| BackgroundDiabetes mellitus, as CHD risk equivalents, has got the extensive consensus. The risks of cardiovascular complications are markedly increased. Diabetic cardiomyopathy (DCM), which occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality. However, the underlying mechanism of DCM has not been clarified, so there was no a targeted treatment strategy.Therefore, it is important to choose an appropriate diabetic animal model for each of diabetes when diabetic cardiomyopathy research. In the present, experimental models of diabetes can be obtained by high-fat diet and/or chemical induction, or the use of spontaneous or genetically derived animal strains. The main limitations of these methodologies include four major parts. The first one is that genetic models cannot completely mimic human disease, so the extrapolation of relationships seen with human disease to rat is not straightforward. The second one is that the effect of metabolism-associated gene knockout on DCM cannot be excluded which could not be intervened. The third one is that an excess of chemical results in type1diabetes. The last one is that long-term high fat diet induced diabetic models take a long time, and can not be sustained over the long term.Thus, the present study focuses on the establishment of diabetic rat model induced by high-fat diet combined with a small dose of STZ. Then monitoring weight, blood pressure, blood glucose, blood lipids, glucose tolerance, insulin tolerance and ultrasound biomicroscopy (UBM) were performed to detect the initiation and development of DCM, investigating the feasibility of establishing the type2diabetic rat model.Objectives 1. To confirm the feasibility of establishing diabetic rat model induced by high-fat diet combined with a small dose of STZ;2. To confirm the feasibility of establishing diabetic cardiomyopathy rat model induced by high-fat diet combined with a small dose of STZ.Methods1. Generation of Diabetes.Sixty male Sprague-Dawley (SD) rats (120-140g) were purchased from the experimental animal center of Shandong University of Traditional Chinese Medicine (Jinan, China). After1week of acclimatization, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IP ITT) were performed. Then the rats were randomized into4groups:control, chow+streptozotocin (STZ), high-fat diet (HF) and diabetes mellitus (DM). HF and DM groups were fed a high-fat diet (34.5%fat,17.5%protein,48%carbohydrate, Beijing HFK Bio-Technology Co. Ltd, China), and the other2groups received normal chow. Four weeks later, IPGTT and IP ITT were performed again, and blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index [ISI=ln(FBG×FINS)-1] was calculated. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (Sigma, St. Louis, MO)(27.5mg·kg-1intraperitoneally [ip] in0.1mol/1citrate buffer, pH4.5) to rats with insulin resistance. Rats in the chow+STZ group received the same dose of STZ. The control and HF groups received citrate buffer (ip) alone. One week after STZ administration, rats with FBG>11.1mmol/1in2consecutive analyses were considered the diabetic rat model. After16weeks of diabetes, rats were sacrificed. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee.2. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT).Glucose tolerance was assessed by IPGTT after rats fasted for12h. A bolus of glucose (1g/kg ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60, and120min and tested for glucose. Plasma glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The mean area under the receiver operating characteristic curve (AUC) was calculated for glucose. To evaluate insulin tolerance, IPITT was performed after rats fasted for4h. A bolus of insulin (1unit/kg ip) was administered, and blood samples were taken for glucose measurement as described above.3. Blood analyses.After rats fasted overnight, we collected jugular blood. Total cholesterol (TC), triglycercide levels (TG) and fasting blood glucose (FBG) were analyzed with use of the Bayer1650blood chemistry analyzer (Bayer, Tarrytown, NY, USA). Free fatty acids (FFA) concentrations were measured using an enzymatic test kit (CSB-E0877Or, HuaMei BIO-TECH, Wuhan, CHINA). Fasting insulin level was measured by ELISA. ISI was calculated.4. Measurements of blood pressures and heart rate.Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan).5. Echocardiography.Echocardiography involved use of the Vevo770imaging system (VisualSonics, Inc, Toronto, Canada). Images were obtained from2-D, M-mode, pulsed-wave (PW) Doppler and tissue Doppler imaging (TDI). All measurements were performed by the same observer and were the average of6consecutive cardiac cycles. Wall thickness and left ventricle dimensions were obtained from a long-axis view at the level of chordae tendineae. Diameters of left ventricular end-diastole (LVEDd), end-diastolic posterior wall (LVPWd) and septum thickness (IVSd), as well as ejection fraction (LVEF) and fractional shortening (FS) were measured according to the American Society of Echocardiography guidelines. The mitral-valve pulsed Doppler recordings were obtained from the apical four-chamber view. After pulsed Doppler, we evaluated transmitral flow velocity variables, including peak E, peak A, and the E/A ratio. Isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT) and ejection time (ET) were measured and were used to calculate the Tei index (Tei index=IVCT+IVRT/ET). TDI of the mitral annulus was obtained from the apical four-chamber view. We analyzed early (E') and late diastolic velocity (A') and calculated E'/A' and E/E'.For quantitative analysis of integrated backscatter (IBS) of the left ventricle, we used a commercially available software package (acoustic densitometry; Phillips Medical Systems, Netherlands).2-D echocardiographic images, including left ventricle long-axis and apical four-chamber views were obtained. The following variables were measured:time-averaged integrated backscatter (IBS), standardized integrated backscatter (IB%) and cyclic variation of integrated backscatter (CVIB).6. Hemodynamic measurement.Rats under deep anesthesia underwent hemodynamic measurement. A fluid-filled catheter was advanced from the right carotid artery into the left ventricle, and the left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) was measured.Results1. General characteristics of diabetic ratsThe rats in DM group had the highest values of water intake, food intake and urine volume as compared with the other3groups (P<0.01～P<0.001). Heart weight and HW/BW were significantly higher in the DM group than control group (P<0.01). No significant differences were seen in body weight in DM group when compared to the control.2. Glucose and insulin tolerance after a4-week HF dietThe HF and DM groups showed impaired glucose tolerance on IPGTT as compared with the control and chow+STZ groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the HF and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity. The DM and HF groups showed the higher mean AUC on IPITT. Thus, the DM group showed insulin resistance after a4-week HF diet.3. Glucose and insulin tolerance at12weeks after the onset of diabetesThe DM group showed impaired glucose tolerance on IPGTT as compared with the control and HF groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the chow+STZ and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity; especially blood glucose levels in DM group were significantly elevated at all time points as compared with the control (P<0.05～P<0.01).The DM group showed the highest mean AUC on IPITT when compared to the other3groups.4. Glucose and insulin tolerance at the end of the experimentThe DM group showed impaired glucose tolerance on IPGTT as compared with the other3groups; especially blood glucose levels were significantly elevated at all time points as compared with the control (P<0.05). The DM group showed the highest mean AUC on both IPGTT and IPITT. Similarly, the chow+STZ and HF groups showed higher mean AUC on both IPGTT and IPITT as compared with the control (P<0.05).5. TC, TG, FFA, and FBG concentrationsAfter4weeks of a high-fat diet, serum TG and FFA levels were significantly higher in the HF and DM groups than control and chow+STZ groups (P<0.05). The ISI was markedly decreased in the HF and DM groups (P<0.05). Insulin resistance appeared at week4in rats fed a HF diet. One week after STZ injection, FBG was remarkably elevated in the DM group and remained elevated until the end of the experiment. ISI consistently decreased in the DM group after the onset of diabetes. Simultaneously, in the DM group, serum TC, TG, and FFA levels were maintained