The Effects And Mechanisms Of Free Fatty Acids On Blood Pressure And Cardiac Function In Rats

Background:Metabolic syndrome (MS) is a clustering character of cardiovascular multifactorial risk. Multifactorial cardiovascular risk is more harmful than only one factor. The environmetal and inherited factors are believed to contribute to the etiology of MS. How to estimate cardiovascular metabolism danger, for MS, reveal its key link of morbidity, and reduce cardiovascular and cerebrovascular events caused by MS is the hot spot researched about MS now and is also to trigger the point of the present project.Because MS is a syndrome including several risk factors, thus it is very important to seek an animal model that can precisely imitate human pathology and physiology process and clinical symptoms of MS. With the age changing and glycogen metabolism road, MS model animal induced by high fat diet and made up from fat is similar to human. Therefore, MS model of rat induced by high fat diet was the ideal animal model reflecting metabolizing mechanism.Cardiovascular events are the most important ending points of MS, and MS has influence on cardiac function of all components. Studies suggest that, the rise of free fatty acids (FFA) levels is closely related to heart dysfunction of MS. The Increasing of FFA blood levels can cause insulin resistance in peripheral tissues, and in insulin resistance state, the multiple risk factors such as hypertension and hyperinsulinemia can lead to the damage on heart function. When free fatty acids of blood circulation increased, exceeding storage capacity of adipose tissue and the organizational ability of FFA oxidation, excessive FFA deposit in cardiac in the form of triglyceride (TG) and then injure the cardiac, known as "lipotoxicity Heart Disease". The relation between radio activation of RAAS and myocardial TG deposition is a current research direction. RAAS beside the myocardium played an important role in the process of remodeling myocardium and changing heart function. But the report about how long-term high fat feeding caused RAAS activation is still limited, whether it is related to the increasing of myocardial deposition of TG?Many studies have confirmed that lipid metabolism disorders and obesity are the initiating factors for triggering MS, thus our group used the animal experiment method to prepare fat diet of which the main component was lard, and chose the intervention as 24 weeks of feeding time in order to build an animal model with human's MS characteristics. We studied deeply the characteristics of each component of MS, and concerned the compound actions among obesity, blood pressure, hyperinsulinemia, insulin resistance, lipid, FFA and other factors, and investigated the relationship between the RAAS activation and the increasing of myocardial deposition of TG.Objective:1. To establish the MS model of SD rats used high-fat diet.2. To investigate the characteristic of MS components.3. To estimate the cardiac functional by the Echocardiographic studies.4. To study the mechanism of the cardiac function damage.Method:Ninety-two Sprague-Dawley male rats aged 8 weeks old, were randomly divided into two groups, including a high-fat diet group (MS) and a normal diet group (NC). After 24 weeks feeding, the following items were detected:?Blood pressure, body weight and blood sugar were recorded weekly.?Hyperinsulinemic-euglycemic clamp technique was performed to evaluate insulin resistance.?Fasting serum (FFA), TG and myocardial TG contents in left ventricle heart were measured respectively.?)IPGTT was performed.?Echocardiographic studies were performed. the maximum velocity of myocardial contraction (+dP/dt max) and the maximum velocity of myocardial diastole (-dP/dt max) of intra cardiac pressure were measured by physiological polygraph.?Cardiac muscles were stained by wolf red chromosome and observed by polarized light microscope.?The protein expression levels of AT1,AT2,Ang?I in myocardium of left ventricle were analyzed by the methods of immunohistochemistry.?The expression of ACE, AT1 and AT2 mRNA in myocardial tissues were also determined respectively by quantification real-time PCR.Result: 1.Blood pressureCompared with the rats in NC group, the weight in the MS group were increased by 31.5% (P<0.01). The systolic BP was increased significantly(155.4±6.6 mmHg vs.l32.1±5.3mmHg,P<0.01).2.Contents of fasting insulin, FFA, serum TG and hyperinsulinemic-euglycemic