Effect And Mechanism Of Atherogensis And Myocardial Energy Metabolism Induced By Satiation And Caloric Restriction

BackgroundTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a multi-functional cytokine, which has been shown to have potent anti-tumor actions by inducing cell apoptosis. In addition, TRAIL is also involved in modulating the pathophysiological processes of cardiovascular, metabolic and autoimmune diseases. TRAIL is expressed as a transmembrane protein, which can be further cleaved by cysteine proteinases at the cell surface, yielding the soluble form of TRAIL (sTRAIL). Soluble TRAIL retains the biological activities of the full length ligand. The major source of sTRAIL in the body is the activated monocytes and neutrophils.Several studies have suggested that there may be an association between production of TRAIL and metabolic disorders in humans.In adipocytes, TRAIL inhibits insulin-stimulated glucose uptake and de novo lipogenesis, effects that appear to be mediated by caspase-dependent degradation of peroxisome proliferator-activated receptor (PPAR)y, but unrelated to alterations in insulin signaling or NF-κB activation. In a recent study, we showed that TRAIL treatment in mouse and human cultured macrophage cells increased the expression of scavenger receptor (SR)-AI and -BI, and this effect was accompanied by concomitant increase in lipid internalization and foam cell formation.Hypercholesterolemia is the foremost risk factor for atherogenesis, and the close relationship between elevated plasma cholesterol and vascular inflammation highlights the importance of impaired lipoprotein metabolism in the etiology of atherosclerosis. During atherogenesis, macrophages migrate into the subendothelial space and internalize oxidized low-density lipoproteins (LDL), resulting in formation and accumulation of cholesterol-laden foam cells. This process represents a central pathophysiological mechanism responsible for the initiation of atherosclerotic lesion formation.. As a risk factor for cardiovascular disease, the presence of hyperlipidemia caused more and more people’s attention. Hyperlipidemia, mainly due to lipid metabolism dysfunction, leading to one or more serum biochemical parameters of lipid index is higher than the normal parameters. Its main clinical manifestations are serum total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) higher than normal and (or)high-density lipoprotein cholesterol (HDL-C) below the normal level,which often offends old people. However, with the pressure of life and the improvement of the dietary nutrition increasing, more and more drinking and smoking, The prevalence of hyperlipidemia Show an increased trend, and patients appeared more and more younger.Experimental studies indicate that TRAIL may have important regulatory roles in the pathogenesis of metabolic disorders including obesity, diabetes and hypercholesterolemia. Some authors reported that a higher concentration of sTRAIL was associated with increased body adiposity in healthy subjects and in patients with metabolic syndromes. In a small cohort of 33 familial hypercholesterolemia patients and 10 healthy controls, Holven and colleagues found that the expression level of TRAIL in peripheral blood mononuclear cells was increased in patients as compared to controls, and this change was accompanied by parallel increases in the expression of other inflammatory markers such as TNF-α receptors and CD40, indicating a proinflammatory phenotype. Moreover, the basal and stimulated release of TNF-a was also concomitantly augmented in hypercholesterolemic patients. Similarly, it has been reported that serum TRAIL concentrations exhibit positive correlations with total cholesterol, LDL-cholesterol and triglycerides, but have an inverse correlation with HDL-cholesterol in humans. After controlling other confounding factors, the association between TRAIL and LDL-cholesterol remains to be significant. Moreover, in a study in Asian people, Choi et al. reported that the serum TRAIL concentration in healthy postmenopausal women was significantly higher in the upper than the lower quartile of the low density lipoprotein cholesterol (LDL-C) level。In another study in patients with metabolic syndromes, it was found that sTRAIL was positively correlated with total cholesterol (TC) and LDL-C。