预IR/MS的可能机制
1、高盐高脂饮食诱导的IR/MS大鼠胰腺组织中磷酸化的PKB活性减少,而螺内酯处理后其活性提高。
2、在高盐高脂饮食诱导的IR/MS大鼠胰腺组织中,p38的磷酸化明显增强,而增加螺内酯后MAPK磷酸化得到了抑制。
结论
1、高盐高脂饮食诱导的代谢综合征大鼠的胰腺组织醛固酮含量明显增加。
2、螺内酯(ALD拮抗剂)能够明显改善高盐高脂饮食诱导的代谢综合征,改善胰岛β细胞凋亡。
3、螺内酯干预代谢综合征的可能机制:干预PI3-K和MAPK信号通路,减轻代谢综合征的胰导β细胞凋亡。
OBJECTIVE:
The lack of specific and efficient therapies is a major cause for the high morbility observed in the insulin resistence(IR) and metabolic syndrome(MS). Recently, much research interest was directed towards the role renin-angiotensin system (RAS) played in the pathogenesis of IR/MS. In the last decades,most mainly showed solicitude for the potentially deleterious effects of angiotensin II.Nevertheless,recent observations have suggested that increased aldosterone(ALD) levels are much more association with IR/MS .Therefore, this study was undertaken to clarify the relation of IR/MS and ALD by using a rat fed fat and salt-enriched diet model. We also investigated the impacts of ALD antagonist (namely MR inhibitor spironolactone ) on IR/MS and its’mechanisms. In addition, to further elucidate the mechanisms, of spironolactone prevent pancreatic gland injury (pancreatic isletβ-cell apoptosis).
METHODS:
Part 1. MS rat model assessments
In total, 36 animals were used in this study, 12 animals in each group. The rats were divided into three groups in randomized manner:(?) control with normal salt diet (0.5% NaCl, NS), (П)fat and salt-enriched diet(49%fat+4%NaCl,FS), (Ш)FS+ spironolactone (FS+S 80mg/kg/day).Spironolactone was dissolved in the drinking wa -ter.Treatment with different salt and fat diet and drugs was started at 3 weeks of age and lasted 12 weeks. The right carotid artery was cannulated for direct blood pressure measurement. Mean blood pressure (MBP) was calculated using the systolic and diastolic blood pressure values.This method consistents with our previous study. Analysis of ALD content in the head of pancreatic gland extracts were performed using enzyme linked immuno sorbent assay(ELISA) kit according to the manufactu- rer’s instructions,while INS was measured in plasma sample .Experimental samples were harvested from each group after 12 weeks.
Other remaining plasma samples were obtained for total cholesterol concentra- tion(TC)、triglyceride (TG)、high density lipoprotein cholesterol (HDL-C)、low density lipoprotein cholesterol (LDL-C)、blood glucose(GLU)、plasma sodium (Na+) and potassium(K+) concentrations measurement.
Metabolic syndrome rat model was assessed by measuring BP, IR index , and blood lipid after 12 weeks; experimental samples were harvested from each group. IR index was proformed by the format of Homeostasis Model Assessment of IR(HOMA-IR index), the index derived from fasting insulin and glucose(fasting insulin X fasting glucose/22.5)[1].
Part 2. Pancreatic gland injury assessments
In a separate set of animals (n = 12 per group), the mentioned treatments (NS, FS, and FS+S) were performed.Animals were then sacrificed after 12 weeks using a lethal dose of sodium pentobarbital,and pancreatic glands tissues were harvested.The middle of pancreatic gland were removed and immediately fixed in 10% phosphate-buffered saline-buffered formalin for 24 h, rinsed in phosphate -buffered saline for 1 h, and then stored in 70% ethanol. The middle of pancreatic glands were embedded in paraffin and sectioned at 4μm. For histology, the sections were deparaffinized to water and continued with immunohistochemistry(IH).Sections were incubated with anti-insuline(1:50) antibodies.After washing, slides were incubated with secondary antibodies conjugated with HRP. Apoptotic Cell Death was evaluated by Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL).TUNEL assay was performed using In Situ Cell Death Detection Kit per manufacturer’sinstructions(Roche). TUNEL-positive and -negative cells were counted in 5 random fields from each group. Results are expressed as number of TUNEL-positive cell/total cells x 100%.