clampCompared with the rats in NC group, the content of fasting insulin, serum TG and plasma FFA were increases by 300%,43.75%and 105% respectively(P<0.01) in the MS group.Blood sugar tended to be stable after 60 min in three groups. In the MS group, a significant decrease in GIR was recorded (P<0.01).Note:The MS model was established successfully by long term high-fat diet.3.Cardiac function3.1 Left ventricle weight index (LVMI)The LVMI in MS group was increased significantly compared with NC group (P <0 .01).3.2Doppler UCGCompared with NC group, EF, FS and CI were decreased significantly by 10.6%, 49.5% and 19.8% respectively(P<0.01).3.3 (+dP/dt max) and(-dP/dt max)Compared with NC group, (+dP/dt max) and(-dP/dt max) were decreased by 30% respectively (P<0.01).Note:The cardiac function of MS model were damaged.5. Comparison of myocardial TG contents in left ventricle heartMyocardial TG contents in MS group were higher than that in NC group(P <0.01).Note:Excessive FFA deposit in cardiac in the form of TG.5.Organic pathologyThe heart's wolf red chromosome showed large cardiac muscle cells, arranged disorder, thicken inside wall of small artery, and narrow lumen. With polarized light microscope, we can watch a mass of collagen fiber around cardiac muscle cell, giving priority to I type collagen fiber.6.The study of RAAS 6.1 serum angiotensin II and aldosterone levelsAfter 24 weeks of feeding, the serum angiotensin II and aldosterone levels in MS group were significantly increased by 124% and 40.64% respectively(P<0.01).6.2 immunohistochemistry detectionResults of immonohistochemistry showed that the expression of Ang?and AT1 in MS group was remarkably enhanced than NC group, brownish yellow colored site targeted in cytoplasm was significantly increased, with IOD [Ang?NC:(2.11±.23)×104 vs MS:(4.91±0.35)×104, P<0.01], [AT1 NC:(2.87±0.27)×104 vs MS:(4.84±0.47)×104, P<0.01] respectively.Results of immonohistochemistry showed that the expression of AT2 in MS group was remarkably weakened than that in NC group, brownish yellow colored site targeted in cytoplasm is significantly decreased, with IOD [(4.51±0.39)×104 vs. (3.23±0.32)×104,P<0.01].6.3 Results of Real-time Detection.The expression of Ang, ACE, Renin, AT1 mRNA in myocardial tissues in MS group was increased by 133.6%(P<0.01),102.8%(P<0.01),27.4% (P<0.05) and 7.4%(P>0.05),compared with the NC group respectively.The expression of AT2 mRNA in myocardial tissues in MS group was decreased by 71.4%(P<0.01)compared with NC group.Analysis of correlation between the contents of serum TG and Ang mRNA:they were positively correlation (r=0.467, P<0.05).Analysis of correlation between the content of myocardial TG and AT2 mRNA: they were negatively correlation (r=-0.376, P<0.05).Conclusion:1.An animal model miming human's MS characteristics is successfully established by a 24 weeks high-fat diet.2. The cardiac functional is estimated to be damaged by the Echocardiographic studies.3. The mechanism of the damaged cardac function is related to the activation of RAAS caused by myocardial TG ectopic sedimentary. Background:The pathogenesis of hypertension is complicated. Hypertension has been divided into primary and secondary hypertension traditionally, with the former accounted for more than 90%. More and more researchs show that blood pressure is only a key link of hypertension-related diseases. Pool first proposed the concept of metabolic hypertension in 1993, such hypertension often coexist with metabolic risk factors, and has a clear causal relationship with metabolic abnormalities, and reject the secondary hypertension, in additionnal, improving metabolic risk factors contributes to control blood pressure.Besides conventional antihypertensive drugs, Reducing metabolic hypertension should be also focused on improving insulin sensitivity, correcting glucose and lipid metabolism disorders and so on. The ideal drugs can reduce blood pressure, and they also should have beneficial effects to metabolic disorders, insulin resistance and atherosclerosis. Many studies have confirmed that TZDs drugs can lower blood pressure. However, whether TZDs drugs could be used as antihypertensive drugs still have many theoretical and practical problems to be resolved, one of which is whether these drugs have protective action to cardiovascular.In the part?,we have successfully established an animal models with human's MS characteristics by a 24 weeks high-fat diet. The