Research On Association Of Adipose Tissue Macrophage Polarization Mediated By TRB3 With Diabetic Coronary Disease

BackgroundDiabetes is the major risk factor of coronary artery disease (CAD), and the characteristics of CAD in diabetic CAD patients are as follows:high incidence, more serious and vulunerable plaques, more extensive, diffuse, and distal CAD and the poor effect of clinical therapy. The mortality is 2- to 4-fold higher in diabetic CAD patients compared with non-diabetic CAD patients. However, the mechanism underlying the aggravation of diabetic CAD remains imcompletely understood. This has highlighted the importance and urgency of studying the mechanism of diabetic atherosclerosis and exploring therapeutic options.Visceral adipose dysfunction plays a vital role in atherosclerotic heart disease. Epicardial adipose tissue (EAT), a special kind of visceral adipose tissue with its close proximity to coronary arteries, interacts directly with the coronary arteries and myocardium through paracrine or vasocrine pathway. Cardiac CT scan revealed that frequencies of incident coronary events increased with the amount of EAT. Clinical studies have shown that EAT dysfunction is frequently observed in diabetic individuals, manifested as the increase of EAT thickness and the release of high levels of pro-inflammatory mediators such as MCP-1 and leptin. Therefore, we speculate that EAT dysfunction at the crossroad of diabetes and CAD might participate in aggravation of diabetic CAD.Macrophages infiltration in adipose tissue initiates the chronic inflammation of adipose tissue, which plays an important role in adipose dysfunction. Macrophage consists of inflammatory M1 macrophage and anti-inflammatory M2 macrophage. Compared with the increase in the number of macrophages, the increase in M1/M2 macrophages ratio was more closely associated with chronic inflammation in adipose tissue, diabtetes and CAD. The impaired glucose metabolism and dyslipidemia caused by diabetes in turn aggravates adipose tissue dysfunction. Therefore, the increase in M1/M2 macrophages ratio might play a key role in the vicious circle between EAT dysfunction and diabetes.PPARy, one of the nuclear receptor superfamily, is a key regulator of macrophage polarization. PPARy-deficient macrophages are resistant to M2 polarization; Obesity and/or inflammatory stress can lead to a switch in the phenotype of adipose tissue macrophages from M2-like to M1 in the absence of PPARy-mediated pro-inflammatory gene repression. Tribbles homolog 3 (TRB3), inhibiting PPARy, is considered a potential inducer of insulin resistance. Several studies on the Q84R polymorphism of TRB3 have provided initial evidence that individual carriers for 84R exhibit a higher risk for cardiovascular diseases. Therefore, TRB3/PPARγ might be a key signal pathway involved in the vicious circle between diabetes and adipose tissue dysfunction by regulating M1/M2 macrophages ratio.In this study, we obtained adipose tissues during cardiac surgery from CAD patients with or without diabetes, and explored the correlation between M1/M2 macrophages ratio in adipose tissues and the severity of CAD. Objectives1.To explore the differences in the numbers of macrophages, M1/M2 macrophages ratios in EAT and subcutaneous white adipose tissue (SWAT) between non-diabetic and diabetic CAD patients;2. To explore the association of M1/M2 macrophages ratios in EAT and SWAT with the severity of CAD;3. To detect the changes of the protein levels of TRB3 and PPARy in adipose tissues in diabetic CAD.Methods1. Study population:A total of 60 patients with CAD undergoing coronary artery bypass graft surgery were studied. A group of 30 subjects were type 2 diabetic patients according to the American Diabetes Association, and either on diet or drug therapy (DM+CAD group). A group of 30 subjects were nondiabetic patients (CAD group). Informed consent was obtained from all subjects based on a protocol approved by the Ethics Committee of Qi Lu Hospital, Shandong University. At the beginning