at higher levels than the control (P<0.05) during diabetes. Thus, the diabetic model induced by a HF diet and low-dose STZ was characterized by insulin resistance, moderate hyperglycemia and hyperlipidemia resembling the state of chemical diabetes in humans.6. Blood pressures and heart rate:there were no differences in SBP, DBP and MAP among4groups during the experimental process. HR was higher in HF group compared with the control and chow+STZ groups at the end of the experiment.7. Left ventricle (LV) dysfunction assessed by echocardiographyWe evaluated EF, fractional shortening (FS), E/A, and E'/A' to investigate changes in systolic and diastolic function. Similar to the pattern in humans, in rats, diastolic dysfunction precedes systolic dysfunction, beginning from2to3months after the induction of DM. In our study, at6weeks after the onset of diabetes (at week11), the DM rats showed a moderate decrease in E/A and E'/A' as compared with the control (P<0.05); LVEF and FS were impaired from week17. At the end of the experiment, LVEF, FS, E/A, and E'/A' were further decreased in the DM group, with the reduction in E/A and E'/A' more pronounced. LVEF, FS, E/A, and E'/A' were also reduced in the chow+STZ group compared with the control (P<0.05) at the end of the experiment. LVEDd was the highest in the DM group. Additionally, LVEDD, IVRT, IVCT, and Tei index were significantly increased in DM group compared with the control(P<0.01-P<0.001) at the end of the experiment.The DM group showed increased IB%and decreased cyclic variation of integrated backscatter (CVIB) in the IVS, LV posterior and lateral walls as compared with the control group (P<0.05) from6weeks after the onset of diabetes (at week11). And this deteriorated at the end of the experiment.8. ElectrocardiogramThere was obvious arrhythmia in DM groups at the end of experiment.9. CatheterizationTo further confirm the LV diastolic dysfunction, LVEDP was measured by cardiac catheterization. DM rats had the highest LV pressure. The chow+STZ and HF groups showed higher LV pressure as compared with the control (P<0.01～P<0.001). In summary, both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function.Conclusions1. The combination of high-fat diet and low-dose streptozotocin would successfully establish type2diabetic rat model, resembling human diabetes mellitus;2. Both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function;3. The type2diabetic rat model was appropriate for gene intervention. BackgroundDiabetic cardiomyopathy (DCM), which occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality. Different pathophysiological stimuli are involved in its development and mediate tissue injury leading to left ventricle (LV) systolic and diastolic dysfunction. Insulin resistance is considered to play a causal role in the pathogenesis and development of DCM. Insulin resistance is associated with increased LV mass and deterioration of LV diastolic function. However, the underlying relevance of insulin resistance leading to altered myocardial structure remains incompletely understood.Tribbles3(TRB3) is a pseudokinase with increased activity in response to fasting that binds to and inhibits the activation of the serine-threonine kinase Akt in the liver. Indeed, humans with a gain-of-function mutation in TRB3show increased insulin resistance and diabetes-associated complications. These observations have led to the suggestion that TRB3elevation contributes to insulin resistance. TRB3also serves as a molecular switch and regulates the activation of the three classes of mitogen-activated protein kinases (MAPKs). TRB3binds to and regulates MAPK kinase, thus controlling the activation of MAPK by incoming signals. However, the TRB3/MAPK signal-transduction pathway has not been investigated in vivo on cardiac tissues directly.Akt and MAPK are the most important pathways involved in "selective" insulin resistance, and activated MAPK contributes to the development of cardiac fibrosis. So we hypothesized that upregulated TRB3induced by insulin resistance might participate in the pathophysiological process of DCM.Objectives1. We established the type2DCM model and determined the relationships among TRB3expression, cardiac remodeling, and cardiac function in the model.2. To further elucidate the role of TRB3in DCM, we used