cardiac functional of MS model was estimated to be damaged by the Echocardiographic studies. We conclude that the damaged cardiac function are related to the activation of RAAS caused by myocardial TG ectopic sedimentary. In this part, we treated the MS models by pioglitazone in the dose of lOmg.kg-1.d-1 to investigate the drug's effct on the metabolic hypertension and the damaged cardiac functional.Objective1. To investigate the effects and mechanism of pioglitazone on the metabolic hypertension.2. To study the cardiac functional by the Echocardiographic studies and estimate whether pioglitazone could reverse the impaired cardic function.Method: Animals were divided into four groups, including a normal diet group (NC,n=11), a normal diet plus pioglitazone intragastric administration (NP,n=8), a high-fat diet plus saline intragastric administration (MC,n=6), a high-fat diet plus pioglitazone intragastric administration (MP,n=8), After 6 weeks intervention, the following items were detected:?Blood pressure, body weight and blood sugar were recorded weekly.?Hyperinsulinemic-euglycemic clamp technique was performed to evaluate insulin resistance.?Fasting serum FFA, TG and myocardial TG contents in left ventricle heart were measured.?24h urinary sodium was measured.?Echocardiographic studies were performed. the maximum velocity of myocardial contraction (+dP/dt max) and the maximum velocity of myocardial diastole (-dP/dt max) of intracardiac pressure were measured by physiological polygraph.?Cardiac muscles were stained by wolf red chromosome and observed by polarized light microscope.Results:1. Body weightThere was no statistically significant difference in body weight between NC and NP group before and after pioglitazone intervention (P>0.05).Compared with the rats in MC group, the weight in the MP group increased slightly (P>0.05) in the early 2 weeks, but at the end of the experiment, there was no statistically significant difference in the body weight(P>0.05).Note:Pioglitazone did not continue to gain weight.2. Blood pressureThere was no statistically significant difference in blood pressure between NC and NP group before and after pioglitazone intervention (P>0.05).There was no statistically significant difference in blood pressure between MC and MP group before pioglitazone intervention (P>0.05). The blood pressure begin to decrease obviously after 2 weeks. At the end of the experiment, there was no statistically significant difference in the body weight (P>0.05).Note:Pioglitazone could decrease metabolic hypertension obviously.3. Hyperinsulinemic-euglycemic clampBlood sugar tended to be stable after 60 min in three groups.There was no statistically significant difference in GIR between NC and NP group before and after pioglitazone intervention (P>0.05).There was no statistically significant difference in GIR between MC and MP group before pioglitazone intervention (P>0.05). The GIR was increased significantly (11.36±1.0 mg/kg/min vs 6.71±0.6 mg/kg/min, P<0.05) after pioglitazone intervention.Note:Pioglitazone could ameliorate IR.4.24h urinary sodiumThere was no statistically significant difference in 24h urinary sodium between NC and NP group before and after pioglitazone intervention (P>0.05).There was no statistically significant difference in 24h urinary sodium between MC and MP group before pioglitazone intervention (P>0.05). The 24h urinary sodium was increased significantly (0.33±0.03mmol/24h vs.0.93±0.13mmol/24h, P<0.05) after pioglitazone intervention.Note:Pioglitazone could improve the role of sodium and water retention.5. Contents of fasting insulin, FFA, serum TGCompared with the rats in MC group, the content of fasting insulin, serum TG and plasma FFA were decreased by 42.7%,20.2% and 20.6% respectively (P<0.01) in the MP group.In the MS group, a significant decreasion in GIR was recorded (P<0.01).Notes:Pioglitazone could ameliorate hyperinsulinemia, decrease high free fatty acids and improve IR.6. Cardiac function6.1 Left ventricle weight index (LVMI)There was no statistically significant difference in LVMI between NC and NP group after pioglitazone intervention(P>0.05).There was no statistically significant difference in LVMI between MC and MP group after pioglitazone intervention (P>0.05).6.2 Doppler UCGThere was no statistically significant difference in Doppler UCG between NC and NP group after pioglitazone intervention (P>0.05).Compared with MC group, EF, FS and CI are