Therefore, we should do further research to clarify the relationship between sTRAIL and hyperlipidemia, in order to accurately define role of TRAIL in the pathogenesis of human atherosclerosis which has great clinical significanceObjectivesIn the present study, we examined the relationship between serum TRAIL and various biochemical parameters in 352 generally healthy subjects who underwent the routine annual health check. We measured the serum TRAIL concentration and levels of total and low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and so on, to investigate the correlation between them. In the present study, we explore whether the relationship was consistent regardless of age, gender and the status of adiposity.Methods1. Study subjectsAll subjects were recruited sequentially at the hospital’s Health Check Centre. These subjects were generally normal individuals registered to undertake their routine annual health check, but not outpatients consulting for specific disorders. A brief checklist was given to each subject and individuals with a known history of diabetes mellitus, autoimmune disease, inflammatory disease and/or coronary heart disease were excluded. There were no restrictions on other conditions such as age and sex.2. Sample processing and data collectionThe blood biochemical tests were performed in the Central Clinical Laboratory of the hospital following standardized procedures. The blood samples were tested, which included triglycerides,total cholesterol, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).Other physiological data were obtained from the participants’ health check reports.3. Measurement of sTRAIL with ELISAVenous blood of 0.5 ml of was obtained after overnight fasting from each subject. Serum samples were prepared by centrifugation and Samples were stored at -80℃ for later use. The serum concentration of sTRAIL was measured with commercial ELISA kits purchased from various suppliers. Assays were carried out following the manufacturer’s protocols。4. Statistical analysisData are presented as mean±standard error of the mean (SEM). Data analysis was performed using GraphPad Prism. The mean data between two groups were analyzed with Mann-Whitney test or unpairedt-test. Spearman’s correlation analysis was used to test the correlation between two parameters. A value ofP<0.05 was considered as statistically significant. Categorical data were examined with Chi-square test. All statistical tests were 2-sided.Results1. Baseline characteristics of study subjectsA total of 392 subjects were recruited sequentially, and we had complete data for 352. The age of the subjects ranged from 30 to 68 years. In this cohort, about 15% had a blood pressure that exceeded the normal value. In about half of the subjects, the total cholesterol level was higher than the normal value (5.2 mmol/L). According to the standard commonly used within China,>45% of these subjects were overweight (body mass index, BMI>24) or obese (BMI>28).2. The variability of the results might be related to the different kits usedWe first tested whether different ELISA kits could generate variable results. We tested a set of 16 samples with three different commercial kits. While two kits produced roughly comparable data, the third kit produced different results. Hence in the following experiments, we chose the kit from R&D Systems, which was more frequently used in previous studies. However, it is noted that the different absolute sTRAIL values as determined by different kits are of little impacts on the conclusions from these studies.3. Using gender, BMI, age as a cutting-off value, the sTRAIL concentration was significantly higher in the TC-high groupUsing TC level of 5.2 mmol/Las a cutting-off value, the subjects were divided into two groups, namely those with normal (TC-Norm group) and elevated (TC-high group) blood total cholesterol levels. We demonstrated that the sTRAIL concentration was significantly higher in the TC-high group (P<0.001). We also tested the difference in sTRAIL concentration using TC of 5.7mmol/L as the cutting-off value. We confirmed that the sTRAIL concentration was still significantly higher in the TC-high group (126.8 ± 6.7) than the TC-Norm group (113.6 ± 4.1) (P<0.005). To verify whether such a relationship between sTRAIL and TC held in different subgroups of the subjects, we first made comparisons in