Part 3. MR protein and PI3-K、MAPK signaling pathway studies
Nuclear protein extraction—Nuclear protein extraction was performed as described by Chaturvedi et al.[2]. Briefly, anesthetized rats from each group were sacrificed after 12 weeks, and pancreatic gland tissues were collected, grinded, lysed in cell lysis buffer (10 mM HEPES, 10 mM NaCl,1 mM EDTA,0.1 mM EGTA,10μM DTT, 20μg/mL leupeptin, 20μg/mL aprotinin, and 500μg/mL benzamidine). Fifteen minutes later, the samples were centrifuged at 12,000g for 5 min at 4℃. The supernatant is cytosol protein.These protein were aliquoted, and stored at -80℃. All nuclear protein extractions were performed on ice with ice-cold reagents.
Western blot analysis—The tail of pancreatic gland tissues were removed and immediately frozen in liquid nitrogen and stored at -80℃until analysis. Protein levels in pancreatic glands were evaluated by Western blot as previously described [3]. For MR detection, cytosolic extracts were prepared as stated above. After cytosolic protein extraction from the pancreatic gland of rat, protein was quantified using a BCA assay kit, and 20ug was loading in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). After electrophoretic transfer, membranes were incubated with rabbit monoclonal antibody specific to MR (Abcam, USA) in Tris-buffered saline with Tween buffer at 4℃overnight. Membranes were incubated with anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (HRP), followed by exposure to ECL chemiluminescent substrate and digital scanning in Image Station 2000. After MR blotting, reprobed with GAPDH monoclonal antibody (1:1000) and HRP-conjugated secondary antibody. The intensities of the bands were quantified using Quantity One software.
For Akt/Protein Kinase B Kinase Activity and p38MAPK phosphorylation detection, membranes were incubated with a phospho-PKB rabbit polyclonal antibody (Santa Cruz), a rabbit monoclonal anti-phospho-p38MAPK(Santa Cruz),respectiv- ely.The membranes were washed and incubated with secondary mouse anti-rabbit antibody HRP (Santa Cruz), and quantitative densitometry was performed by using a computer-based image analysis system.
RESULTS:
Part 1. Spironolactone attenuated occurrence of MS after FS diet
As shown in Table 1, plasma concentration of TG, TC ,LDL-C except HDL-C were significantly elevated at FS diet group after12 weeks in rat ,compared with NS group(P<0.05). When MS animals were treated with Spironolactone, the levels of TG, TC and LDL-C were markedly reduced.In contrast ,HDL-C was increased,but had no significant difference in three group. In addition,the FS-fed rats displayed impaired glucose tolerance as was expressed by a significant insulin resistence levels of blood glucose and insulin following HOMA-IR index( P < 0. 01 vs.NS group). The above index changes nearly consistent with MS, including BP increasing , Insulin resistance, and hyperlipoidemia were measured in FS diet rat. Body weight of FS group was increased compared with NS group in the study period (P<0.01). Interestingly, plasma sodium and potassium concentrations had also no significant difference.
Part 2. Spironolactone attenuated pancreatic isletβ-cell apoptosis of MS after FS diet
There were no pathological changes in the pancreatic gland tissue from NS animals (Fig. 3A),while high-power sections (X400) demonstrated the reduction of insuline expression changes after FS administration(Fig. 3B). Spironolactone treatm- ent(Fig. 3C) improved the expression of insulince compared with untreated FS group rat.Correspondingly, TUNEL staining further comfirmed the cause of reduction of insuline expression. As shown in Figure 3E,there was 2.5-fold increase of apoptotic cells in FS group sections compared with NS group (Fig.3D) (P<0.01 vs.NS group),which was substantially improved after spironolactone treatment(Fig.3F) (P<0.01vs.FS group). Thus, early treatment of rat with spironolactone showed a protective effect through observed pathological changes on FS-induced IR/MS.
Part 3. Spironolactone inhibited the activation of pancreatic gland MR and phosphorylation of p38MAPK、but recused activity of PI-3K Signaling Pathways
To determine the effect of spironolactone on MR activation, nuclear extracts from three group the tail of pancreatic gland tissues were assayed for non-selective MR binding activity by using Western blot analyse as previously described. Figure 4 shows that FS-induced activation of MR was inhibited significantly by spironolactone treatment (P<0.01 vs. FS).