of the surgical intervention, EAT was taken from the anterior wall of the left ventricle. SWAT was taken from the subcutaneous fat on the sternum.2. Assessment of the severity of CAD:using the Gensini scoring system through coronary arteriography;3. Serum parameters of CAD patients:fasting glucose, total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c) and insulin;4. Pathology analyses:HE staining; Immunochemistry assay was performed to detect macrophage (F4/80), M1 macrophage (CD11c), M2 macrophage (CD206) and calculate M1/M2 macrophages ratio; Immunochemistry assay was performed to detect pro-inflammatory factors including MCP-1, TNFa, IL-6 and anti-inflammatory factors IL-10;5. Real-time PCR:The mRNA of FAS, Perilipin, ATGL, HSL, Adiponectin and leptin were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green method;6. Western bot:The proteins of GLUT4, IRS1, TRB3 and PPARy were detected by western blotting.Results1. Patient Characteristics:(1) There were no significant differences in age, gender, body weight and blood pressure between CAD group and DM+CAD group; The DM+CAD group showed significantly higher waist circumference (WC) (90.67 ± 7.07 vs 95.8 ± 7.43, P=0.008), hip circumference (HC) (95.43 ± 5.83 vs 98.27 ± 4.81, P=0.044) and body mass index (BMI) (24.45 ± 2.41 vs 25.84 ± 2.41, P=0.029);(2) Compared with non-diabetic CAD patients, CAD patients with diabetes showed significantly higher Gensini score (74.13 ± 41.1vs 114.73 ± 54.47, P=0.002).2. Serum parameters of CHD patients:There were no significant differences in TC and LDL-c between CAD group and DM+ CAD group; Compared with non-diabetic CAD patients, CAD patients with diabetes showed significantly higher fasting glucose (4.91 ± 0.53 vs 7.41 ± 1.99, P<0.001), TG (1.33 ± 0.63 vs 1.74 ± 0.84, P=0.024) and FFA (65.73 ± 36.21 vs 96.50 ± 67.90, P=0.034), accompanied by lower HDL-c (1.15 ± 0.23 vs 1.03 ±0.19, P=0.0024)3. Pathology analyses:(1)The epicardial adipocyte was smaller than subcutaneous adipocyte, but both of EAT and SWAT showed varying and significant increases in adipocyte size in diabetic subjects;(2)The numbers of infiltrating macrophages in EAT and SWAT were significantly increased in the diabetic CAD group than CAD group (8.0382 ± 0.79003 vs 14.993 ± 1.87488, P=0.003; 5.7918 ± 0.66575 vs 9.4504 ± 1.23046, P=0.007). Interestingly, the numbers of not only M1 macrophages but also M2 macrophages were significantly increased in EAT and SWAT in the diabetic group. However, the degree of increased M1 macrophages was greater than that of increased M2 macrophages in adipose tissue in diabetic CAD patients. CAD patients with diabetes showed significantly increased M1/M2 macrophages ratio in EAT and SWAT (particularly EAT) (0.8893 ± 0.06104 vs 2.3085 ± 0.30445, P<0.001; 0.8759 ± 0.11247 vs 1.8729 ± 0.17126, P<0.001).(3) The MCP-1, IL-6 and TNFα, pro-inflammatory cytokines secreted by M1 macrophages, were up-regulated in EAT (4.30-fold,3.42-fold and 2.49-fold respectively, P<0.05 for all) and SWAT (2.43-fold,2.02-fold and 1.60-fold respectively, P<0.05 for all) in the DM+ CAD group compared with CAD group;(4) The IL-10, anti-inflammatory cytokine produced by M2 macrophages, were also up-regulated in EAT (1.78-fold, P= 0.004) and SWAT (1.43-fold, P= 0.044) in diabetic patients, but the degrees of the increases of anti-inflammatory cytokines were lower than that of pro-inflammatory cytokines in EAT and SWAT in CAD patients with diabetes.