Effects And Mechanisms Of Compound RapA Therapy On Type 2 Diabetes Goto-kakizaki Rats

Numerous studies have shown that serine/threonine kinase kinase Akt, also known as protein kinase B (PKB), is an important upstream signal molecules associated with growth factors, cytokines, and other cellular stimulations, which leads to downstream signaling activation in vivo. Abnormal activation or inhibition of activation of Akt has participated in a number of important pathophysiological processes of complex diseases, including atherosclerosis, type 2 diabetes and cancer. Akt family consists of three mammalian isoforms:Akt1, Akt2, Akt3, the encoding genes of isoforms in the three different gene loci, which have similar conserved domain in functional structure. The coding regions of the Akt family genes were comparised, the similarity to 85%. In principle, Akt, about 480 amino acid residues from the NH2 to the COOH end of the order of the end of PH domain, catalytic domain and regulatory domain are involved in regulating Akt activation. Aktl, recruited to the plasma membrane followed by the PH domain binding to the cell membrane phosphatidylinositol triphosphate (PIP3), has effect on phosphorylation of the catalytic domain Thr308 and Ser473. Akt in mammals are expressed in various tissues; Aktl in most mammalian tissues; Akt2 isforms is mainly high in the insulin-sensitive organs such as liver, skeletal muscle, adipose tissue and heart et al; Akt3 isoform is mainly rich in the male reproductive system and brain.Diabetes (Diabetes mellitus, DM) is a kind of chronic progressive diseases, including type 1 diabetes and type 2 diabetes, gestational diabetes and other genetic diabetes. Type 2 diabetes which accounts for 90% of the diabetic patients, the incidence numbers are increasing every year. Diabetes as a disease class is the result of its final outcome in all body organs involved, leading to organ dysfunction caused by tissue complications, severe cases can cause death. Cardiac damage or diabetic cardiomyopathy is a kind of damage to cardiac dysfunction, but not including coronary artery disease and hypertension for the combined features of the disease. In general, hyperglycemia, hyperlipidemia and hyperinsulinemia in diabetic myocardial damage are main important rist factors in the pathogenesis of diabetic cardiomyopathy, and they also induced a series of adverse reactions, eventually lead to cardiac fibrosis and collagen deposition. Focus on improving these risk factors, from the fifties of last century, that a large number of experimental and therapeutic drugs appear, such as guanidine hydrochloride acid, acarbose, recombinant human insulin (embedded trace the release of the body) and the AICAR, but Thees drugs themselves are to control the positioning of certain risk factors, target organ damage has occurred and complications, the treatment effect is not very optimistic. Akt kinase in the heart of the level of metabolism and the role of survival has been increasingly clear. However, in specific ways to improve diabetes ultimately lead to myocardial damage or the occurrence of diabetic cardiomyopathy, the incidence of death or the development of heart failure, Akt agonist compounds RapA therapeutic effect and mechanism of the official concerned in this study. This compound is the first found from the strains and confirmed that there are no reports abroad.Through this study, we assume that at the onset of type 2 diabetes of stable period, tissues and organs have not yet obvious damage and complications. By use of long-term oral compound RapA in the Goto-kakizaki rats, we would study the compound effects on insulin resistance and heart under the influence for the heart, such as cardiac fibrosis, myocardial mitochondrial function and apoptosis. In vitro studies, to determine whether compound RapA could activate Akt kinase directly or through the activation of upstream molecules of Akt kinase by small interfering RNA silencing, we use that free fatty acids could lead to myocardial oxidative stress, loss of mitochondrial function and