To assess the effect of MAPK、PI3-K signaling pathways, Western blot analysis with antibodies specific to the phosphorylated forms of p38 MAPK(p-p38 MAP- K)/ativity of PI-3K (phosphorylation-PKB) were conducted.Results showed that spironolactone treatment after FS-diet inhibited the phosphorylation of p38 MAPK (Fig. 5A, upper),but recused activity of PI3-K(Fig. 6). There was nochange seen in total cellular levels of p38 MAPK(Fig. 5B, middle), showing that spironolactone did not have nonspecific effects on protein levels. Phosphorylation–PKB can stand for ativity of PI-3K. Western blot analysis showed that MS rat pancreatic gland tissues exhibited a decreasing of phosphorylation–PKB ativity (Fig.6) ,wheres in FS spironolactone-treated can rescue its activation.Collectively, these results indicate that spironolactone interferes with the cell apoptosis through a mechanism dependent, at least in part, on their ability to suppress MR expression and signal transduction through p38 MAPK,but recuse PI3-K pathways.
CONCLUSIONS :
1. IR/MS in rat models is manifested as an increased secretion of ALD.
2.The ALD inhibitor( namely MR inhibitor), spironolactone, can prevent the manifestation in IR/MS after FS diet, and significantly improved pancreatic isletβ-cell apoptosis.
3.The possible mechanisms of the prevention pancreatic isletβ-cell apoptosis by spironolactone are: the intervention of PI3-K and MAPK signaling pathway that reduced the IR/MS pancreatic isletβ-cell apoptosis.
由于目前尚无特异有效的防治方法,胰岛素抵抗(insulin resistence,IR)和代谢综合征(metabolic syndrome,MS)的发病率一直居高不下。最近,肾素-血管紧张素系统(Renin-angiotensin system,RAS)引起关注,有证据提示RAS与IR/MS存在重要关联。既往集中于血管紧张素II(angiotensin II,Ang II)的研究,但近年来醛固酮(aldosterone,ALD)备受关注,为系统研究ALD与IR/MS的关系,本研究的目的在于:①建立以高盐高脂诱导的IR/MS动物模型;②以观察IR/MS中胰腺ALD的变化规律,并通过ALD拮抗剂干预观察动物病情和预后的改变;③在此基础上,进一步研究ALD拮抗剂螺内酯干预IR/MS细胞凋亡通路的机制。
研究内容和方法
第一部分: MS动物模型的评价
36只3周龄雄性Wistar大鼠随机分为3组(每组12只):(Ⅰ)正常盐组(0.5% NaCl, NS),(Ⅱ)高盐高脂组(49%fat+4% NaCl,FS), (Ⅲ)高盐高脂+螺内酯组(螺内酯80mg/kg/day, FS+S)。螺内酯溶于饮用水中,在观察12周后,测体重(WB),颈总动脉插管直接测血压,血浆测血脂指标(TC,TG,LDL-C,HDL-C),测血清胰岛素和血糖值,根据稳态胰岛素抵抗指数(HOMA-IR index:fasting insulin Xfasting glucose/22.5)[1])评价是否存在胰岛素抵抗,根据代谢综合征的诊断标准及统计学方法进行组间比较评价模型是否构建成功。
第二部分:在IR/MS模型中胰腺组织的ALD变化
建立以高盐高脂诱导的IR/MS动物模型,在观察12周后分离出胰腺组织,胰腺头部采用酶联免疫吸附试验(ELISA)方法测定ALD浓度的变化及Western blotting检测盐皮质激素受体(mineralocorticoid receptor,MR)的变化。
胰腺中部行病理检查观察胰岛β细胞变化,在此基础上,着重观察是否存在细胞凋亡。采用TUNEL检查凋亡细胞。及比较ALD拮抗剂作用后的效果。
第三部分:ALD拮抗剂干预IR/MS的可能机制
收集NS组、FS组和FS+S组的大鼠胰腺组织,分别提取胰腺尾部组织细胞提取总蛋白,采用Western blotting方法检测胰腺组织中丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)和磷脂酰肌醇3激酶/蛋白激酶B (PI3-K /Akt)信号通路的变化。
研究结果
第一部分:MS动物模型的评价
高盐高脂能够诱导大鼠在12周后出现典型的IR/MS改变:
①高血压:FS组较NS组大鼠血压明显增高(BP:189.43±12.83 vs 153.65±20.53,P<0.01);
②血脂异常:FS组较NS组大鼠血脂明显增高(TC:1.51±0.24 vs. 0.88±0.12, TG:0.97±0.54 vs. 0.31±0.17, LDL-C: 0.90±0.23 vs. 0.53±0.23,三者均P<0.05,但HDL-C:0.83±0.17 vs. 0.89±0.13,P>0.05);
③胰岛素抵抗:FS组大鼠胰岛素抵抗指数较NS组明显升高(HOMA-IR index:2.45±0.36 vs. 1.84±0.41,P<0.01);
④体重增加:FS组较NS组大鼠体重明显增加(BW:326.45±6.84 vs.303.3±9.13,P<0.01)。
该模型指标符合代谢综合征诊断标准,IR/MS模型构建成功。
第二部分:在IR/MS模型中胰腺组织的ALD变化