(6) The INFγ, M1 macrophage polarization inducer, were significantly increased in EAT and SWAT in diabetic patients (859.2363 ± 81.27950 vs 2438.2843 ±441.08642, P=0.015; 787.0618 ± 73.38717 vs 1634.7678 ± 303.67604, P=0.022), and the M2 macrophage polarization inducer including IL-4 (691.7181 ± 49.64617 vs 1086.2468 ± 122.75524, P=0.014; 644.4628 ± 69.21432 vs 996.8646 ± 74.17361, P=0.006) and IL-13 (864.3688 ± 103.17058 vs 1403.1810 ± 172.13152, P=0.023; 686.7570 ± 143.05874 vs 1126.0514 ± 116.3674, P=0.038) were also significantly increased in EAT and SWAT in CAD patients with diabetes;4. Multivariate linear regression model for Gensini score is as follows: Gensini score= 0.454×(M1/M2 macrophages ratio in EAT)+0.392×LDL-c, M1/M2 macrophages ratio in EAT (P=0.012) and LDL-c (P=0.028) were enrolled, among which M1/M2 macrophages ratio in EAT and LDL-c were risk factors;Multivariate linear regression model for M1/M2 macrophages ratio in EAT is as follows:M1/M2 macrophages ratio in EAT=0.631 ×fasting glucose +0.370× WC, fasting glucose (P<0.001) and WC (P=0.001) were enrolled, among which fasting glucose and WC were risk factors;5. Real-time PCR:Compared with non-diabetic CAD patients, the mRNA levels of FAS (1.00000 ± 0.00000 vs 3.71400 ± 0.57180, P=0.029; 1.00000 ± 0.00000 vs 2.4557 ± 0.44288, P=0.044) and leptin (1.00000 ± 0.00000 vs 5.53510 ± 1.07432, P=0.013; 1.00000 ± 0.00000 vs 3.699030 ± 0.52269, P<0.0001) were significantly increased, whereas the mRNA levels of Perilipin (1.00000 ± 0.00000 vs 0.55770 ± 0.11265, P=0.017; 1.00000 ± 0.00000 vs 0.76250 ± 0.10055, P=0.046), ATGL (1.00000 ± 0.00000 vs 0.50700 ± 0.10337, P=0.009; 1.00000 ± 0.00000 vs 0.64070 ± 0.06643, P=0.006), HSL (1.00000 ± 0.00000 vs 0.20020 ± 0.05518, P<0.0001; 1.00000 ± 0.00000 vs 0.60580 ± 0.10979, P=0.023) and Adiponectin (1.00000 ± 0.00000 vs 0.643 ± 0.11482, P=0.021; 1.00000 ± 0.00000 vs 0.7205 ± 0.10295, P=0.035) were significantly decreased in EAT and SWAT in CAD patients with diabetes.6. Western blot:Compared with non-diabetic CAD patients, the proteins of IRS1 (0.44120 ± 0.06505 vs 0.21960 ± 0.03455, P=0.013; 0.53570 ± 0.08059 vs 0.29790 ± 0.04345, P=0.027) and GLUT4 (0.92950 ± 0.07987 vs 0.64970 ± 0.05241, P=0.015; 1.01070 ± 0.04565 vs 0.86180 ± 0.03942, P=0.009) were significantly decreased, whereas TRB3 (0.68150 ± 0.12607 vs 1.15790 ± 0.18196, P=0.035; 0.48040 ± 0.10520 vs 0.81890 ± 0.09720, P=0.040) was significantly increased accompanied by down-regulated PPARy (0.93050 ± 0.11089 vs 0.49890 ± 0.04768, P=0.010; 1.09010 ± 0.09385 vs 0.75260 ± 0.08006, P=0.021) in EAT and SWAT in CAD patients with diabetes.Conculusions1. Compared with non-diabetic CAD patients, the numbers of macrophages including M1 and M2 macrophages and M1/M2 macrophages ratio were significantly increased in EAT and SWAT in CAD patients with diabetes, with severe CAD;2.M1/M2 macrophages ratio in EAT was positively associated with Gensini score;3.The increase of M1/M2 macrophages ratio in EAT played an important part in the aggravation of CAD in CAD patients with diabetes;4. Compared with non-diabetic CAD patients, TRB3 was significantly increased accompanied by down-regulated PPARy in CAD patients with diabetes, suggesting that TRB3/PPARγ signaling pathway might play a vital role in the aggravation of CAD in CAD patients with diabetes;.BackgroundDiabetes has been recognized as an equivalence of coronary artery disease (CAD). However, the mechanism underlying the development and aggravation of atherosclerosis in patients with diabetes remains incompletely understood. Clinical studies demonstrated that obesity was an independent risk factor of insulin resistance (IR) and cardiovascular disease. The epidemic of obesity is closely associated with the incidence of diabetes-related cardiovascular disease. However, there are no effective drugs to treat obesity. Rajesh and Ajay have confirmed that activation of immune system activity was capable of treating obesity and diabetes. Immune system and metabolism are integrated, in which large numbers of immune cells reside in adipose tissue contribute to the pathogenesis of adipose tissue inflammation, leading to obesity-related metabolic dysfunction. However, the underlying mechanism has not yet been fully elucidated.Adipose tissue inflammation involves abnormal adipose tissue macrophages (ATMs) accumulation. Accumulating evidences suggests that adipocytes secrete a variety of bioactive molecules such as MCP-1, contributing to macrophages infiltration. Adipocytes and macrophages colocalize in adipose tissue in obesity. ATMs consist of classically activated Ml macrophages