decreased significantly by7.1%, 67.2%?7.9% respectively(P<0.01) in MS group.6.3 (+dP/dt max) and(-dP/dt max)There was no statistically significant difference in (+dP/dt max) and (-dP/dt max) between NC and NP group after pioglitazone intervention (P>0.05).Compared with MC group, (+dP/dt max) and(-dP/dt max) were ameliorated (P <0.01).Note:The damaged cardiac function of MS models were ameliorated after pioglitazone intervention.7. Myocardial TG contentsThere was no statistically significant difference in myocardial TG contents between NC and NP group after pioglitazone intervention (P>0.05).Myocardial TG contents in MP group were decreased by 37.1% than MC group (P<0.01).Note:Pioglitazone could ameliorate the excessive FFA deposit in cardiac in the form of TG.8.Organic pathologyThe heart's wolf red chromosome showed large cardiac muscle cells, arranged disorder, thicken inside wall of small artery, and narrow lumen. With polarized light microscope, a mass of collagen fiber around cardiac muscle cell was founded, giving priority to I type collagen fiber, while Pioglitazone could ameliorate cardiac fibrosis.Conclusion:1.Antihypertensive effect of pioglitazone was obvious, although the effect-acting slightly slow, long-term efficacy is more stable. It is speculated that the slow step-down mode of pioglitazone play its effect through an indirect mode, such as improving insulin resistance and/or reducing the compensatory hyperinsulinemia.2.Pioglitazone will not continue to gain weight, and can reduce sodium and water retention, which is related to the reduced compensatory hyperinsulinemia.3.The damaged cardiac function of MS models were ameliorated after pioglitazone intervention, which is confirmedby the use of Doppler UCG and Organic pathology.4.Pioglitazone could reverse the impaired cardic function through ameliorating the excessive FFA deposit in cardiac in the form of TG. Backgroud:In the long-term clinical practice, we notice that blood pressure levels in patients will fall after having diabetes. Although epidemiological data have been confirmed that impaired glucose tolerance and insulin resistance in type 2 diabetes are similar, the former is much higher than the latter in the level of insulin. When pre-diabetes develops to diabetes, the degree of insulin resistance does not weaken, even actually increase, while there is a significant decline in insulin levels. Hence, a view was put forward that the hyperinsulinemia of pre-diabetes may play an important role on the elevation of blood pressure.In the part?,we have successfully established an animal model with metabolic hypertension. In the part II, We demonstrated the antihypertensive effect of pioglitazone and speculated that pioglitazone play its antihypertension effect through an indirect mode, such as improving insulin resistance and/or reducing the compensatory hyperinsulinemia. Various studies have reported on the mechanism of hypertension in diabetic patients with an emphasis on hyperinsulinemia and insulin resistance, but the independent contribution of hyperinsulinemia or insulin resistance in the elevation of blood pressure is not clearly. The purpose of the present study was to determine the role of hyperinsulinemia and insulin resistance in the development of hypertension.The interaction mechanisms between insulin resistance and/or hyperinsulinemia and hypertension are very complex, the second part of the subject has explored that it is mainly related to several factors such as the increased sympathetic nervous system excitability, kidney water and sodium retention, the increased level of ET and the proliferation of vascular smooth muscle cell. In this part, we would explore the mechanism of hyperinsulinemia on elevating blood pressure.Objective:1. To establish animal models including:IR+hyperinsulinemia, IR without hyperinsunemia, IR+ectohyperinsuemia.2. To investigate the effects of hyperinsulinemia per se on the elevation of blood pressure. 3. To explore the mechanism of hyperinsulinemia elevating blood pressure. Method:After 24 weeks of feeding, MS rats were further randomized into:the MS group (treated with high-fat diet), and DM group{injected with STZ for 3 weeks then randomized into two groups:HI group (treated with Lantus for 3 weeks to keep the plasma glucose normal) and DS group (teated with physiological saline for 3 weeks)}. the following items were detected:?Blood pressure and body weight were recorded weekly.?Hyperinsulinemic-euglycemic clamp technique was performed to evaluate insulin resistance.?24h urinary sodium was measured.?Plasma insulin, gulocose, endothelin-1(ET-1), norepinephrine(NE), aldosterone(ALD), angiotensin?