TRB3gene silencing in vivo to explore the mechanisms of TRB3in DCM as a potential target for treatment.Methods1. To design and synthesize4pieces of siRNA against mouse TRB3according to RNAi principle, then construct pGenesil-1.2-TRB3-shRNA plasmid using the most effective siRNA. Next, pShuttle-Basic-EGFP-TRB3-shRNA plasmid would be synthesized. With the shuttle plasmid, the pAdxsi-TRB3-shRNA was constructed.2. Induction of Diabetes.Sixty male Sprague-Dawley (SD) rats (120-140g) were purchased from the experimental animal center of Shandong University of Traditional Chinese Medicine (Jinan, China). The animals were housed at220C with12-h light-dark cycles. After1week of acclimatization, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. Then the rats were randomized into4groups: control, chow+streptozotocin (STZ), high-fat diet (HF) and diabetes mellitus (DM). HF and DM groups were fed a high-fat diet (34.5%fat,17.5%protein,48%carbohydrate, Beijing HFK Bio-Technology Co. Ltd, China), and the other2groups received normal chow. Four weeks later, IPGTT and IPITT were performed again, and blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index [ISI=ln(FBGXFINS)-1] was calculated. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (Sigma, St. Louis, MO)(27.5mg-k9-1intraperitoneally [ip] in0.1mol/1citrate buffer, pH4.5) to rats with insulin resistance. Rats in the chow+STZ group received the same dose of STZ. The control and HF groups received citrate buffer (ip) alone. One week after STZ administration, rats with FBG>11.1mmol/1in2consecutive analyses were considered the diabetic rat model. After16weeks of diabetes, rats were sacrificed. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee.3. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT).Glucose tolerance was assessed by IPGTT after rats fasted for12h. A bolus of glucose (1g/kg ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60, and120min and tested for glucose. Plasma glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The mean area under the receiver operating characteristic curve (AUC) was calculated for glucose.To evaluate insulin tolerance, IPITT was performed after rats fasted for4h. A bolus of insulin (1unit/kg ip) was administered, and blood samples were taken for glucose measurement as described above.4. Blood analyses.After rats fasted overnight, we collected jugular blood. Total cholesterol, triglycercide levels and fasting blood glucose were analyzed with use of the Bayer1650blood chemistry analyzer (Bayer, Tarrytown, NY, USA). Free fatty acids (FFA) concentrations were measured using an enzymatic test kit (CSB-E08770r, HuaMei BIO-TECH, Wuhan, CHINA). Fasting insulin level was measured by ELISA. ISI was calculated.5. Echocardiography and measurement of BP.Echocardiography involved use of the Vevo770imaging system (VisualSonics, Inc, Toronto, Canada). Images were obtained from2-D, M-mode, pulsed-wave (PW) Doppler and tissue Doppler imaging (TDI). All measurements were performed by the same observer and were the average of6consecutive cardiac cycles. Wall thickness and left ventricle dimensions were obtained from a long-axis view at the level of chordae tendineae. Diameters of left ventricular end-diastole (LVEDd), end-diastolic posterior wall (LVPWd) and septum thickness (IVSd), as well as ejection fraction (LVEF) and fractional shortening (FS) were measured according to the American Society of Echocardiography guidelines. The mitral-valve pulsed Doppler recordings were obtained from the apical four-chamber view. After pulsed Doppler, we evaluated transmitral flow velocity variables, including peak E, peak A, and the E/A ratio. Isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT) and ejection time (ET) were measured and were used to calculate the Tei index (Tei index=IVCT+IVRT/ET). TDI of the mitral annulus was obtained from the apical four-chamber view. We analyzed early (E') and late diastolic velocity (A') and calculated E'/A' and E/E'.For quantitative analysis of integrated backscatter (IBS) of the left ventricle, we used a commercially available software package (acoustic densitometry; Phillips Medical Systems, Netherlands).2-D echocardiographic images, including left ventricle long-axis and apical four-chamber views were obtained. The following variables were measured:time-averaged integrated backscatter (IBS), cyclic variation of integrated backscatter (CVIB) and standardized integrated backscatter (IB%).Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan) as previously described.6. Hemodynamic measurement.Rats under deep anesthesia underwent hemodynamic measurement. A fluid-filled catheter was advanced from the right carotid artery into the left ventricle, and the LVED pressure (LVEDP) was measured.7. Histology and morphometric analysis.Paraformaldehyde (4%)-fixed hearts were bisected transversely at the mid-ventricular level, embedded in paraffin, and cut into4-μm sections. A single myocyte was measured with images captured from hematoxylin and eosin (H&E)-stained sections. The myocyte cross-sectional area was assessed under×400magnification within the left ventricle, and a mean was obtained by quantitative morphometry with automated image analysis (Image-Pro Plus, Version5.0; Media Cybernatics, Houston, TX, USA).Dark green-stained collagen fibers were quantified as a measure of fibrosis in Masson's trichrome-stained sections. The collagen volume fraction (CVF) and perivascular collagen area/luminal area (PVCA/LA) were analyzed by quantitative morphometry with automated image analysis (Image-Pro Plus, Version5.0). CVF was calculated as previously reported. Perivascular collagen was excluded from the CVF measurement. To normalize the area of perivascular collagen around vessels with different sizes, the perivascular collagen content was represented as the PVCA/LA ratio. Interstitial and perivascular fibrosis were evaluated by Picrosirius red staining. Sections were stained with0.5%sirius red (Sigma, St. Louis, MO) in saturated picric acid for25min. Collagen stained an intense red color.Myocardial frozen sections (5-μm) were stained with Oil Red O (Sigma, St. Louis, MO) for10min, washed, and then counterstained with hematoxylin for30s. A Nikon microscope (Nikon, Melville, NY) was used to capture the Oil Red O-stained tissue sections.8. Hydroxyproline analysis. The collagen content of myocardial tissue was determined by hydroxyproline assay. Tissue hydrolysate was detected by use of an ELISA kit (F15649, WESTANG BIO-TECH, SHANGHAI, CHINA). Data were expressed as micrograms collagen per milligram dry weight, with the assumption that collagen contains an average of13.5%hydroxyproline.9. Immunohistochemical staining.Paraffin sections underwent immunohistochemistry by a microwave-based antigen retrieval method. The sections were incubated with primary rabbit polyclonal anti-collagen â… , anti-collagen â…¢, anti-TNF-α, and anti-IL-6antibodies (Abcam, Cambridge, MA, USA) overnight, then a matching biotinylated secondary antibody for30min at37℃. Negative controls were omission of the primary antibody. The stained sections were developed with diaminobenzidine and counterstained with hematoxylin. The results were viewed under a confocal FV1000SPD Laser Scanning microscope (Olympus, Japan).10. Quantitative real-time RT-PCR.Total RNA was prepared with the TRIzol reagent (Gibco/Invitrogen, Carlsbad, CA). RT-PCR was performed using the following primers:β-actin forward5'AGA CCT TCA ACA CCC CAG3', reverse5'CAC GAT TTC CCT CTC AGC3'; brain natriuretic protein (BNP) forward5'GGG CTG TGA CGG GCT GAG GTT3', reverse5'AGT TTG TGC TGG AAG ATA AGA3'; TRB3forward5'TGA TGC TGT CTG GAT GAC AA3'; reverse5'GTG AAT GGG GAC TTT GGT CT3'; TNF-α forward5'CAC GCT CTT CTG TCT ACT GA3'; reverse5'GGA CTC CGT GAT GTC TAA GT3'; IL-6forward5'ACC ACT TCA CAA GTC GGA GG3', reverse5'ACA GTG CAT CAT CGC TGT TC3'. Reactions were carried out on a real-time PCR thermocycler (IQ5Real-Time PCR cycler, Bio-Rad), using SYBR green as fluorescence dye. Relative expression analysis involved the2method.11. Western blot analysis.Western blot analysis was as previously described. We used antibodies against TRB3(Calbiochem, La Jolla, CA), collagen â… , collagen â…¢, TNF-α, IL-6(Abcam, Cambridge, MA, USA), phospho-ERK/ERK, phospho-p38/p38MAPK, phospho-JNK/JNK, and phospho-Akt/Akt (Cell Signaling Technology, Beverly, MA), followed by anti-IgG horseradish peroxidase-conjugated secondary antibody. TRB3, collagen â… , collagen â…¢, TNF-α, and IL-6protein levels were normalized to that of β-actin as an internal control and phospho-specific proteins to total protein.12. Gene silencing