and alternatively activated M2 macrophages, and M2 macrophages consist of M2a、M2b and M2c macrophages.Compared with the increase in the number of macrophages, the increase in M1/M2 macrophages ratio was more closely associated with adipose tissue dysfunction. However, the underlying relevance of dysfunctional adipocytes leading to the phenotypic switch of macrophages remains incompletely understood.PPARy, one of the nuclear receptor superfamily, is highly expressed in adipose tissue. PPARy-deficient macrophages are resistant to M2 polarization and PPARy ligands switched high fat diet-induced macrophage M2b polarization toward M2a. Therefore, PPARy is a key regulator of macrophage polarization. Tribbles homolog 3 (TRB3), negatively regulating PPARy transcriptional activity, suppressed adipocyte differentiation and induced IR. To identify the vicious circle between dysfunctional adipocytes and activated macrophages, we constructed IR adipocyte model and explored the interaction between adipocytes function and macrophage phenotypic switches. Then, we used TRB3 gene silencing in vitro to explore the mechanism of TRB3 in the vicious circle between adipocytes dysfunction and macrophages polarization as a potential target for treatment of obesity, diabetes and diabetes-related cardiovascular disease.Objectives1. To construct IR adipocyte model and explore the effects of dysfunctional adipocytes on M1/M2 macrophages ratio and the macrophage phenotype switch between M2a and M2b;2. To explore the effects of the change of M1/M2 macrophages ratio and the macrophage phenotype switch between M2a and M2b on adipocyte function;3. To explore the effects of TRB3/PPARy signaling pathway on macrophage polarization and corresponding adipocyte function changes.Methods1. Construction of IR adipocyte model:3T3-L1 preadipocytes were cultured in DMEM containing 10% FBS, and differentiated in a cocktail containing insulin, isobutylmethylxanthine and dexamethason based on a standard protocol. IR was induced by incubating differentiated 3T3-L1 adipocytes in DMEM containing 25mM glucose,10% FBS and 10nM insulin for 24h;2. M2a macrophage differentiation:M2a macrophage polarization was induced in M2a macrophage conditioned medium (RPMI 1640 full medium containing 20ng/ml of M-CSF and 20ng/ml of IL-4) for 4 days;3. ELISA:Conditioned medium (CM) was collected from adipocytes and the expression levels of INFγ, IL-4 and IL-13 in CM were detected by ELISA;4. Macrophages were stimulated with adipocytes CM:CM were collected from control or IR adipocytes and used to stimulate macrophages for 48h;5. Co-culture of macrophages and adipocytes:Co-culture of adipocytes and macrophages (RAW264.7 or M2a macrophages) in the transwell system were performed for 48h, in which adipocytes were cultured in an insert on a semi-permeable membrane (0.4μm, Corning, NY, USA) and macrophages were grown in the lower chamber of the well;6. Flow cytometry:The ratios of M1 macrophages (CDllc+) and M2 macrophages (CD206+) were analyzed by flow cytometry;7. Real-time PCR:The mRNA of TNFα, IL-10, MR and Dectin-1 were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green method;8. Assessment of macrophage functions:the migration, adhesion and phagocytosis of macrophages were assessed;9. Western bot:The proteins of FASN, ATGL, HSL, GLUT4, IRS-1, TRB3 and PPARy were detected by western blotting.Results1. Construction of IR adipocyte model:adipocytes were incubated with high glucose (25mM) and high insulin (lOnM); IRS-1 protein level was significantly reduced at 24h compared with Oh (0.6783±0.11294 vs 0.2465±0.0796, P<0.01); The protein level of membranous GLUT4 was significantly decreased (1.00±0.00 vs 0.4318±0.09309, P<0.01), whereas cytoplasmic GLUT4 protein level was significantly increased (1.00±0.00 vs 1.7673±0.0975, P<0.001) at 24h compared with Oh; Thus, high glucose and high insulin for 24h decreased IRS-1 protein level and glucose transport, inducing IR in adipocytes.2. Macrophages stimulated with adipocytes CM:(1) The expression levels of macrophage polarization inducers of adipocytes CM:Compared with control adipocytes CM, the expressions of INFy were significantly increased (27.9833±3.7226 vs 76.0333±5.85172, P<0.01) in IR adipocytes CM, as well as the expressions of IL-4 and IL-13 (3.02±0.078156 vs 7.63±0.20518, P<0.05; 14.1567±0.32271 vs 21.5367±0.54229, P<0.001); The increased degree of INFy was greater and INFy/IL-13 ratio was significantly increased in IR adipocytes CM compared with control adipocytes CM (1.9671±0.21447 vs 3.863±0.23059, P<0.001), suggesting that IR adipocytes induced macrophages phenotypic switch.