males and females separately. The mean sTRAIL concentration in males was significantly higher in TC-high than TC-Norm individuals. A similar difference of sTRAIL concentration was also present in females. Then we divided the subjects into two groups according to their BMI, namely<24 (normal bodyweight) and24 (overweight/obese). In these two subgroups, significant differences in mean sTRAIL concentration between TC-high and TC-Norm individuals were consistently detected= Moreover, a significantly higher sTRAIL concentration was also observed in TC-high individuals in different age bands.4. Using a cutting-off value of 3.6 mmol/L of LDL-C, we found that the sTRAIL concentration was significantly higher in the LDL-high group, and the results are different because of gender, age, BMIUsing a cutting-off value of 3.1 mmol/L of LDL-C, we did not detect a significant difference between LDL-low versus LDL-high subjects (113.8 ± 4.6 and 121.8 ± 5.5 pg/ml respectively, P= 0.073). However, using a cutting-off value of 3.6 mmol/L, we found that the sTRAIL concentration was significantly higher in the LDL-high group (P<0.0005). We further confirmed that the mean sTRAIL concentration in males was significantly higher in the LDL-high than LDL-low individuals. However, the difference in females was not significant. In relation to the body adiposity, we found that in both of the normal bodyweight group and the overweight/obese group, the mean sTRAIL concentrations were all significantly higher in LDL-high individuals. In younger people, sTRAIL concentrations were 123.3 ± 7.8 in LDL-high versus 109.2 ± 5.3 in LDL-low individuals (P= 0.0013). In older people, sTRAIL concentrations were 132.3 ± 14.3 in LDL-high versus 118.1 ± 6.0 in LDL-low individuals, of which the difference was not significant (P= 0.1851).5. TC and LDL-C levels in the upper quartile were significantly higher than those in the lower quartileAccording to the level of sTRAIL, we divided into four parts,and compared various physiological and biochemical parameters between subjects in the lower and upper quartiles of the sTRAIL concentration. The mean age was not different between the two quartiles. TC and LDL-C levels in the upper quartile were significantly higher than those in the lower quartile (4.89 ± 0.08 versus 5.28 ± 0.10 for TC,P= 0.0056; 2.89 ± 0.07 versus 3.15 ± 0.09 for LDL-C,P= 0.042), whereas there were no significant differences in triglycerides, HDL-C, SBP or DBP. BMI was also different between the two quartiles.Conclusion1. There is a positive correlation between TC and sTRAIL concentration using gender, BMI, age as a cutting-off value2. Using a cutting-off value of LDL-C, the sTRAIL concentration was significantly higher in the LDL-high group3. In the lower and upper quartiles of the sTRAIL concentration, compare TC and LDL-C between subjects4. TC and LDL-C levels in the upper quartile were significantly higher than those in the lower quartileBackgroundTumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was first discovered as a homologue of cytokines of the TNF family. TRAIL is also known as Apo-2 ligand (Apo-2L) or TNF super family 10 (TNFSF10). TRAIL shows 28% homology to Fas ligand and 23% to TNF-a. Human TRAIL is a 281-amino acid (291 for murine TRAIL) type II transmembrane protein. The membrane-bound TRAIL can be cleaved by cysteine proteases, and subsequently released into circulation, leading to formation of the soluble form of TRAIL. A major source of soluble TRAIL appears to be the activated leukocytes such as monocytes and neutrophils. Soluble TRAIL forms a homotrimer through interactions between the Cys230 residue and the bound zinc ion; this trimerization is essential for TRAIL binding to the cognate receptors and subsequent induction of biological responses. Five different TRAIL receptors have been identified. Of these, TRADL-R1 (also known as death receptor 4, DR4 or TNFRSF10A) and TRAIL-R2 (DR5 or TNFRSF10B) are functional transmembrane receptors that mediate TRAIL-induced cellular responses. In the mouse, only the DR5 receptor, but not DR4, is expressed.TRAIL has potent anti-tumor activities and has entered into clinical trials for cancer therapy. In addition to inducing tumor cell apoptosis, TRAIL shows broad other biological functions both in vitro and in vivo, and these include potential protective effects on