1、在IR/MS大鼠胰腺组织中ALD的含量较NS组明显增高(5722.49±1156.13 vs. 2337.22±1350.09,P<0.01),提示MS模型存在胰腺ALD浓度升高。Western blotting检测ALD结合的受体MR在FS组表达亦升高,提示ALD浓度升高同时也激活了其受体的表达。
2、免疫组化的结果显示,与NS组比较,IR/MS大鼠胰腺组织的胰岛素表达下降;而ALD拮抗剂螺内酯处理后胰岛素表达则明显增强。
3、TUNEL结果显示,与NS组比较,FS组细胞凋亡明显增加,提示可能是胰岛β细胞减少的原因。而ALD拮抗剂螺内酯处理后细胞凋亡明显减少,提示ALD拮抗剂螺内酯对胰腺组织损伤及凋亡有明显保护作用。
第三部分:ALD拮抗剂干预IR/MS的可能机制
1、高盐高脂饮食诱导的IR/MS大鼠胰腺组织中磷酸化的PKB活性减少,而螺内酯处理后其活性提高。
2、在高盐高脂饮食诱导的IR/MS大鼠胰腺组织中,p38的磷酸化明显增强,而增加螺内酯后MAPK磷酸化得到了抑制。
结论
1、高盐高脂饮食诱导的代谢综合征大鼠的胰腺组织醛固酮含量明显增加。
2、螺内酯(ALD拮抗剂)能够明显改善高盐高脂饮食诱导的代谢综合征,改善胰岛β细胞凋亡。
3、螺内酯干预代谢综合征的可能机制:干预PI3-K和MAPK信号通路,减轻代谢综合征的胰导β细胞凋亡。
OBJECTIVE:
The lack of specific and efficient therapies is a major cause for the high morbility observed in the insulin resistence(IR) and metabolic syndrome(MS). Recently, much research interest was directed towards the role renin-angiotensin system (RAS) played in the pathogenesis of IR/MS. In the last decades,most mainly showed solicitude for the potentially deleterious effects of angiotensin II.Nevertheless,recent observations have suggested that increased aldosterone(ALD) levels are much more association with IR/MS .Therefore, this study was undertaken to clarify the relation of IR/MS and ALD by using a rat fed fat and salt-enriched diet model. We also investigated the impacts of ALD antagonist (namely MR inhibitor spironolactone ) on IR/MS and its’mechanisms. In addition, to further elucidate the mechanisms, of spironolactone prevent pancreatic gland injury (pancreatic isletβ-cell apoptosis).
METHODS:
Part 1. MS rat model assessments
In total, 36 animals were used in this study, 12 animals in each group. The rats were divided into three groups in randomized manner:(?) control with normal salt diet (0.5% NaCl, NS), (П)fat and salt-enriched diet(49%fat+4%NaCl,FS), (Ш)FS+ spironolactone (FS+S 80mg/kg/day).Spironolactone was dissolved in the drinking wa -ter.Treatment with different salt and fat diet and drugs was started at 3 weeks of age and lasted 12 weeks. The right carotid artery was cannulated for direct blood pressure measurement. Mean blood pressure (MBP) was calculated using the systolic and diastolic blood pressure values.This method consistents with our previous study. Analysis of ALD content in the head of pancreatic gland extracts were performed using enzyme linked immuno sorbent assay(ELISA) kit according to the manufactu- rer’s instructions,while INS was measured in plasma sample .Experimental samples were harvested from each group after 12 weeks.
Other remaining plasma samples were obtained for total cholesterol concentra- tion(TC)、triglyceride (TG)、high density lipoprotein cholesterol (HDL-C)、low density lipoprotein cholesterol (LDL-C)、blood glucose(GLU)、plasma sodium (Na+) and potassium(K+) concentrations measurement.
Metabolic syndrome rat model was assessed by measuring BP, IR index , and blood lipid after 12 weeks; experimental samples were harvested from each group. IR index was proformed by the format of Homeostasis Model Assessment of IR(HOMA-IR index), the index derived from fasting insulin and glucose(fasting insulin X fasting glucose/22.5)[1].