(2) Macrophage phenotype switches stimulated by adipocytes CM:Compared with the stimulation of Control adipocytes CM, the M1/M2 macrophages ratio was significantly increased by IR adipocytes CM (1.1425±0.18347 vs 5.3525±0.41085, P<0.01);(3) Macrophage functions changes stimulated by adipocytes CM:Compared with the stimulation of Control adipocytes CM, the function of adhesion (15.3333±2.02759 vs 55.0000±5.2915, P<0.01), migration (24.6667±4.91031 vs 124.3333±13.01708, P<0.01) and phagocytosis (14.7267±6.96525 vs 48.4667±4.90453, P<0.05) of macrophages were all significantly increased by IR adipocytes CM.3. Co-culture of macrophages and adipocytes:(1) Co-culture of adipocytes and Raw264.7 macrophages:Compared with Control+ vehichle co-culture group, M1/M2 macrophages ratio was significantly increased in IR+ vehichle co-culture group (1.4236±0.24252 vs 5.8494±0.1611, P<0.05);Compared with Control+ vehichle co-culture group, the functions of adhesion (19.6667±1.45297 vs 55.3333±6.35959, P<0.05), migration (27.3333±6.00925 vs 140.3333±15.60271, P<0.05) and phagocytosis (10.56±3.54056 vs 57.4667±3.92825, P<0.05) of macrophages were all significantly increased in IR+ vehichle co-culture group;(2) Co-culture of adipocytes and M2a macrophages:Compared with Control+ vehichle M2a co-culture group, the expressions of TNFa (1.0000±0.00000 vs 10.1741±0.54125, P<0.05) and IL-10 (1.0000±0.00000 vs 9.8212±1.45653, P<0.05) were significantly increased and the expressions of MR (1.0000±0.00000 vs 0.1085±0.01546, P<0.05) and Dectin-1 (1.0000±0.00000 vs 0.0559±0.02036, P<0.05) were significantly reduced in IR+ vehichle M2a co-culture group; The expression of IL-23 (Ml macrophage) was not detected in IR+ vehicle M2a co-culture group. These observations, taken together, suggested the macrophage phenotypic switch from M2a (TNFαlow, IL-10low, MRhigh, Dectin-1high) to M2b (TNFαhigh, IL-10high, MR, Dectin-1) induced by IRt adipocytes.Compared with Control+ TRB3vehichle M2a co-culture group, the functions of adhesion (14.6667±1.45297 vs 29.0000±5.2915, P<0.05) and migration (8.6667±1.76383 vs 28.0000±3.4641, P<0.05) of macrophages were all significantly increased in IR+ vehichle co-culture group; There were no significant differences in the function of phagocytosis between the two groups (86.13±5.464 vs 96.87±0.94, P>0.05);(3) The effects of M1/M2 macrophages ratio change on adipocyte functions: Compared with Control+ vehichle co-culture group, the protein of FASN (0.2886±0.05638 vs 0.6524±0.01138, P<0.05) was significantly increased, and the proteins of ATGL (0.3015±0.04058 vs 0.0928±0.00907, P<0.05), HSL (0.7425±0.0442 vs 0.3727±0.03422, P<0.05) and IRS-1 (0.7351±0.07402 vs 0.3363±0.02335, P<0.05) were significantly reduced in IR+ vehichle co-culture group, along with increased M1/M2 macrophages ratio, suggesting that the increase of M1/M2 macrophages ratio caused adipocyte dysfunction;(4) The effects of macrophage phenotype switch on adipocyte functions: Compared with Control+ vehichle M2a co-culture group, the protein of FASN (0.3224±0.02885 vs 0.5285±0.07128, P<0.05) was significantly increased, and the proteins of ATGL (0.306±0.033336 vs 0.1462±0.03311, P<0.05), HSL (0.7043±0.03063 vs 0.2392±0.03717, P<0.05) and IRS-1 (0.6836±0.02287 vs 0.3302±0.01423, P<0.05) were significantly reduced in IR+ vehichle co-culture M2a group, along with macrophages polarization from M2a to M2b, suggesting that the macrophages polarization