cardiovascular disease. TRAIL gene deletion enhanced atherogenesis in hyperlipidemic mice, supporting that endogenous TRAIL has vascular protective effects. It remains to be determined, however, whether the vascular protective action of TRAIL is via direct impacts on residential vascular cells, or indirectly by modulating systemic immune functions. In this article, we provided a critical review on the potential relationships between TRAIL and atherosclerosis. We summarized the evidence from a variety of in vitro experiments, indicating that excessive TRAIL may stimulate endothelial cell apoptosis, smooth muscle proliferation and migration, foam cell formation and inflammatory responses. These results raise the possibility that increased TRAIL production may also have potential detrimental effects on vascular inflammation and atherosclerosis. Notably, this notion is not standing alone. Indeed, biphasic effects of TRAIL on the outcome of rheumatoid arthritis and central nervous system inflammation have also been reported. Understanding the pharmacological effects of exogenous TRAIL on vascular inflammation and atherogenesis is clinically important, given that TRAIL emerges to be a promising therapeutic agent for treating cancer, autoimmune and even cardiovascular diseases.Atherosclerosis is an important risk factor of cardiovascular disease. The occurrence of atherosclerosis development process is very complex. In generally speaking, atherosclerosis is the results of the combination of genetic factors and environmental factors. It is thought that autoimmunity plays an essential role in atherogenesis. Autoimmune diseases are associated with an increased risk of atherosclerosis, while atherosclerosis and autoimmune diseases are both characterized by the presence of chronic systemic inflammation. The relationship between TRAIL and atherosclerosis or coronary arterial disease (CAD) in humans has been addressed by several clinical studies. In patients with unstable angina, the serum level of soluble TRAIL was significantly lower than that in patients with stable atherosclerosis or that in healthy control. By contrast, the levels of TNF-aor Fas ligand were not changed. Similarly, Mori and colleagues showed that the serum TRAIL level was significantly decreased in patients with CAD, and there was an inverse correlation between the serum TRAIL level and the severity of disease, suggesting that TRAIL might be a useful biomarker for CAD. Recently, Deftereos and colleagues examined 56 coronary arteries in CAD patients with intravascular ultrasound and measured the TRAIL concentration in coronary blood samples. These authors demonstrated that a lower blood TRAIL concentration was associated with larger coronary plaque volumes. Moreover, there was a significant association between low serum TRAIL levels and the presence of lesions with unstable characteristics.TRAIL gene deletion in micehas provided convincing evidence supporting that endogenous TRAIL is essential in maintaining blood vessel homeostasis and repressing the process of atherogenesis. However, In the mouse, only the DR5 receptor, but not DR4, is expressed. Therefore, we selected the DR5 knockout mice to study the TRAIL of atherosclerotic plaque. In this research, we observe whether there are any changes in atherosclerotic plaque area, in order to further clarify the role of TRAIL/DR5 in atherosclerosis.Objectives1. To observe the body weight、the epididymal fat pads、mesenteric fat pads、 perirenal fat padsvthe concentration of serum cholesterol, Glu, HDL-C,LDL-C and TG in the double knock mice.2. To further clarify the change of atherosclerotic plaque area using ApoE/DR5 and LDLR/DR5 DKO mice.Experiment materials and methods1. AnimalsMale apoE-/- mice at the age of 8-wk were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD (Beijing, China). DR5-/- mice were purchased from Mutant Mouse Regional Resource Centers (MMRRC). LDLR-/- mice were purchased from Model Animal Research Center of Nanjing University(MARC). All animal studies were complied with the Animal Management Rules of the Chinese Ministry of Health and approved by the Ethical and Use Committee of the Qilu Hospital of the Shandong University.2.Creation of ApoE/DR5 and LDLR/DR5 DKO miceapoE-/-、LDLR-/- and DR5-/- mice were separately crossed at the age of 8-wk, the genotype of the offsprings