apoptosis, which was able or unable to protect against damages to cardiac cells.MethodsMaterialsRapA was obtained from Doctor Zhang (North China pharmaceutical Group Corporation, China). A siRNA pool kit for p85alpha of PI3Ks'subunit was purchased from Thermo fisher scientific (Shanghai, China).Human recombinant Insulin was received from Wanbang Pharmaceutical Corporation (Xuezhou, China).Plasma glucose assay kit; total cholesterol quantitation kit, free fatty acids quantitation kit and, kit were obtained from Biovision corporate (Mountain View, California). Luciferase plasmid of p-ARE (antioxidant response element)-luc was provided from Beyotime Institute of Biotechnology (Shanghai, China).Animals, experimental design and drug treatmentAdult male diabetic Goto-Kakizaki rats (Slack, China)(n=40) and age-matched non-diabetic Wistar rats(Slack, China) were used for the study. Animals'houses were maintained in temperature (22±2℃) and light-controlled (12h light/dark cycle) environment; standard rodent high fat diet foods, chow foods and fresh water were provided to age-matched diabetic Goto-kakizaki and non-diabetic Wistar rats respectively.At 20-22 weeks of age, body weight and other physiological indicators (fast insulin, plasma glucose, triglyceride and cholesterol) of diabetic Goto-Kakizaki rats were markedly stable, which were compared with earlier indicators, relative to lean Wistar controls as previously reported.To determine whether RapA agonist would protect against cardiac injuries, appropriate group (n=10) of rats were oral treated for 16 weeks, from age 24 to 40 weeks, with RapA 2.5mg/kg/day and 5.0mg/kg/day in 0.9%NaCl buffer respectively.The investigation conformed to the Guide for Care and Use of laboratory Animals published by the US National Institutes of Health (NIH Publication NO.85-23 revised 1996).All experimental protocols were approved by Tongji medical college committee for Animals care and use.EchocardiographyEchocardiographic analysis was performed using a commercially available echocardiograph (VIVID 7, General Electric) equipped with a 15-MHz linear array ultrasound transducer. Parameters needed for the calculation of cardiac function and dimensions were measured from five systole-diastole cycle at least. According to Teichholz method, short axis view of the left ventricle at the level of the papillary muscles was obtained by the linear transducer as previously described above. Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were measured from LV M-mode tracing (with a sweep speed of 50mm/s) at the papillary muscle level; LV fractional shortening (FS) and ejection fraction (EF), measures of LV systolic function, were calculated from LV M-mode by the following equations:FS%=[(LVEDD-LVESD)/LVEDD]×100EF%=[(LVEDV)-(LVESV)/LVEDV]×100.Measurement of cardiovascular variablesFor measurement of heart beat ratio and left ventricular pressure, a 2.0-French ultra miniature conductance catheter (MPVS400; Millar Instruments, USA) was introduced through the right jugular vein into the left ventricle as described previously.Pathology and Histological analysisThe whole heart was fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin. Paraffin-embedded samples were sliced to 6um-thick sections, which were stained with Hematoxylin-eosin staining and Masson's tri-Chrome staining for detecting collagen. To determine whether RapA could take effect on the size of cardiomyoctyes cross-sectional area and the degree of myocardial fibrosis,10 fields were randomly chosen respectively, and the size of cardiomyocytes was measured by use of Image J 3.0 software, and the cardiac collagen fraction was calculated as the ratio of Masson's trichrome fibrosis area to total myocardium area with the software of Image Pro-plus 4.0.Biochemical measurements and OGTTMethods and measurements for plasma glucose, total cholesterol, triglyceride and free fatty acids were performed and calculated as appropriate kits'manuals and instructions respectively. An oral glucose test (OGTT,5g/kg body weight) was performed after all rats fasted 12 