from M2a to M2b caused adipocyte dysfunction;4. The effects of TRB3 silencing and PPARy inhibitor on macrophage polarization and corresponding adipocytes functions(1) The effects of TRB3 silencing on M1/M2 macrophages ratio and corresponding adipocytes functions:Compared with IR+ vehichle co-culture group, M1/M2 macrophages ratio was significantly reduced in IR+TRB3shRNA co-culture group (5.38494±0.1611 vs 2.7752±0.1482, P<0.05);Compared with IR+ vehichle co-culture group, the protein of FASN (0.6524±0.01138 vs 0.4978±0.02013, P<0.05) was significantly decreased, and the proteins of ATGL (0.0928±0.00907 vs 0.2284±0.02413, P<0.05), HSL (0.3727±0.0342 vs 0.6234±0.03253, P<0.05) and IRS-1 (0.3363±0.02335 vs 0.5457±0.049, P<0.05) were significantly increased in IR+ TRB3shRNA co-culture group, suggesting that TRB3 silencing ameliorated adipocyte dysfunction along with decreased M1/M2 macrophages ratio;(2) The effects of TRB3 silencing on macrophages phenotype switch between M2a and M2b and corresponding adipocytes functions:Compared with IR+ vehichle M2a co-culture group, the expressions of TNFa(10.1741±0.54125 vs 4.078±0.32861, P<0.05) and IL-10 (9.8212±1.45653 vs 4.9922±0.52921, P<0.05) were significantly decreased and the expressions of MR (0.1085±0.01546 vs 0.3037±0.05341, P<0.05) and Dectin-1 (0.0559±0.02036 vs 0.4244±0.03933, P<0.05) were significantly increased in IR+ TRB3shRNA M2a co-culture group; TRB3 silencing significantly decreased macrophage phenotypic switch from M2a to M2b induced by IR adipocytes;Compared with IR+ vehichle M2a co-culture group, the protein of FASN (0.5285±0.07128 vs 0.3591±0.03328, P<0.05) was significantly decreased, and the proteins of ATGL (0.1462±0.03311 vs 0.2508±0.02857, P<0.05), HSL (0.2392±0.03717 vs 0.3945±0.06253, P<0.05) and IRS-1 (0.3302±0.01423 vs 0.4754±0.03142, P<0.05) were significantly increased in IR+ TRB3shRNA M2a co-culture group, suggesting that TRB3 silencing ameliorated adipocyte dysfunction along with decreased macrophage phenotypic switch from M2a to M2b;(3) The effects of PPARy inhibitor on M1/M2 macrophages ratio and corresponding adipocytes functions:Compared with IR+ TRB3shRNA co-culture group, M1/M2 macrophages ratio was significantly increased in IR+ TRB3shRNA +GW9662 co-culture group (2.7752±0.1482 vs 4.65±0.27095, P<0.05), suggesting that PPARy inhibitor reversed the decreased effect of TRB3 silencing on M1/M2 macrophages ratio;Compared with IR+ TRB3shRNA co-culture group, the protein of FASN (0.3564±0.0451 vs 0.6689±0.04726, P<0.05) was significantly increased, and the proteins of ATGL (0.709±0.06562 vs 0.4023±0.07475, P<0.05), HSL (0.7144±0.05866 vs 0.3132±0.10777, P<0.05) and IRS-1 (0.9044±0.09809 vs 0.6102±0.03064, P<0.05) were significantly decreased in IR+ TRB3shRNA +GW9662 co-culture group, suggesting that PPARy inhibitor aggravated IR of adipocytes;(4) The effects of PPARy inhibitor on macrophages phenotype switch between M2a and M2b and corresponding adipocytes functions:Compared with IR+ TRB3shRNA M2a co-culture group, the expressions of TNFα (1.0000±0.00000 vs 3.6803±0.18148, P<0.05) and IL-10 (1.0000±0.00000 vs 3.3744±0.64555, P<0.05) were significantly increased and the expressions of MR (1.0000±0.00000 vs 0.2635±0.03888, P<0.05) and Dectin-1 (1.0000±0.00000 vs 0.4154±0.04681, P<0.05) were significantly decreased in IR+ TRB3shRNA+ GW9662 M2a co-culture group, suggesting that PPARy inhibitor promoted macrophage switch from M2a to M2b;Compared with IR+ TRB3shRNA M2a co-culture group, the protein of FASN (0.2906±0.0402 vs 0.4728±0.02693, P<0.05) was significantly increased, and the proteins of ATGL (0.725±0.03671 vs 0.3835±0.04293, P<0.05), HSL (1.0795±0.11401 vs 0.7006±0.05019, P<0.05) and IRS-1 (0.6893±0.01518 vs 0.3655±0.06295, P<0.05) were significantly decreased in IR+ TRB3shRNA +GW9662 M2a co-culture group;5. The interaction between TRB3 and PPARγ:Compared with Oh, TRB3 protein levels were significantly increased at 12h,24h and 48h in macrophage with the treatment of IR adipocytes CM (0.6437±0.04636 vs 