were DR5+/- apoE+/-. Then heterogygous mice were mated with each other. We selected DR5-/- apoE-/- and DR5+/+ apoE-/-, DR5-/- LDLR-/-and DR5+/+ LDLR-/- mice by genotyping.3.Treatment groupsMice at the age of 8-wk were randomly divided into four groups:DR5-/-apoE-/- and DR5+/+ apoE-/-,DR5-/- LDLR-/- and DR5+/+ LDLR-/- .Every group was separately fed a high-calorie diet and normal diet for 16 weeks.4.Tissue Sample CollectionFor the collection of blood samples, animals were fasted overnight, anesthetized with 0.8% pentobarbital sodium. Blood was intracardially collected using a syringe though the heart-tips. Followed sacrificing animals, various tissurs, heart and arteries were dissected. For the analysis of mRNA and protein expression, samples were immediately placed in liquid nitrogen and then the kept in -80℃ until use. For histological analysis, samples were fixed with 4% paraformaldehyde overnight, followed by washing with lx PBS several times.5.Blood Lipid AnalysisAfter collection of blood samples, plasma from each mouse was prepared and kept at -80℃ until use. Total plasma levels of cholesterol (CHO), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), Glucose (GLU) and high-density lipoprotein cholesterol (HDL-C) were measured using an automatic biochemical analyzer (Roche, Germany).6.Histological StainingTissue samples were stained with Oil-Red O using a standard protocol.7.Statistical AnalysisAll experiments were repeated at least three times. Data are presented as mean± standard deviation (SD). Data analysis was performed with unpaired t-test or one-way ANOVA followed by post hoc Newman-Keuls test as appropriate. P<0.05 was considered as statistically significant.Results1. The weight of mice increased significantlyThe double knock mice were fed a a high-calorie diet and normal diet for 16 weeks.We observed the weight of HF groups was significantly higher than that of CF groups. A gain of the weight expressed in the DR5-/- apoE/-/ HF group was the highest proportion. while the DR5+/+ apoE-/- HF group was the lowest. However, that was not related to the genotype.2. The mass of the epididymal fat pads、mesenteric fat pads、perirenal fat pads in HF group of DKO mice increased significantly compared with the CF groupWe compared the pads of the epididymal, mesenteric, perirenal fat in in HF group of DKO mice with the the CF group after 16weeks, and the results of fat mass were significant. Firstly the fat mass of HF group were higher than the control group in DKO mice, no matter the genotyping of the mice. Secondly, in the HF group, there were no significant difference in fat mass between the DR5-/- DKO mice and DR5+/+ DKO mice, and so were in the CF group.3. The results of serum samples3.1 The result of serum CHO in different double knockout miceThe intra-group compairson is that the level of serum CHO in HF group was higher than in CF group, no matter the genotyping of the mice. However after the high caloric diet, there were no significant difference between before and after the DR5 knockout. The serum CHO of DR5+/+ LDLR-/- and DR5-/- LDLR-/- DKO mice in HF group exerts higher concentration than in the CF group, P<0.0001. However the level of CHO in DR5+/+ LDLR-/- DKO mice was much lower than that in DR5-/-LDLR-/- DKO mice, P<0.0001.3.2 The result of serum Glu in different double knockout miceThe level of serum Glu in DR5-/- apoE-/- DKO mice HF group was lower compaired with that in CF group, while the level in DR5-/- LDLR-/- DKO mice HF group was significantly higher than in CF group. In addition, the serum Glu of DR5+/+ apoE-/- DKO mice CF group was higher than that of DR5-/- LDLR-/- DKO mice CF group, and the other groups did not achieve statistical significance.3.3 The result of serum HDL-C in different double knockout miceCompaired with the DR5-/- apoE-/- DKO mice CF group, the level of serum HDL-C in the HF group significantly decreased. And under the condition of a normal diet, the concentration of serum HDL-C in the DR5+/+ apoE-/- DKO mice was obviously lower than that in the DR5-/- apoE-/- DKO mice. However, on the basis of the LDLR-/- double knock mice, the level of serum HDL-C in DR5+/+ LDLR-/- DKO HF mice was elevated, while under the condition of the same diet, only the level of serum HDL-C in DR5+/+ LDLR-/- DKO mice was significantly lower. Besides, the test groups had no significant