of TRB3.Sixty rats were randomized to receive TRB3-siRNA or vehicle treatment. Gene silencing occurred immediately after the appearance of LV diastolic dysfunction. After12weeks of diabetes, both E/A and E'/A' were<1.0as assessed by echocardiography in DM rats. Then animals were injected via the jugular vein with2.5×1010plaque-forming units of an adenovirus harboring TRB3gene (TRB3-siRNA) or a control empty virus (vehicle). Adenovirus transfer was repeated in2weeks. According to our present and previous studies, TRB3level was increased in HF-diet-fed and high fructose-fed rats. Previous studies have shown that insulin resistance was a hallmark of obesityand metabolic syndrome. In light of the interaction between TRB3and insulin resistance, we also investigated the effect of TRB3-siRNA on HF-diet-induced cardiac injury. Four weeks after first adenovirus injection, rats were sacrificed. The heart was excised and weighed.Results1. Generation of type2DCM model(1) General characteristics of diabetic ratsAs expected, the rats in DM group had the highest values of water intake, food intake and urine volume as compared with the other3groups (P<0.01～P<0.001). Heart weight was significantly higher in the DM group than control and chow+STZ groups (P<0.01).(2) Glucose and insulin toleranceâ‘ After a4-week HF diet, insulin resistance was confirmed by IPGTT and IPITT. By IPGTT, the levels of blood glucose in the DM group were significantly higher at week4than at baseline at all time points tested except15min. The AUC across the time for glucose level was higher at week4than at baseline (29.08±1.27vs.25.09±0.73, respectively, P<0.05). Similarly, IPITT revealed impaired insulin sensitivity. Thus, the DM group showed insulin resistance after a4-week HF diet.â‘¡At12weeks after the onset of diabetes (at week17), The DM group showed impaired glucose tolerance on IPGTT as compared with the control and HF groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the chow+STZ and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity; especially blood glucose levels in DM group were significantly elevated at all time points as compared with the control (P<0.05～P<0.01).The DM group showed the highest mean AUC on IPITT when compared to the other3groups.â‘¢At the end of the experiment, the DM group showed impaired glucose tolerance on IPGTT as compared with the other3groups; especially blood glucose levels were significantly elevated at all time points as compared with the control (P<0.05). The DM group showed the highest mean AUC on both IPGTT and IPITT. Similarly, the chow+STZ and HF groups showed higher mean AUC on both IPGTT and IPITT as compared with the control (P<0.05).(3) TC, TG, FFA, and FBG concentrationsAfter4weeks of a high-fat diet, serum TG and FFA levels were significantly higher in the HF and DM groups than control and chow+STZ groups (P<0.05). The ISI was markedly decreased in the HF and DM groups (P<0.05). Insulin resistance appeared at week4in rats fed a HF diet. One week after STZ injection, FBG was remarkably elevated in the DM group and remained elevated until the end of the experiment. ISI consistently decreased in the DM group after the onset of diabetes. Simultaneously, in the DM group, serum TC, TG, and FFA levels were maintained at higher levels than the control (P<0.05) during diabetes. Thus, the diabetic model induced by a HF diet and low-dose STZ was characterized by insulin resistance, moderate hyperglycemia and hyperlipidemia resembling the state of chemical diabetes in humans. (4) Left ventricle (LV) dysfunction assessed by echocardiography and catheterizationWe evaluated EF, fractional shortening (FS), E/A, and E'/A' to investigate changes in systolic and diastolic function. Similar to the pattern in humans, in rats, diastolic dysfunction precedes systolic dysfunction, beginning from2to3months after the induction of DM. In our study, at6weeks after the onset of diabetes (at week11), the DM rats showed a moderate decrease in E/A and E'/A' as compared with the control (P<0.05); LVEF and FS were impaired from week17. At the end of the experiment, LVEF, FS, E/A, and E'/A' were further decreased in the DM group, with the reduction in E/A and E'/A' more pronounced. LVEF, FS, E/A, and E'/A' were also reduced in the chow+STZ group compared with the control (P<0.05) at the end of the experiment. LVEDd was the highest in the DM group.To further confirm the LV diastolic dysfunction, LVEDP was measured by cardiac catheterization. DM rats had the highest LV pressure. The chow+STZ and HF groups showed higher LV pressure as compared with the control (P<0.01～P<0.001). In summary, both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function.