Part 2. Pancreatic gland injury assessments
In a separate set of animals (n = 12 per group), the mentioned treatments (NS, FS, and FS+S) were performed.Animals were then sacrificed after 12 weeks using a lethal dose of sodium pentobarbital,and pancreatic glands tissues were harvested.The middle of pancreatic gland were removed and immediately fixed in 10% phosphate-buffered saline-buffered formalin for 24 h, rinsed in phosphate -buffered saline for 1 h, and then stored in 70% ethanol. The middle of pancreatic glands were embedded in paraffin and sectioned at 4μm. For histology, the sections were deparaffinized to water and continued with immunohistochemistry(IH).Sections were incubated with anti-insuline(1:50) antibodies.After washing, slides were incubated with secondary antibodies conjugated with HRP. Apoptotic Cell Death was evaluated by Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL).TUNEL assay was performed using In Situ Cell Death Detection Kit per manufacturer’sinstructions(Roche). TUNEL-positive and -negative cells were counted in 5 random fields from each group. Results are expressed as number of TUNEL-positive cell/total cells x 100%.
Part 3. MR protein and PI3-K、MAPK signaling pathway studies
Nuclear protein extraction—Nuclear protein extraction was performed as described by Chaturvedi et al.[2]. Briefly, anesthetized rats from each group were sacrificed after 12 weeks, and pancreatic gland tissues were collected, grinded, lysed in cell lysis buffer (10 mM HEPES, 10 mM NaCl,1 mM EDTA,0.1 mM EGTA,10μM DTT, 20μg/mL leupeptin, 20μg/mL aprotinin, and 500μg/mL benzamidine). Fifteen minutes later, the samples were centrifuged at 12,000g for 5 min at 4℃. The supernatant is cytosol protein.These protein were aliquoted, and stored at -80℃. All nuclear protein extractions were performed on ice with ice-cold reagents.
Western blot analysis—The tail of pancreatic gland tissues were removed and immediately frozen in liquid nitrogen and stored at -80℃until analysis. Protein levels in pancreatic glands were evaluated by Western blot as previously described [3]. For MR detection, cytosolic extracts were prepared as stated above. After cytosolic protein extraction from the pancreatic gland of rat, protein was quantified using a BCA assay kit, and 20ug was loading in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). After electrophoretic transfer, membranes were incubated with rabbit monoclonal antibody specific to MR (Abcam, USA) in Tris-buffered saline with Tween buffer at 4℃overnight. Membranes were incubated with anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (HRP), followed by exposure to ECL chemiluminescent substrate and digital scanning in Image Station 2000. After MR blotting, reprobed with GAPDH monoclonal antibody (1:1000) and HRP-conjugated secondary antibody. The intensities of the bands were quantified using Quantity One software.
For Akt/Protein Kinase B Kinase Activity and p38MAPK phosphorylation detection, membranes were incubated with a phospho-PKB rabbit polyclonal antibody (Santa Cruz), a rabbit monoclonal anti-phospho-p38MAPK(Santa Cruz),respectiv- ely.The membranes were washed and incubated with secondary mouse anti-rabbit antibody HRP (Santa Cruz), and quantitative densitometry was performed by using a computer-based image analysis system.
RESULTS:
Part 1. Spironolactone attenuated occurrence of MS after FS diet
As shown in Table 1, plasma concentration of TG, TC ,LDL-C except HDL-C were significantly elevated at FS diet group after12 weeks in rat ,compared with NS group(P<0.05). When MS animals were treated with Spironolactone, the levels of TG, TC and LDL-C were markedly reduced.In contrast ,HDL-C was increased,but had no significant difference in three group. In addition,the FS-fed rats displayed impaired glucose tolerance as was expressed by a significant insulin resistence levels of blood glucose and insulin following HOMA-IR index( P < 0. 01 vs.NS group). The above index changes nearly consistent with MS, including BP increasing , Insulin resistance, and hyperlipoidemia were measured in FS diet rat. Body weight of FS group was increased compared with NS group in the study period (P<0.01). Interestingly, plasma sodium and potassium concentrations had also no significant difference.
Part 2. Spironolactone attenuated pancreatic isletβ-cell apoptosis of MS after FS diet
There were no pathological changes in the pancreatic gland tissue from NS animals (Fig. 3A),while high-power sections (X400) demonstrated the reduction of insuline expression changes after FS administration(Fig. 3B). Spironolactone treatm- ent(Fig. 3C) improved the expression of insulince compared with untreated FS group rat.Correspondingly, TUNEL staining further comfirmed the cause of reduction of insuline expression. As shown in Figure 3E,there was 2.5-fold increase of apoptotic cells in FS group sections compared with NS group (Fig.3D) (P<0.01 vs.NS group),which was substantially improved after spironolactone treatment(Fig.3F) (P<0.01vs.FS group). Thus, early treatment of rat with spironolactone showed a protective effect through observed pathological changes on FS-induced IR/MS.