0.7816±0.03266 vs 1.0100±0.0975 vs 1.2375±0.08942, P<0.05); On the contrary, compared with Oh, PPARγ protein levels were significantly decreased at 24h and 48h in macrophage with the treatment of IR adipocytes CM (0.3363±0.02182 vs 0.3929±0.01287 vs 0.1844±0.011, P<0.05); The interaction between TRB3 and PPARy was analyzed by a coimmunoprecipitation method and found that the combination between TRB3 and PPARy was significantly increased by IR adipocytes. Silencing TRB3 increased the expressions of PPARγ in co-culture system (0.1762±0.02853 vs 0.28829±0.00807, P<0.05), along with decreased M1/M2 macrophages ratio. These observations, taken together, suggest that TRB3/PPARγ signaling pathway played an important role in the transformation of macrophage polarization.Conclusions1. Compared with control adipocytes, IR adipocytes promoted the increase of M1/M2 macrophages ratio and macrophage polarization from M2a to M2b;2. The increase of M1/M2 macrophages ratio and macrophage polarization from M2a to M2b promoted adipocyte dysfunction;3.TRB3 gene silencing improved adipocyte dysfunction through the inhibition of the increase of M1/M2 macrophages ratio and macrophage polarization from M2a to M2b;4.TRB3/PPARγ signaling pathway played an important role in the transformation of macrophage polarization.BackgroundThe epidemic study shows that the number of diabetic patients is growing at an alarming rate and China will be one of the two countries owning most of diabetic patients in the world till 2030. Diabetes is the major risk factor of many diseases including atherosclerotic heart diseases. Insulin resistance is a major characteristic of type 2 diabetes. Adipose tissue is the initial site of insulin resistance. This has highlighted the importance and urgency of studying the mechanism of insulin resistance of adipose tissues and providing the theoretical basis for therapy of diabetes-related diseases.Adipose tissue consists of white and brown adipose tissues (WAT and BAT). Most of the previous studies focused on the roles of WAT or BAT in insulin resistance respectively. However, few studies have compared the differences of the roles of adipose tissues in different regions of the same organism in insulin resistance. WAT consists of visceral and subcutaneous adipose tissues (SWAT). Compared with SWAT, visceral white adipose tissue is more associated with insulin resistance. It has been reported that BAT is also associated with insulin resistance. The unbalance of production of pro-inflammatory and anti-inflammatory cytokines in adipose tissues is associated with insulin resistance. The expression levels of inflammatory cytokines were determined by the balance of M1 macrophages and M2 macrophages. However, the underlying mechanism of macrophage polarization remains unknown.Recently six transmembrane protein of prostate 2 (STAMP2 or STEAP4) has been reported as a counterregulator of inflammation and insulin resistance. Wellen reported that the visceral depot had a much stronger phenotype than the subcutaneous depot in STAMP2 deficiency in STAMP2-/- mice. But there is no report about STAMP2 expression in brown adipose. STAMP2 deficiency markedly increased macrophages infiltration in adipose tissues, but whether it is responsible for the macrophage polarization shift remains a question.With the aim of evaluating the influence of STAMP2 on inflammation and macrophages infiltration of adipose tissues in different regions of diabetic animals, we constructed type 2 diabetic ApoE-/-/LDLR-/- mice model with STAMP2 gene overexpression in vivo, and studied the effects of STAMP2 overexpression on macrophages infiltration and polarization, inflammatory adipocytokines expression and corresponding signal pathway. We hypothesized that STAMP2 might play a major role in the mechanism of macrophage polarization shift, by which activation of STAMP2 improved insulin resistance.Objectives1. To explore the expressions of STAMP2 in different regions of adipose tissues in diabetic ApoE-/-/LDLR-/- mouse;2. To explore the