hours at least. Blood was drawn from the tail vein at 0,30,60,90 and 120 min after the oral glucose treatment. Whole blood glucose concentrations were measured by the Johnsons'Lifescan Ultra 2 meter instrument (Shanghai, China).Fast insulin assayPlasma insulin levels were determined by Rat/mouse Insulin ELISA kit (St.Charles, Millipore).Insulin signaling in vivoMale diabetic Goto-kakizakti rats and age-matched non-diabetic Wistar rats were fed with the above mentioned HFD and chow foods for 36 weeks by the oral treatment of RapA (12wk) and saline, respectively. Body weights of all rats were measured before the day for injection of human recombinant insulin. Rats were fasted 12 hours at least and intraper-i toneally injected with insulin (10U/kg body weight) or equal volume of 0.9% NaC1. After 30 min, rat tissues were immediately excised, frozen in liquid nitrogen for 1-2 hours at least, and kept at-80℃until homogenization and other analysis.Western blotsWestern blotting was performed as previously described.Detection of NADPH oxidase activity, MDA assay, Superoxide Dismutase (SOD) ActivityNADPH oxidase activity, MDA and superoxide dismutase (SOD) activity were measured by the colorimeric assay method with commercial kit (Biovision Inc., USA) according to the manufacturer's instructions. Caspase activity assayCaspase 3 and caspase 9 activity assay were performed as above mentioned.Evalution of cAMP in vivo and invitroCyclic AMP in vivo and invitro was evaluated using cAMP assay kit (Cellsignaling, MA, USA) according to the assay protocol.Primary cardiac cell cultureA method of isolation of rat neonatal cardiomyocytes was as previously reported. The isolated cardiomyocytes were grown and maintained as recommend-ed at 37℃in 5% CO2 in DMEM modified to contain 4Mm L-glutamine,4.5g/L glucose, 1mM sodium pyruvate, 2.0g/L sodium bicarbonate,3.0g/L HEPES, added 10% fetal bovine serum (FBS, Gibco, USA) and appropriate antibiotics.Determination of IC50 and AKT kinasc activity in primary cardiac cellsTo determine the cytotoxic activity and apoptosis dose of RapA, cell proliferation reagent WST-1 (Roche, USA) was used for the measurement of IC50% with the treatment of com-pound RapA in primary cardiac cells. AKT kinase activity in vitro was measured by AKT activity kit (Cellsignaling, MA, USA) as the instruction of the manufacture. Calculation of IC50 in vitro was by use of Excel 2007 (Microsoftware, USA).Measurement of ROS in vitro and apoptosis assayPalmitic(C 16:0) was provided from Sigma Aldrich (Shanghai, China).BSA (fatty acids-free) was purchased from Roche (Shanghai, China). A method of preparation of free fatty acids was performed as previously reported. Primary cardiomyocytes (2.0-4.0x105 cells/ml) were incubated with 10-20uM DCF-DA (Invitrogen, USA) for 30-40 min at 37℃. The DCF fluorescence was measured by FACS (Beckman) with ex/em wavelength of 485/525nm. The percentage of neonatal cardiac cells apoptosis was confirmed using Mitochondrial Membrane potential/Annvexin V apoptosis kit (Invitrogen, USA) and the annvexin V-FITC apoptosis Detection kit (Sigma, China). Stained cells were analyzed as above mentioned. Luciferase assayReport gene assay was performed as previously described.Statistical analysisData are means±standard deviation (SD). Differences between groups were assessed using Student's t test or ANOVA followed by Least-significant Difference, Neuman-Keuls and Bonferroni post hoc testing, where appropriate. P<0.05 indicated significance. All experiments were carried out at least 3 times.ResultsMetabolic and physiological characteristics in control and diabetic Goto-kakizaki rats After the 12-week oral treatment of compound RapA, there was no significantly difference on body weight between the untreated and the compound-treated RapA Goto-kakizaki rats, however, the body weight of the control non-diabetic wistar rats was significantly higher than that of other groups, at age of 36 weeks, fast glucose, insulin, triglyceride and total cholseterols levels were significantly higher in diabetic than in control