Part 3. Spironolactone inhibited the activation of pancreatic gland MR and phosphorylation of p38MAPK、but recused activity of PI-3K Signaling Pathways
To determine the effect of spironolactone on MR activation, nuclear extracts from three group the tail of pancreatic gland tissues were assayed for non-selective MR binding activity by using Western blot analyse as previously described. Figure 4 shows that FS-induced activation of MR was inhibited significantly by spironolactone treatment (P<0.01 vs. FS).
To assess the effect of MAPK、PI3-K signaling pathways, Western blot analysis with antibodies specific to the phosphorylated forms of p38 MAPK(p-p38 MAP- K)/ativity of PI-3K (phosphorylation-PKB) were conducted.Results showed that spironolactone treatment after FS-diet inhibited the phosphorylation of p38 MAPK (Fig. 5A, upper),but recused activity of PI3-K(Fig. 6). There was nochange seen in total cellular levels of p38 MAPK(Fig. 5B, middle), showing that spironolactone did not have nonspecific effects on protein levels. Phosphorylation–PKB can stand for ativity of PI-3K. Western blot analysis showed that MS rat pancreatic gland tissues exhibited a decreasing of phosphorylation–PKB ativity (Fig.6) ,wheres in FS spironolactone-treated can rescue its activation.Collectively, these results indicate that spironolactone interferes with the cell apoptosis through a mechanism dependent, at least in part, on their ability to suppress MR expression and signal transduction through p38 MAPK,but recuse PI3-K pathways.
CONCLUSIONS :
1. IR/MS in rat models is manifested as an increased secretion of ALD.
2.The ALD inhibitor( namely MR inhibitor), spironolactone, can prevent the manifestation in IR/MS after FS diet, and significantly improved pancreatic isletβ-cell apoptosis.
3.The possible mechanisms of the prevention pancreatic isletβ-cell apoptosis by spironolactone are: the intervention of PI3-K and MAPK signaling pathway that reduced the IR/MS pancreatic isletβ-cell apoptosis.
引文
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2.Chaturvedi MM, Mukhopadhyay A, Aggarwal BB: Assay for redox-sen- sitive transcription factor. Methods Enzymol. 2000; 319:585-602.
3. Ding G, Franki N, Kapasi AA, Reddy K, Gibbons N, Singhal PC. Tubular cell senescence and expression of TGF-beta1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats. Exp Mol Pathol .2001;70:43-53.
4. Ford ES, Giles WH, Mokdad AH. Increasing prevalence of the metabolic syndrome among u.s. Adults. Diabetes Care. 2004 ;27:2444-9.
5. Dekker JM, Girman C, Rhodes T, Nijpels G, Stehouwer CDA, Bouter LM, Heine RJ. Metabolic syndrome and 10-year cardiovascular disease risk in the Hoorn Study. Circulation. 2005;112:666-73.
6. McNeill AM, Rosamond WD, Girman CJ, Golden SH, Schmidt MI, East HE, Ballantyne CM, Heiss G. The metabolic syndrome and 11-year risk of incident cardiovascular disease in the atherosclerosis risk in communities study. Diabetes Care. 2005;28:385-390.
7.Vasan RS, Evans JC, Larson MG, Wilson PWF, Meigs JB, Rifai N,Benja- min EJ, Levy D. Serum aldosterone and the incidence of hypertension in nonhypertensive persons. N Engl J Med. 2004;351:33- 41.
8. Freel EM, Connell JM. Mechanisms of hypertension: the expanding role of aldosterone. J Am Soc Nephrol. 2004;15:1993-2001.
9. Rocchini AP, Katch VL, Grekin R, Moorehead C, Anderson J. Role foraldosterone in blood pressure regulation of obese adolescents. Am J Cardiol. 1986;57:613-18.
10.Gaddam KK, Pimenta E, Husain S. Aldosterone and cardiovascular dise- ase. Curr Probl Cardiol. 2009 ,34:51-84.
11.Wehling M. Effects of aldosterone and mineralocorticoid receptor blockade on intracellular electrolytes. Heart Fail Rev. 2005;10:39-46.
12.Lavoie JL, Sigmund CD. Minireview: overview of the renin-angiotensin system-an endocrine and paracrine system. Endocrinology, 2003;144:2179-2183.