(Ang?) levels were measured.?The protein expression levels of AT1R, ET-1 in aortas were analyzed by the methods of immunohistochemistry.?AT1R, ET-1, ETAR mRNA expression in aortas were evaluated by quantification real time-PCR.Results:1. Blood pressure and heart rateAfter injection of STZ, there was a significant reduction in SBP (20.9±3.3mmHg) (P<0.05), There was no statistically significant difference in heart rate after STZ intervention (P>0.05).There was a significant increase in SBP in HI group (17.5±3.2mmHg) (P<0.01) after injection of Lantus for 3 weeks. There was a statistically significant difference in heart rate after Lantus intervention (P<0.05).2. Hyperinsulinemic-euglycemic clampBlood sugar tended to be stable after 60 min in three groups.There was no statistically significant difference in GIR among the MS, DM, DS and HI groups.Note:The IR did not change during the fluctuation of Blood pressure.3.Comparison of the content of fasting insulinAfter injection of STZ, there was a significant reduction in fasting insulin (32.45±6.59?IU/mL vs.119.63±14.86?IU/mL, P<0.01), While there was a significant increase in fasting insulin (120.20±20.57?IU/mL vs.38.86±7.82?IU/mL, P <0.01) after injection of Lantus for 3 weeks.Note:The change of MS rat blood pressure and insulin level was synchronous.4.24h urinary sodium After injection of STZ, there was a significant decreased in 24h urinary sodium (0.59±0.09 mmol/24h vs.0.26±0.03mmol/24h, P<0.05), While there was a significant decrease in 24h urinary sodium (0.33±0.05 vs.0.62±0.07mmol/24h, P<0.05) after injection of Lantus for 3 weeks.Note:The change of MS rat 24h urinary sodium, blood pressure and insulin level was synchronous.5.Contents of ET-1, NE, ALD, Ang?5.1 ET-1The levels of ET-1 in MS group were higher than that in NC group (2.29±0.13 pmol/1 vsl.57±0.07pmol/l, P<0.05). After injection of STZ, there was a significant reduction in ET-1 levels (1.56±0.08pmol/l vs.2.26±0.13pmol/l, P<0.05), While it was increased significantly (2.23±0.19 pmol/l vs.l.58±0.09pmol/l, P<0.05) after injection of Lantus for 3 weeks.5.2 NEThe levels of NE in MS group were higher than NC group (319.4±61.4pg/ml vs 105.5±19.7pg/ml, P<0.05). After injection of STZ, there was a significant reduction in NE levels (120.2±27.1 pg/ml vs 325.4±66.4pg/ml, P<0.05), While it was increased significantly (232.3±52.8pg/ml vs.115.6±21.7pg/ml, P<0.05), after injection of Lantus for 3 weeks.5.3 Ang?The levels of Ang?in MS group were higher than that in NC group (201.5±23.1 pg/ml vs.89.0±12.2pg/ml, P<0.05).There was no statistically significant difference in Ang?before and after STZ intervention (P>0.05).There was no statistically significant difference in Ang?before and after Lantus intervention (P>0.05).5.5 ALDThe levels of ALD in MS group were higher than that in NC group (1349.6±42.4 pg/ml vs 957.9±49.2pg/ml,P<0.05).There was no statistically significant difference in ALD before and after STZ intervention (P>0.05).There was no statistically significant difference in ALD before and after Lantus intervention (P>0.05). 6. immunohistochemistry detection6.1 Results of immonohistochemistry showed that the expression of AT1R and ET-1 in MS group were remarkably enhanced than those in NC group, brownish yellow colored site targeted in cytoplasm was significantly increased.6.2 Results of immonohistochemistry showed that the expression of ET-1 in HI group was remarkably enhanceed than that in DS group, while there was no statistically significant difference in AT1R between DS and HI group.7. Results of Real-time detection.After 6 months of high fat diet feeding, AT1R, ET-1, ETAR mRNA expression in aortas were significantly increased (P<0.05). STZ administration decreased ET-1 and ETAR mRNA expression. The expression of ET-1 and ETAR mRNA in aortas in HI group were increased respectively by 30.2%(P<0.05) and 58.6%(P<0.05) compared with DS group, While there was no significant difference in the AT1R expression between the above two groups.Conclusion:1. The change of MS rat blood pressure and insulin level is synchronous, the hyperinsulinemia per se rather than the insulin resistance leads to the blood pressure elevation.2. The plausible mechanisms on the elevation blood pressure of hyperinsunemia