difference.3.4 The result of serum LDL-C in different double knockout miceAfter 16 weeks of high caloric diet, the concentration of serum LDL-C in DR5+/+ apoE-/- DKO mice elevated significantly, while that in CF group had not statistical significance. In the other DKO mice, that is to say the DR5+/+ LDLR-/- and DR5-/- LDLR-/-, the level in HF group were obviously superior compaired with the CF group. Under the same condition of the diet, the level of serum LDL-C in HF group of DR5+/+ LDLR-/- DKO mice was statistically lower than in CF group.3.5 The result of serum TG in different double knockout miceThe concentration of serum TG in HF group of DR5-/- apoE-/- was reduced than in CF group, while that in HF group of DR5+/+ LDLR-/- and DR5-/- LDLR-/- DKO mice was significantly higher than in the CF group. Under the same condition of the nomal diet, there is no significant difference in serum cholesterol levels between before and after gene knockout. Whereas under the condition of high calorie diet, the TG level in DR5+/+ LDLR-/- DKO mice was much lower than in DR5-/- LDLR-/- DKO mice.4. Vascular plaque area changed greatly in different double knockout mice Under the same conditions in the diet, vascular plaque were smaller in DR5-/-background mice after stained with Oil-Red O. On one hand, Moreover, the area of aortic arch plaques in DR5-/- apoE-/- HF group were significantly reduced compaired with the DR5+/+ apoE-/- mice,and the same situation happened in the DR5-/-LDLR-/- mice. on the other hand, no matter what kind of diets, the plaque areas of descending aorta-abdomens aorta in DR5-/- apoE-/- HF group were much smaller than that of DR5+/+ apoE-/- mice,but there was no significant difference in DR5-/-LDLR-/- mice and DR5+/+ LDLR-/- mice.Conclusion1.The mass of the epididymal fat pads、mesenteric fat pads、perirenal fat pads in HF group of DKO mice increased significantly compared with the CF group. And the concentration of the serum cholesterol,HDL-C,LDL-C and TG in the double knock mice were changed, and the results of HF group were higher than that of CF group.2. Under the condition of a high calorie diet, atherosclerotic plaque area in the ApoE/DR5 and LDLR/DR5 DKO mice was significantly smaller..BackgroundIn the above chapters, we showed that the influence of high-calorie diet for the body. Accelerating constantly in the rhythm of life, more and more people choice high calorie diet, leading to cardiovascular diseases such as diabetes and other metabolic syndrome and hypertension. And this choice seriously affected the people’s health. As saying in yangshenglun,’people should have a balanced diet to avoid getting sick, so that they can keep themselves healthy longer’. And this article suggested a diet was closely related to longevity. However, a balanced diet does not mean overeating. Traditional Chinese medicine believes that eating well mainly eat moderately. Ancient health expert Gehong suggested that people eat moderately and abstemiously. In ancient China, many ancient health experts presented one concept: Bigu, which means a way of keeping in good health.The balanced diet, in our study that is a calorie restriction diet. Modern research has found that caloric restriction have beneficial effects on the body. Colman et al had found that caloric restriction delayed disease onset and mortality in rhesus monkeys. Of course, many epidemiological studies have reported that caloric intake is related to the incidence of several chronic diseases, including cardiovascular disease, cancer, diabetes, as well as neurodegenerative disorders. Studies of Okinawan centenarians support the view that a low-calorie diet can increase prospects for good health and longevity in humans. A recent study of self-selecting volunteers on CR also noted remarkably low values on risk factors for cardiovascular disease. These studies are valuable and clearly suggestive of the relationship between caloric intake and aging but are no substitute for experimental evidence.The researchers have focused on the family longevity genes to investigate the relationship between caloric restriction diet and life. We chose Sirtl as the research object, to discuss the change of sirtl and the influence and mechanism on myocardial glucolipid metabolism and cardiac function with the condition of caloric restriction diets.Objectives1. To