rats. Furthermore, compared with the untreated Goto-kakizaki rats group, the compound treatment (2.5mg/kg.d-1,5.0mg/kg.d-1) tended to decrease the levels of fast plasma insulin, fast plasma glucose and total cholesterol. The RapA-treatment did not take effect on fasting triglyceride levels between the untreated and the compound-treated groups. The untreated and compound treated GK rats exhibited significantly higher heart rate compared with the wistar control, but left ventricular pressure did not have difference between the untreated and the RapA administration (2.5mg/kg.d-1,5.0mg/kg.d-1) groups(p>0.05).The oral administration of compound RapA enhances oral glucose tolerance in the diabetic Goto-kakizaki rats partlyEnhancement of glucose tolerance is one of the most critical standards for determining the effectiveness of drugs for diabetic cardioprotecting; we performed OGTT to evaluate whether the compound RapA could inhibit damage of glucose tolerance. At the appropriate time point the levels of whole blood glucose in the diabetic Goto-kakizaki groups were still greatly higher compared with those of the non-diabetic control group. For 16-week treatment (5.0mg/kg.d-1) of compound RapA to Goto-kakizaki rats, the levels of glucose obtained 0,30,60 and 90 min in the diabetic GK rats after glucose intake were significantly lower than that in the untreated GK rats (5.12±0.94 vs.7.62±1.10 p<0.05; 11.36±1.68 vs.14.60±2.47 p<0.05; 15.86±1.97 vs.20.82±1.74 p<0.05 and 18.04±1.48 vs. 22.22±1.83 p<0.05, respectively). Furthermore, the lower dose treatment (2.5mg/kg.d-1) of compound RapA to the diabetic GK rats in the levels of whole blood glucose was significantly lower than those of the untreated diabetic and the higher dose administration rats at 60 and 90 min (15.58±2.37 vs.20.82±1.74 p<0.05 and 17.38±3.32 vs.22.22±1.83 p<0.05; 15.58±2.37 vs.15.86±1.97 p<0.05 and 17.38±3.32 vs.18.04±1.48 p<0.05, respectively). This data indicate that the oral administration of compound RapA attenuated damage of glucose tolerance and has anti-hyperglycemic effect on Goto-kakizaki rats.Reducement of diabetic cardiac fibrosis by use of compound RapANo differences in ratios of heart weight to body weight (Hw/Bw) and the cardiomyocyte size in a cross-sectional area were noted between the wistar control group and the diabetic GK groups.Furthermore, heart cross-sections were stained with Masson Trichrome for the determination of the extent of cardiac fibrosis at age of 40 weeks with and without the oral administration of compound RapA. Morphologically, collagen deposition tended to increase in response to long term diabetes but was inhibited by compound RapA. On quantitative analysis of perivasculum and interstitial fibrosis in the heart by fibrosis area fraction, compound RapA (2.5mg/kg per day,5.0mg/kg per day) significantly decreased perivasculum and interstitium fibrosis compared with the untreated GK rats (5.218±0.409 vs.6.182±0.373 and 4.532±0.331 vs.6.182±0.373 p<0.05; 8.77±0.65 vs.9.81±0.68 and 4.32±0.68 vs.9.81±1.35 p<0.05, respectively).Compound RapA ameliorates dysfunction of diabetic heartEvaluation of echocardiography exhibited that the EF %(ejection fraction) and FS %(fractional shortening percentage) diameters in the oral treatment (2.5mg/kg per day, 5.0mg/kg per day) of diabetic GK groups at age of 40 weeks were markedly larger (70.20±1.17 vs.66.43±3.20 p<0.05,77.71±2.37 vs.66.43±3.20 p<0.05; 34.80±0.75 vs. 32.29±2.25 p<0.05,36.29±2.12 vs.32.29±2.25 p<0.05, respectively), however, there were no markedly differences in the indicators of IVS, LVPW, LVd and LVs.Activation of Akt kinase and insulin resistance are enhanced and attenuated by oral administration of compound RapAOur results showed that the compound RapA promoted myocardial Akt Ser473 and Akt Thr308 phosphorylation in the treated diabetic GK rats but not in the control GK rats following intravenous insulin markedly, expectedly, Ser473 and Thr308 phosphorylation of Akt were significantly increased by the use of compound RapA without the stimulus of insulin (Figure 4). Ser307 and Ser612 