were the sodium retention, hypertonicity of the sympathetic nerve system and the increased endothlin levels induced by insulin. The role of insulin on increasing blood pressure tend to be an accelerator.3. Doctors need to aware and avoid the elevation blood pressure due to hyperinsulinemia in clinical practice. Backgroud:Numerous studies support a link between insulin resistance (IR) and hypertension, however, the mechanisms are largely unknown. There is some evidence that elevated plasma free fatty acids (FFA) may be involved in this process. For instance, the levels of FFA in hypertensive patients were higher than those in healthy subjects. Whereas in a Paris Prospective Study, FFA level was an independent risk factor for hypertension. Further, resistance to the FFA-lowering action of insulin was severely impaired in abdominally obese hypertensive patients, and correlated with blood pressure (BP) independently of insulin and insulinmediated glucose disposal. Experimentally, rats that were fed a high-fat diet for 8 weeks had increased plasma FFA and a 12mmHg increase in systolic BP, endothelium-dependent vasodilation (EDV) and nitric oxide (NO) production are impaired when FFA are increased by an Intralipid+heparin infusion. Furthermore, EDV can be induced by impaired eNOS activity and reduced NO production. However, the relationship between the elevation of BP caused by FFA and the impairment of EDV, which can also be caused by FFA, has not been clarified. The aim of this study was to not only examine whether the FFA induced elevation of BP was related to the FFA-induced impairment of EDV but also to determine the role of oxidative stress in mediating FFA-induced BP elevation.Objective:To investigate whether FFA can induce elevation of blood pressure and the possible relationship and mechanism between insulin resistance and FFA-induced hypertension.Materials and Methods:Male Sprague-Dawley rat models were established and classified into three groups:the control group (NC group, n=20), the FFA group(n=22), and the N-acetylcysteine group(NAC group, n=23). The NC group received a 4h infusion of saline (18?l/min), the FFA group received a 4h infusion of 20% Fat Emulsion (Intralipid,18?l/min)+heparin (0.72IU/min), and the NAC group received a 4h infusion of Intralipid (18?l/min)+heparin (0.72IU/min) and NAC (2.76?mol/kg/min).?Blood pressure was recorded using intraarterial recording method.?An organ chamber experiment was performed to determine EDV/EIV.?The hyperinsulinemic-euglycemic clamp technique was performed to investigate insulin resistance.?FFA, ROS, GSH and NO2-/NO3-levels were measured in plasma.?eNOS mRNA expression in endothelial cells was evaluated by real time-PCR.Results:1.The level of serum FFAs was elevated compared with the NC group (P<0.05).2.In the FFA group, the Intralipid+heparin infusion resulted in a significant increase, at the end of the 4h infusion, systolic (11.5±2.2mmHg) and diastolic (6.0±1.2mmHg) BP increased. In the NAC group, systolic and diastolic BP remained the same.3.In FFA group, the aortic rings tended to demonstrate impaired EDV in response to ACh. There was no difference of EDV response in the NAC and NC group.4.The GIR was decreased significantly in FFA group compared with NC group (5.3±0.6 mg/kg/min vs 13.4±0.5 mg/kg/min, P<0.01). There was no significant difference between the NC group and NAC group.5.ROS concentrations in the FFA group were higher than in the NC group (864.3±135.4 U/mL vs.452.4±90.5 U/mL, P<0.05), and GSH concentrations in the FFA group were lower than in the NC group (153.1±55.9 mg/L vs.171.9±60.5 mg/L, P<0.05).6.In the FFA group, NO2-/NO3-levels were significantly reduced (14.3±1.9?mol/l vs.19.3±2.9?mol/1, P<0.05) and eNOS mRNA expression was significantly decreased by 41.2% compared with the NC group. NAC administration increased NO2-/NO3-levels (17.9±1.6?mol/1 vs.14.3±1.9?mol/l, P<0.05) and eNOS mRNA expression compared with the FFA group.Conclusions:Our study suggests that elevated FFA concentrationscan directly and independently reduce endothelial NO production and eNOS mRNA expression and activity, which may impair EDV, and thus increase BP. Further, blood vessel endothelial cell oxidative stress may play an important role in this link. Although the mechanism of FFA-induced hypertension remains to be defined, these data suggest that clinical strategies to reduce FFA concentrations would have beneficial cardiovascular effects.