examine the mechanism the of sirtlprotien level change under the condition of caloric restriction diets.2. To explore the effect and mechanism of myocardial glucolipid metabolism and cardiac function with the condition of caloric restriction diets.Materials and Methods1. The subjects and animal grouping Male C57BL/6 mice at the age of 8-wk were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co. LTD (Beijing, China). Mice were randomly divided into two groups:normal diets group (CF) and caloric restriction diets group (CR),10 mice in each group.2. Echocardiographic measurementsUltrasound echocardiography was performed after 16 weeks. Under general anesthesia with 1-2% isoflurane, the procedure was conducted using a high resolution echocardiography system (Vevo770, Visual Sonics, Canada) with a 35-MHz transducer. Two-dimensional parasternal long-and short-axis images of the LV were recorded. Using the M-mode function, the LV end-diastolic diameter (LVIDd), LV end-systolic diameter (LVIDs), inter-ventricular septum end-diastolic thickness (IVSTd) and LV posterior wall end-diastolic thickness (LPWTd) were measured. LV fractional shortening (FS) ejection fraction (FS) and corrected LV mass were automatically calculated by the echocardiographic system.3. Real-time quantitative PCRReal-time PCR was carried out using Taqman Gene Expression primer-probe sets as described. The 18S RNA was used as the house-keeping gene. The relative quantification method was used for data analysis. Briefly, ΔCt was calculated as the Ct value of the test gene minus that of the house-keeping gene. ΔΔCt was calculated as the ΔCt value of a given gene in treated groups minus that in control group. The relative gene expression level (fold) was expressed as the calculated value of 2-ΔΔCt.4. Western bloting analysisTissues were homogenized in lysis buffer, then total protein samples were separated by SDS-PAGE and electro-transferred to nitrocellulose membranes. The membrane was blocked with 5% nonfat milk and incubated with specific primary antibodies. Secondary HRP-conjugated antibodies were developed with ECL Prime reagents and detected with a LAS-4000 luminescent image analyzer.5. Statistical analysisData are presented as mean±standard error of the mean (SEM). Data analysis was performed using GraphPad Prism. The mean data between two groups were analyzed with Mann-Whitney test or unpairedt-test. Spearman’s correlation analysis was used to test the correlation between two parameters. A value of P<0.05 was considered as statistically significant.Results1. Caloric restriction increase the level of Sirtl protein expression via PI3K/AKT signaling pathwayTo clarify the level of Sirtl protein expression in the cardiac tissue, we used SDS-PAGE and western blotting, and demonstrated that Caloric restriction increase the level of Sirtl protein expression significantly. Simultaneously, the level of PI3K protein expression in CR group was significantly higher than that in CF group. And there was a significant rise in expression of target protein AKT in CR group.2. The levels of myocardial glucolipid metabolism related genes expression were elevated significantly under the condition of caloric restrictionUsing the RT-PCR, we detected the myocardial glucolipid metabolism related genes. The result showed that the mRNA level of pyruvate dehydrogenase kinase 4 in CR group increased significantly compared with CF group. And genes correlated with lipid metabolism, such as CD36, MCAD, CTPlb had a similar result:the mRNA level of them increased significantly.3. There was no significant difference in level of cardiac function between the two groups.The results of echocardiographic measurements showed that the left ventricular posterior wall(LVPWd) was less thickness in CR group than that in CF group, However the differences in left ventricular end diastolic diameter (LVIDd),left ventricular internal diameter (LVIDs), ejection fractions (EF%) and fraction shortening(FS%) was not statistically significant between the two groups.ConclusionsThrough research we found that caloric restriction increase the level of Sirt1 protein expression via PI3K/AKT signaling pathway, leading mRNA level of myocardial gluco-lipid metabolism related genes expression increasing. And there was no significant difference in level of cardiac function between the two groups.