(5) Pathology characteristics of diabetic ratsThe HW/BW ratio was25%higher in the DM than control group (P<0.01), and LV myocyte size was40%and31%higher, respectively, than the control and HF groups (P<0.001). Furthermore, the relative mRNA expression of BNP, a marker of LV hypertrophy, was higher in DM than control and HF rats (P<0.05).The DM group showed cardiac fibrosis, with a diffuse, small, patchy and nonuniform pattern, as well as destroyed and disorganized collagen network structure in the interstitial and perivascular areas. CVF and collagen content were higher in the DM than other groups (P<0.05～P<0.001), as was PVCA/LA (P<0.001). These histological changes were confirmed by echocardiographic results. The DM group showed increased IB%and decreased cyclic variation of integrated backscatter (CVIB) in the IVS, LV posterior and lateral walls as compared with the control and HF groups (P<0.05). Similarly, CVF, collagen content and PVCA/LA were significantly increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).Coincident with cardiac dysfunction and hypertrophy, myocardial lipid analysis revealed striking myocardial accumulation of triglycerides in diabetic rats. DM rats had higher oil red O staining areas than other groups. The mRNA expression levels of TNF-α and IL-6were significantly higher in DM group as compared with the control (P<0.05, P<0.01). The protein expression levels of TNF-α and IL-6were significantly increased in DM group as compared with the other three groups. Likewise, the protein expression of TNF-a and IL-6content was increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).(6)Immunohistochemistry and western blot analysis revealed the protein expression of collagen â… and â…¢ content increased in the DM group, and the ratio of collagen â… /â…¢ significantly elevated as compared with the control group (216±16%vs.100±6%, respectively,P<0.001). Likewise, the protein expression of collagen â… and â…¢ content was increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).These results show the established type2DCM model, with insulin resistance, severe LV dysfunction and myocardial remodeling.(7) Activated TRB3/MAPK signaling pathway in DCMCardiac TRB3mRNA and protein expression was significantly increased in DM rats. We detected Akt expression in the myocardium. The phosphorylation of Akt was significantly lower in the DM group than the control and HF groups.Accompanied by TRB3overexpression, the phosphorylation of ERK1/2and JNK was markedly increased, whereas that of p38MAPK was decreased, which suggested that the TRB3/MAPK signaling pathway participates in the pathogenesis of DCM.2. TRB3gene silencing reverses DCM(1) Detection of cardiac and hepatic TRB3expression by western blot analysis after gene silencingCompared with vehicle treatment, TRB3-siRNA treatment conferred downregulated mRNA and protein expression of cardiac TRB3. The mRNA and protein expression of hepatic TRB3was also downregulated as compared to the vehicles.(2) TRB3gene silencing ameliorated metabolismWater intake and urine volume were decreased in the DM group with TRB3-siRNA treatment. The elevated serum levels of TC, TG, FFA and FBG were greatly reduced after4-week transfection. (3) TRB3gene silencing improved glucose tolerance and insulin sensitivityWith TRB3gene silencing, ISI was higher in the DM TRB3-siRNA group than vehicle (-5.17±0.14vs.-5.67±0.14, respectively, P<0.05). With TRB3-siRNA treatment, IPGTT and IPITT results showed blood glucose level significantly lower for TRB3-siRNA treated rats than vehicle group (P<0.05). The AUC for glucose and insulin was lower for the DM than vehicle group (P<0.05). The TRB3-siRNA HF group showed lower mean AUC on both IPGTT and IPITT than vehicle group (P<0.05).(4) Recovery of cardiac function in DCM after TRB3silencingDuring4-week follow-up after TRB3-siRNA treatment, FS and E'/...
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