13.Leung PS, de Gasparo M. Involvement of the pancreatic renin-angiotens -in system in insulin resistance and the metabolic syndrome. J Cardiometab Syndr. 2006;1:197-203.
14.Leung PS, de Gasparo M. Involvement of the pancreatic renin- angio tensin system in insulin resistance and the metabolic syndrome. J Cardiometab Syndr. 2006;1(3):197–203.
15.Goodfriend TL, Egan BM, Kelley DE. Plasma aldosterone, plasma lipoproteins, obesity and insulin resistance in humans. Prostaglandins Leukot Essent Fatty Acids. 1999;60:401-5.
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2.Chaturvedi MM, Mukhopadhyay A, Aggarwal BB: Assay for redox-sen- sitive transcription factor. Methods Enzymol. 2000; 319:585-602.
3. Ding G, Franki N, Kapasi AA, Reddy K, Gibbons N, Singhal PC. Tubular cell senescence and expression of TGF-beta1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats. Exp Mol Pathol .2001;70:43-53.
4. Ford ES, Giles WH, Mokdad AH. Increasing prevalence of the metabolic syndrome among u.s. Adults. Diabetes Care. 2004 ;27:2444-9.
5. Dekker JM, Girman C, Rhodes T, Nijpels G, Stehouwer CDA, Bouter LM, Heine RJ. Metabolic syndrome and 10-year cardiovascular disease risk in the Hoorn Study. Circulation. 2005;112:666-73.
6. McNeill AM, Rosamond WD, Girman CJ, Golden SH, Schmidt MI, East HE, Ballantyne CM, Heiss G. The metabolic syndrome and 11-year risk of incident cardiovascular disease in the atherosclerosis risk in communities study. Diabetes Care. 2005;28:385-390.
7.Vasan RS, Evans JC, Larson MG, Wilson PWF, Meigs JB, Rifai N,Benja- min EJ, Levy D. Serum aldosterone and the incidence of hypertension in nonhypertensive persons. N Engl J Med. 2004;351:33- 41.
8. Freel EM, Connell JM. Mechanisms of hypertension: the expanding role of aldosterone. J Am Soc Nephrol. 2004;15:1993-2001.
9. Rocchini AP, Katch VL, Grekin R, Moorehead C, Anderson J. Role foraldosterone in blood pressure regulation of obese adolescents. Am J Cardiol. 1986;57:613-18.
10.Gaddam KK, Pimenta E, Husain S. Aldosterone and cardiovascular dise- ase. Curr Probl Cardiol. 2009 ,34:51-84.
11.Wehling M. Effects of aldosterone and mineralocorticoid receptor blockade on intracellular electrolytes. Heart Fail Rev. 2005;10:39-46.
12.Lavoie JL, Sigmund CD. Minireview: overview of the renin-angiotensin system-an endocrine and paracrine system. Endocrinology, 2003;144:2179-2183.
13.Leung PS, de Gasparo M. Involvement of the pancreatic renin-angiotens -in system in insulin resistance and the metabolic syndrome. J Cardiometab Syndr. 2006;1:197-203.
14.Leung PS, de Gasparo M. Involvement of the pancreatic renin- angio tensin system in insulin resistance and the metabolic syndrome. J Cardiometab Syndr. 2006;1(3):197–203.
15.Goodfriend TL, Egan BM, Kelley DE. Plasma aldosterone, plasma lipoproteins, obesity and insulin resistance in humans. Prostaglandins Leukot Essent Fatty Acids. 1999;60:401-5.
16. Goodfriend TL, Kelley DE, Goodpaster BH, Winters SJ. Visceral obesity and insulin resistance are associated with plasma aldosterone levelss in women. Obes Res. 1999;7:355–362.
17.Hayden MR, Sowers JR.Pancreatic renin-angiotensin-aldosterone system in the cardiometabolic syndrome and type 2 diabetes mellitus.J Cardiometab Syndr.2008;3:129-31.
18.Bentley-Lewis R, Adler G, Perlstein T, Seely EW, Hopkins PN, Williams GH, Garg R. Body mass index predicts aldosterone production in normotensive adults on a high-salt diet. J Clin Endocrinol Metab. 2007;92:4472-75.
19.Shimamoto K, Shiiki M, Ise T, Miyazaki Y, Higashiura K, FukuokaM,Hirata A, Masuda A, Nakagawa M, Iimura O. Does insulin resistance participate in an impaired glucose tolerance in primary aldosteronism.J Hum Hypertens. 1994;8:755–759.
20. Petrasek D, Jensen G, Tuck M, Stern N. In vitro effects of insulin on aldosterone production in rat zona glomerulosa cells. Life Sci. 1992;50:1781-87.