differences of macrophages and M1/M2 macrophages ratios in different regions of adipose tissues in diabetic ApoE-/-/LDLR-/- mouse;3. To explore the effects of STAMP2 overexpression on macrophages infiltration and M1/M2 macrophages ratios in different regions of adipose tissues in diabetic ApoE-/-LDLR-/- mouse.Methods1. Construction of diabetic ApoE-/-/LDLR-/- mouse model:thirty two 3-week-old male ApoE-/-/LDLR-/- mice were randomly divided into Control group (n=16) and high-fat diet group (n=16). Then, the mice of high-fat diet group were fed on high-fat diet, after 6 weeks, those mice showing insulin resistance were injected once with low dose of STZ (75mg/kg) intraperitoneally. At age 11 weeks, mice with similar degrees of hyperglycemia and body weight were regarded as diabetic mouse model;2. Groups of animal experiment:At age 20 weeks, ApoE-/-/LDLR-/- mice were divided into Control+ Vehicle group (n=6), Control+ STAMP2 (n=10), DM+ Vehicle (n=6) and DM+ STAMP2(n=10);3. IPGTT test:mice were fasted for 10-16h before glucose tolerance tests. Intraperitoneal injection glucose load was administered at 2g/kg of body weight. Glucose levels were measured from tail bleeds with a glucometer at Omin, 15min,30min,60min and 120min after glucose administration;4. Monitor body weight of ApoE-/-/LDLR-/- mice every two weeks;5. Pathology analyses:HE staining; Immunochemistry assay was performed to detect macrophages (F4/80+), Ml macrophages (CD11c+), M2 macrophages (CD206+) and calculate M1/M2 macrophages ratio; Immunochemistry assay was performed to detect pro-inflammatory factors including MCP-1, TNFα, IL-6 and anti-inflammatory factors IL-10; Oil red O staining was used to detect lipid content in liver;6. Western bot:The proteins of STAMP2, p-JNK and JNK were detected by western blotting.Results1. Construction of diabetic ApoE-/-/LDLR-/- mouse model:At age 3 weeks, there were no significant differences in the levels of blood glucose between the control and diabetic mice confirmed by IPGTT. After 6-week high-fat diet, the levels of blood glucose in the diabetic group were significantly higher at week 9 than the control group at 15min,30min,60min and 120min (P<0.05 for all conditions). The AUC across the time for glucose level was higher at week 9 than baseline (,P<0.05 for all conditions). At age 11 weeks, compared with the control group, the levels of blood glucose in the diabetic group were significantly elevated at Omin,15min, 30min,60min and 120min (P<0.05 for all conditions). The AUC across the time for glucose level was higher at week 11 than week 9.The mean body weight was significantly higher for the diabetic group than the control group at the age of 9,20,22 and 24 weeks (P<0.05 for all conditions).2. The expressions of STAMP2 protein in adipose tissues in ApoE-/-/LDLR-/-mouse(1) Compared with Control group, endogenous STAMP2 expression was significantly decreased in epididymal white adipose tissue (EWAT) and BAT in diabetic ApoE-/-/LDLR-/- mice (0.9596 ± 0.0268 vs 0.5416 ± 0.06812, P=0.002; 1.0120 ± 0.10051 vs 0.6134 ± 0.03813, P=0.004); there were no significant differences in STAMP2 expression in SWAT between the two groups (0.0214 ± 0.00474 vs 0.0162 ± 0.00151, P>0.05);(2) Compared with Control+Vehicle group, the expressions of STAMP2 were significantly elevated in EWAT and BAT in Control+STAMP2 (0.9596 ± 0.0268 vs 1.2114 ± 0.04363,P=0.019; 1.0120 ± 0.10051 vs 1.1233 ± 0.05992,P=0.020); there were no significant differences in STAMP2 expression in SWAT between the two groups (0.0214 ± 0.00474 vs 0.0271 ± 0.00729, P>0.05);(3) Compared with DM+ Vehicle group, the expressions of STAMP2 were significantly elevated in EWAT and BAT in DM+ STAMP2 group (0.5416 ± 0.06812 vs 0.7783 ± 0.09189, P=0.028; 0.6134 ± 0.03813 vs 0.8475 ± 0.06312, P=0.044); there were no significant differences in STAMP2 expression in SWAT between the two groups (0.0162 ± 0.00151 vs 0.0189 ± 0.00197, P>0.05);3. The effect of STAMP2 overexpression on adipose tissue morphology(1) Compared with Control group, the adipocyt...