| Partâ… The establishment of isolated rat heart insulinresistant modelObjective To investigate the effect of 10,20,30 and 50U/Linsulin on the myocardial glycometabolism of isolated rat heart andestablish the insulin-resistant model of isolated rat heart.Methods Thirty Sprague-Dawley rats were randomized into fivegroups according to the concentration of insulin in the buffer,the concentration are 10,20,30 and 50U/L.The rats were killed and their hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfused with Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Thecoronary effluent was collected at 30 and 60min,and theconcentration of glucose in the coronary effluent was determinedto calculate the glucose uptake and evaluate the glycometabolismof myocardial.Results The glucose intake of myocardial are2.73±0.21,1.72±0.26,1.59±0.23,1.56±0.16,1.39±0.27mmol/Lat 30min,and2.63±0.19,1.59±0.21,1.49±0.18,1.38±0.20 and1.23±0.22mmol/Lat60min.Compared with the control group,the rat hearts stimulatedwith hyperinsulinism group had a significantly depression ofglucose uptake relied upon the level of insulin in the buffer(P＜0.05).Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin,the glucose uptake of myocardial depressed,and thephenomenon of myocardial insulin resistance significantly existed. Partâ…¡The study of myocardial damage on insulinresistance of isolatedheartObjective To observe the damage of myocardial on isolated ratheart after stimulated with hyperinsulinism.Methods Thirty Sprague- Dawley rats were randomized into fivegroups according to the concentration of insulin inthe buffer,the concentration are 10,20,30 and 50U/L.The rats were killedand their hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfused with Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Heartrate and left ventricular±dp/dtmax were measured and recordedusing computerized data acquisition system.The coronary effluentwas collected at 60min,and the concentration of cardiac creatinekinase (CK) and cardial troponin I(cTn-I) were detected;theapoptosis index (AI) by TUNEL staining was caculated;and thestructural change of myocardial was observed by light and electronmicroscope.Results (1)After stimulated 60min,left ventricular -dp/dtmaxdepressed compared with the control group (P＜0.01),but HR not,demonstrated that heart function cut down,especially left ventricular diastolic function.(2) The lever of CK and cTn-I inthe coronary effluent was significantly higher the controlgroup(P＜0.01) (3)The value of apoptosis increased in insulinstimulated group than those in the control group(P＜0.01),indicatedcardiomyocyte apoptosis decreated.(4)10U/L insulin groupmyocardium observed by light microscope,cardiac muscle fibersarrayed mussily and dropsied more than the control group,and inultramicrostructurethe density of chondriosome reduced,cristaecollapsed,even disappeared,the pathematology changes occurrencedon the myocardial structure.Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin for 60min,the myocardial insulin resistance existed,the heart function depressed,released more cardiac creatase,cardiomyocyte apoptosis increased,thestructure changed,and themyocardium got damaged.Partâ…¢The mechanism of myocardial insulin resistanceon isolated rat heart Objective To investigate the mechanism of myocardialinsulinresistance on isolated rat heart after stimulated withhyperinsulinism from prereceptor,receptor and postreceptor.Methods Thirty Sprague- Dawley rats were randomized into fivegroups according to the concentration of insulin in the buffer,theconcentration are 10,20,30 and 50U/L.The rats were killed andtheir hearts were taken out to be mounted onto a Langendorffperfusion apparatus to be perfusedwith Krebs-Henseleit buffer inthe presence of various concentration of insulin for 60min.Afterperfused for 60min,the myocardium was obtained,and the contentof insulin receptor (InsR) mRNAandglucosetransporter 4 (GLUT-4)mRNA by RT-PCR,InsR protein and GLUT-4 protein was detected byWestern blotting method,the eNOS and ET-1 by immunohistochemistry.Results (1) Compared with the control group,the content of InsRmRNA and protein decreased (P＜0.01),demonstrated the expressionof InsR mRNA and protein were both down regulated after stimulatedwith hyperinsulinism for 60min.(2) The content of GLUT-4mRNAandprotein decreased either,compared to the control group(P＜0.01),demonstrated the expression of GLUT-4 mRNA and protein decreasedtoo,the myocardial glucose transport diminished and the glucose intake degrade.(3) The levels of eNOS protein descend while thelevels of ET-1 raise in 10U/L insulin group(P＜0.01),purportedendothelial dysfunction existed,the aeteria coronariacontracted,blood flow reduced,result in insulin and glucose transport tointerstitial substance decreased,metabolic product cumulated,aggraved myocardial insulin resistance.Conclusion Stimulated the isolated rat heart with 10,20,30 and50U/L insulin for 60min,the myocardial endothelial function disord,and the expression of InsR and GLUT-4 decreased.The changesoccurred on prereceptor,receptor and postreceptor,and thephenomenon of myocardial insulin resistance significantly existed.Partâ…£The study on i nsul in resistance on cardiocyteinduced with hyperinsulinsmObjective To establish insulin resistant myocardial cell modelby induced primary cultured cardiocyte with hyperinsul insm,andinvest i gated the effect of 10-6mol/L insulin on cardiocyte insulinreceptor and pathematology change. Methods Cardiomyocytes of neonatal mice were in vitro harvested,culture and purified by trypsin digestion and differentialattachment technique.Cardiomyocytes were incubated in 10-8,10-7,10-6,10-5 mol/L insulin for 24 hours.The insulin inducedcardiomyocytes were incubated for another 24 hours by DMEM,finallythe culture fluid was obtained,and the concentration of glucosewas determined;3H-D-glucose incorporation experiment was adopt toevaluate the insulin sensitivity of cardiomyocytes;the content ofmyocardial insulin receptor (InsR) mRNA was detected by RT-PCRmethod,and the InsR protein detected by Western blotting;inadditional,the structural change of myocardial cell was observedby electron microscope.Results (1)When the concentration of insulin raised from 10-8mol/Lto 10-6mol/L,the glucose consumption of the cardiomyocytesdecreased gradually,but when the concentration raised to 10-5mol/L,the glucose consumption decreased no more.It demonstrated thatafter stimulated with insulin for 24h,the cardiomyocytes glucoseintake reduced,insulinresistanceexisted,and the best interferedconcentration is 10-6mol/L.(2) The amounts of incorporation of3H-D-glucose in 10-6mol/L insulin group cardiomyocytes were lower than the control group stimulated with various concentration ofinsulin,and the amounts of both groups raised upon theconcentration of insulin.(3) After stimulated thecardiomyocyteswith 10-6mol/L insulin,the content of InsR mRNA and proteindecreased compared to the control group(P＜0.01).(4) 10-6mol/Linsulin group cardiomyocytes changed significantly under theelectron microscope than the control group ones:dropsied more,andthe chondriosome reduced,cristaecollapsedor even disappeared.Conclusion Stimulated cardiomyocytes with hyperinsulinism wasable to induce a state of insulin resistance,the best interferedconcentration is 10-6mol/L,the insR mRNA and protein were both downregulated,and ultramicrostructure changed. |
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