phosphorylation of insulin receptor substrate-1 were significantly decreased in the treated GK rats'hearts compared to the untreated GK rats. Furthermore, similar to the effect of oral administration of compound RapA on Akt Ser473 and Thr308 phosphoylation, Tyr1152/1153 Insulin Receptor beta and Tyr895 Insulin Receptor substrate-1 phosphorylation were significantly increased in hearts of the treated GK rats relative to the untreated GK control. These data demonstrated that the cardio-protecting effect of compound RapA attenuated insulin resistance in Goto-kakizaki rats.AS160 phosphorylation and translocation of cytoplasmic GLUT4 to membrane are promoted by the stimulus of compound RapAFollowing in vivo intravenous injection of insulin, we observed markedly effects on phosphorylation of Akt's downstream substrate AS 160 after 16 weeks oral treatment of compound RapA to the diabetic Goto-kakizaki rats. Insulin resistance-mediated glucose uptake to inhibit phorsphorylation of Akt and its downstream target AS 160 led us to believe that GLUT4 translocation was significantly impaired in the diabetic Goto-kakizaki rats compared with the treated GK rats, and the levels of translocation of GLUT4 to myocardium membrane were no difference between the lower dose (2.5mg/kg per day) treatment group and the higher dose (5.0mg/kg per day) treatment group.Effect on Akt's upstream signaling molecules-p85 and p110alpha subunits of PI3K in vivoTo examine whether Akt's upstream signaling molecules can be activated by compound RapA, we assessed p110alpha and p85 subunits of PI3K activation in heart lysates. At the stimulus status of insulin, p-p85 subunit of PI3K was evaluated in hearts from the lower dose (2.5mg/kg per day) and the higher dose (5.0mg/kg per day) treated GK rats compared with the GK control rats; furthermore, similar to the effect on Akt kinase, Tyr458 phosphorylation of p85 subunit of PI3K was significantly increased by use of compound RapA (2.5mg/kg.d-1, 5.0mg/kg.d-1) without the administration of insulin (Figure 6A). Unexpectedly, expression of pllOalpha subunit of PI3K in the GK rats with treatment of compound RapA was lower than those of the untreated GK control rats in vivo. These results showed that compound RapA can affect the activation of p85 subunit of PI3K-Akt signaling pathway for protecting heart damage not via pllOalpha subunit of PI3K in the diabetes status.Compound RapA activates Akt kinase's downstream signaling molecules relative to mitochondrial dysfunction in diabetic cardiac myocytes.To test whether compound RapA protects the heart from long term diabetes mediated by its activation of GSK3α/β, the total expression and phosphorylation of GSK3α/βwere determined from the hearts of the wistar control, the untreated GK control and the treated GK rats. The results showed that compound RapA significantly suppressed Ser21/9 phosphorylation of GSK3α/βinduced by diabetes in the treated GK control rats compared with the untreated diabetic rats; furthermore, we found that compound RapA induced a significant decrease of GSK3a/p phorsphorylation between the non-diabetic wistar group and the treated GK group (Figure 7A), it was an effect that was unexpected. It was observed that phorsphorylation of PKCεchanged by compound RapA was similar to changes of GSK3α/βin vivo. To detect whether cyclic AMP activity was reduced by compound RapA, we examined the concentrations of myocardial cyclic AMP as above method. These results demonstrated that relative cyclic AMP activity in GK control rats was significantly higher (43.07±5.91 vs.6.35±1.89) compared with the non-diabetic wistar rats (Figure 7C); expectedly, the lower dose (2.5mg/kg.d-1) and the higher dose (5.0mg/kg.d-1) treatment were markedly lower (32.52±5.91 vs.43.07±5.91,26.07±2.63 vs. 43.07±5.91, respectively) compared with the untreated GK rats.The results obtained in the previous experiment promoted us to evaluate whether compound RapA could indirectly or directly affect expression of NADPH oxidase and NADPH oxdiase activity induced by mitochondrial dysfunction in long term