Study Of Aldosterone In Metabolic Syndrome

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 assessmentsIn 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 assessmentsIn 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 studiesNuclear 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 dietAs 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 dietThere 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 PathwaysTo 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.