21.Wang CC, Goalstone ML, Draznin B.Molecular mechanisms of insulin resistance that impact cardiovascular biology. Diabetes.2004;53:2735-2740.
22.Callera GE, Touyz RM, Tostes RC, Yogi A, He Y, Malkinson S, Schiffrin EL. Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src. Hypertension. 2005 ;45:773-9.
23.Chen C, Liang W, Jia J, van Goor H, Singhal PC, Ding G. Aldosterone induces apoptosis in rat podocytes: role of PI3-K/Akt and p38MAPK signaling pathways.Nephron Exp Nephrol. 2009;113:e26-34.
24.Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, Palensky J, Wittes J. The Randomized Aldactone Evaluation Study Investigators.The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med. 1999;341:709 -717.
25.Pitt B, White H, Nicolau J, Martinez F, Gheorghiade M, Aschermann M, van Veldhuisen DJ, Zannad F, Krum H, Mukherjee R, Vincent J.Eplerenone reduces mortality 30 days after randomization following acute myocardial infarction in patients with left ventricular systolic dysfunction and heart failure. J Am Coll Cardiol. 2005;46:425-431.
26.Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, Bittman R, Hurley S, Kleiman J, Gatlin M. The Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study Investigators.Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med. 2003;348:1309-1321.
27.Zeng ZH(曾昭华), Luo BH, Gao YJ, Su CJ, He CC, Yi JJ, Li N, Lee RM.,Control of vascular changes by renin-angiotensin-aldosterone system in salt-sensitive hypertension. Eur J Pharmacol. 2004;503:129-33.
28.Zeng ZH(曾昭华), Luo BH, Gao YJ, Su CJ, He CC, Li N, Lee RM. Arterial structural changes in hypertensive rats induced by capsaicin and salt loading. Clin Exp Pharmacol Physiol. 2004;31:502-5.
29.曾昭华; Robert; K.W.Lee;罗碧辉; Gao YJ;何兆初;苏诚坚.一种新的高盐致高血压动物模型及其血管重构改变[J].中国临床药理学与治疗学. 2005.01.26; 10: 24-28.
30. Lastra-Lastra G, Sowers JR, Restrepo-Erazo K, Manrique-Acevedo C, Lastra-González G. Role of aldosterone and angiotensin II in insulin resistance: an update. Clin Endocrinol (Oxf). 2009;71:1-6.
31. Gaddam KK, Pimenta E, Husain S. Aldosterone and cardiovascular disease. Curr Probl Cardiol. 2009 ,34:51-84.
32.Fujita T. Aldosterone and CKD in metabolic syndrome.Curr Hypertens Rep.2008,10 :421-3.
33. Toshiro Fujita. Aldosterone in salt-sensitive hypertension and metabolic syndrome .J Mol Med (2008) 86:729-34.
34. Bochud M, Nussberger J, Bovet P, Maillard MR, Elston RC,Paccaud F, Shamlaye C, Burnier M. Plasma aldosterone is independently associated with the metabolic syndrome. Hypertension, 2006,48:239-45
35.Ogihara T, Asano T, Fujita T. Contribution of salt intake to insulin resistance associated with hypertension. Life Sci. 2003 ,73:509-23.
36.Fujita T. Mineralocorticoid Receptors, Salt-Sensitive Hypertension, and Metabolic Syndrome. Hypertension. Hypertension. 2010 ;55:813-8.
37.Ouvrard-Pascaud A, Sainte-Marie Y, Bénitah JP, Perrier R, Soukaseum C, Cat AN, Royer A, Le Quang K, Charpentier F, Demolombe S, Mechta-Grigoriou F, Beggah AT, Maison-Blanche P, Oblin ME, Delcayre C, Fishman GI, Farman N, Escoubet B, Jaisser F. Conditional mineralocorticoid receptor expression inthe heart leads to life-threatening arrhythmias. Circulation. 2005; 111: 30 25-33.
38.Amaravadi R, Thompson CB. The survival kinases Akt and Pim as potential pharmacological targets. J Clin Invest. 2005;115:2618-24.
39. Saldeen J, Lee JC, Welsh N. Role of p38 mitogen-activated protein kinase (p38 MAPK) in cytokine-induced rat islet cell apoptosis. Biochem Pharmacol. 2001 ;61:1561-9.
40. Khoo S, Cobb MH.Activation of mitogen-activating protein kinase by glucose is not required for insulin secretion.Proc Natl Acad Sci USA.1997;94: 559 9-604.
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