diabetes. We assessed expressions of subunits of NADPH oxdiase in heart lysates, including gp91 phox, p67phox, p47phox and p40phox. Expressions of gp91 phox and p47phox in the treated higher dose group were significantly decreased compared with the untreated GK group; it was no difference between the lower dose group and the GK control. Oppositely, expressional levels of p67phox and p40phox were lower in lower dose group than those of GK control and the higher dose group respectively; furthermore, it did not differ between the GK control and the higher dose group in levels of expression of p67phox and p40phox. Expressions of NADPH oxdiase in the treated GK rats approached but did not return to the normal state of the wistar rats. We exhibited that relative NADPH oxidase activity in the oral treatment (2.5mg/kg per day, 5.0mg/kg per day) of diabetic GK groups at age of 40 week were significantly downregulated (2.96±0.08 vs.3.44±0.25,2.53±0.24 vs.3.42±0.25, respectively). As the purpose of evaluating the effect of antioxidant by compound RapA, we investigated relative SOD activity and level of MDA in heart tissue. Figure 7E, indicating myocardial relative SOD activity, showed that it was downregulated by 4-fold in GK control group and enhanced by 2-fold and 3-fold in the lower dose (2.5mg/kg per day) and the higher dose (5.0mg/kg per day),respectively. Myocardial MDA level was increased by 1.7-fold in the GK control and inhibited by 14% and 24% in two dose treatment group, respectively.To explore whether compound RapA can inhibit apoptosis of mitochondria induced by ROS, we examined myocardial Bax, Bad, Bcl-2, cleaved caspase-3 and cleaved caspase-9 expression by western blotting. Western blotting showed that decreases in Bad, cleaved caspase-3 and cleaved caspase-9 expression in the treated GK rats including the lower and the higher dose groups compared with those of untreated GK rats; furthermore, compound RapA did not reduce the expression of Bax in treated GK rats. Similar to the effect on expression of cleaved caspase-3 and caspase-9, relative activities of caspase-3 and caspase-9 were markedly lower in treated GK rats than those in GK control rats and showed dose dependent way.Thus, we tended to believe that compound RapA had anti-ROS-formation effect and inhibit the progression of apoptosis of cardiac mitochondria in long term diabetes.The compound RapA activates p44/42MAPK (ERK1/2) and inhibits JNKERK (p44/42MAPK) is an important molecule for protecting diabetic heart in the pathological development of myocyte apoptosis and cardiac fibrosis; oppositely, activation of JNK could inhibit restoration of diabetic cardiac function and increase cardiac apoptosis and fibrosis. So, we examined the activation levels of ERK and JNK in hearts of GK rats with or without treatment of compound RapA and the non-diabetic wistar control rats. The results demonstrated that Thrl85/Tyrl87 phosphorylation of ERK1/2 were significantly upregulated compared with the GK control group without the oral administration of compound RapA for 16 week (Figure 7H); furthermore, by the effect of compound RapA, the activation levels of ERK1/2 in hearts of two oral treatment GK group approached the normal status of phosphorylation of ERK1/2 in the non-diabetic wistar rats. Phosphorylation levels of cardiac JNK1 was significantly lower in the diabetic GK rats treated with 5.0mg/kg per day of compound RapA for 16 week compared with the GK control rats, however,2.5mg/kg per day for 16 week of compound RapA did not affect the phosphorylation of cardiac JNK1 in appropriate diabetic group. Compound RapA inhibits ROS generation and apoptosis mediated by FFA in primary cardiac cells not via PI3K's subunit-p85aFFA (free fatty acids) is closely involved in the development of type 2 diabetes, which generates reactive oxygen species in heart and finally leads to myocardium apoptosis. According to the results above, to test whether compound RapA could take effect on activation of AKT for protecting diabetic heart directly, we assessed the expression of PI3K's p85αsubunit with p85αgene siRNA pool in primary cardiac cell. The results showed that p85αprotein expression was unaltered in cells exposed to a negative control siRNA sequence; however, siRNA pool against PI3K's p85αsubunit reduced protein expression markedly compared with cardiac cells transfected with negative control siRNA after 48 hours. Relative intracellular ROS levels generated by FFA (100UM) for 48h were markedly increased (305.67±12.73 vs.5.77±0.36, p<0.05) compared with the negative control in primary cardiac cells; Furthermore, compound RapA (5UM) for 48h added to primary cells reduced relative ROS levels markedly (204.42±6.07 vs.305.67±12.73), compared with the FFA (100UM) group. In accordance with compound RapA treatment, siRNA pool-mediated silence of PI3K's p85a in cardiac cells displayed no significantly difference on relative intracellular ROS levels (204.42±6.07 vs.198.02±11.76, p<0.05) compared with the FFA(100UM)+RapA(5UM) group.The apoptotic effect of ROS was partly mediated by transcriptional factors responded to ROS. To study whether compound RapA could inhibit transcriptional factor's activity induced by ROS, a luciferase reporter gene construct containing antioxidant response element was transfected into primary cardiac cells with or without compound RapA or p85α-siRNA. Similar to the results above, relative luciferase activities of ARE plasmid of the FFA (100UM)+RapA group and the FFA (100UM)+RapA+p85α-siRNA group were significantly higher (1.42±0.08 vs.0.61±0.12,1.24±0.07 vs.0.61±0.12) than those of the FFA (100UM) group. These data suggested that compound RapA could reduce generation of ROS-mediated by free fatty acids and activities of transcriptional factors induced by ROS by activation of AKT kinase directly not via its upstream signal molecule-PI3K's subunit p85a in vitro.Previously reports have indicated that ROS mediated by FFA could upregulate apoptosis level of cardiac cells in vitro. Here, we determined whether FFA-induced apoptosis of primary cardiac cells is prevented by compound RapA with or without the treatment of p85α-siRNA in vitro. Two days after the co-treatments of FFA and compound RapA and transfection of p85a siRNA, levels of mitochondrial and cellular apoptosis were assessed by FACS; the methods of FACS were performed as described above. Stimulation of cardiac cells with FFA (100UM) significantly induced the cellular and mitochondrial apoptosis by 5.9-and 4.2-fold, respectively, furthermore, compound RapA largely blocked the cellular and mitochondrial apoptotic effect of free fatty acids, expectedly, and addition of p85a-siRNA did not prevent the anti-apoptotic effect of compound RapA. These results suggested that the anti-apoptotic effect of compound RapA was mediated in a large part through activation of AKT kinase directly not via its upstream signal molecule-PI3K's subunit p85a.DiscussionThis study was undertaken to examine the therapeutic effect of compound RapA on the cardiac damage in the long term diabetes. The important findings in the study were:(i) compound RapA reduces metabolic levels of risk factors, including fast plasma insulin, fast plasma glucose, total cholesterol and free fatty acids (FFA); (ii) the damaged glucose tolerance induced by type 2 diabetes may well be ameliorated by the oral treatment of compound RapA in part; (iv) compound RapA protects against the cardiac dysfunction induced by cardiac fibrosis; (iv) cardiac fibrosis induced by insulin resistance could be weakened by the administration of compound RapA; (Ⅴ) compound RapA inhibits insulin resistance and enhances the senstivity of the stimulation of insulin, and activates the PI3K-Akt signaling pathway, including the phosphorylation of Akt, membrane translocation of GLUT4; (Ⅵ) mitochondrial dysfunction and apoptosis induced by reactive oxygen species in myocardium may well be suppressed by compound RapA; (Ⅶ) the apoptotic effect of free fatty acid in vivo may de repressed by compound RapA through the activation of Akt kinase diretly not via the upstream signaling molecule-PI3-kinase's subunit p85α.