Inhibition Of Lipoapoptosis By Adiponectin In Pancreatic Beta Cells And It’s Mechanism

Part1Pancreatic β-cell lipoapoptosis and it’s mechanismObjectives: To confirm that lipotoxicity induce pancreatic β-cellapoptosis and investigate it’s possible apoptotic pathways.Methods: Pancreatic β cells were cultured in vitro, and divided intosix groups randomly:①Control group (BSA): Min6cells were cultured inDMEM medium containing0.5%BSA for24hours.②Palmitate group(PA): Min6cells were cultured in DMEM medium containing0.5%BSAand0.4mmol/l palmitate for24hours.③Oleate group(OA): Min6cellswere cultured in DMEM medium containing0.5%BSA and0.4mmol/loleate for24hours.④Z-LEHD-FMK(LEHD): Min6cells werepreincubated with40uM Z-LEHD-FMK for2hour, then cultured inDMEM medium containing0.5%BSA and0.4mmol/l palmitate for24hours.⑤Z-IETD-FMK(IETD): Min6cells were preincubated with40uMZ-IETD-FMK for2hour, then cultured in DMEM medium containing0.5%BSA and0.4mmol/l palmitate for24hours.⑥Z-LEHD-FMK andZ-IETD-FMK group(LEHD/IETD): Min6cells were preincubated withboth Z-IETD-FMK(40uM) and Z-LEHD-FMK (40uM) for2hour, then cultured in DMEM medium containing0.5%BSA and0.4mmol/lpalmitate for24hours.Annexin V-of Cy3apoptosis detection kit was used for cell apoptosis,and western blotting for caspase-3,8,9expression.Results: Compared with control group and oleate group, Cleavedcaspase3expression increased in palmitate group (p <0.05). The number ofapoptotic cells increased significantly in palmitate group (p <0.01).Cleaved caspase8(p <0.05) and cleaved caspase9(p <0.01) both increasedin palmitate group, with high level for cleaved caspase9. Z-LEHD-FMK(Caspase9inhibitor) significantly reduced cleaved caspase3expression (p<0.05), there was a trend for Z-IETD-FMK (Caspase8inhibitor) with nostatistical differences. Z-LEHD-FMK (Caspase9inhibitor) combiningZ-IETD-FMK (Caspase8) couldn’t completely inhibited cleaved caspase3expression.Conclusion: Lipotoxicity induced pancreatic β cell apoptosis byactivating both intrinsic and extrinsic apoptotic pathways, while theintrinsic pathway may play a major rolein it. Cleaved Caspase3activationinduced by lipotoxicity may also be partly independent of Caspase8andCaspase9expression. Part2Inhibition of lipoapoptosis by adiponectin and it’smechanismObjectives: To confirm that adiponectin has anti-apoptotic propertiesand explore it’ mechanism.Methods: Pancreatic β cell lines were cultured in vitro, and dividedinto three groups.①Control group (BSA): Min6cells were cultured inDMEM medium containing0.5%BSA for24hours.②Palmitate group(PA): Min6cells were cultured in DMEM medium containing0.5%BSAand0.4mmol/l palmetate for24hours.③Adiponectin group(AD): Min6cells were preincubated with5ug/ml adiponectin for2hours, then culturedin DMEM medium containing0.5%BSA and0.4mmol/l palmitate for24hours.Annexin V-of Cy3apoptosis detection kit was used for cell apoptosis,and western blotting for caspase-3,8,9and BAX、BCL-2expression.Results: Compared with palmitate group, the number of apoptosiccells was significantly lower in adiponectin group (p <0.01). Cleavedcaspase3,9expression significantly decreased in adiponectin group (p<0.05). Caspase8expression also decreased in adiponectin group, but withno statistical differences. BAX expression decreased in adiponectin group,with BCL-2expression increasement (p <0.05).Conclusion: Adiponectin has anti-lipotoxicity properties byinhibiting both intrinsic and extrinsic apoptotic pathways, however inhibition of intrinsic apoptosic pathways may play a major role in it. Part3Diabetic intervention effect of recombinant lentiviral vectorexpressing mouse adiponectin proteinObjectives: To construct recombinant lentiviral vectors expressingmouse adiponectin. To increase blood adiponectin level by recombinantlentiviral vectors in diabetes. To investigate diabetic intervention effect ofadiponectin.Methods: To construct recombinant lentiviral vectors expressingmouse full-length adiponectin protein (with green fluorescent protein).Multiple injections of small doses of streptozotocin plus high-fatdiet induced type2diabetes in mice. Diabetic mice were divided into twogroups randomly.(1) Control group (Control): Mice were fed on normaldiet for6weeks (2) Diabetic mice group: After fasting for16hours, micewere intraperitoneally injected with STZ (55mg/kg body weight, once dailyfor5days). If random blood glucose level exceeded16.7mmol/L, diabeticmodels were considered to be successful. Mice were fed on high-fat diet.4weeks later, diabetic mice were divided into three groups randomly:(1)Saline injection group (Saline,): Mice received100ul saline injection by tailvein,then continued high-fat diet for2weeks.(2) Adiponectin interventiongroup (Plenti-Acdc-EGFP): Mice received injection of100ul recombinant lentiviral vectors expressing mouse adiponectin by tail vein, thencontinued high-fat diet for2weeks. The virus titer was1x108ifu.(3)Vector injection group (Plenti-EGFP): Mice received injection of100ulempty viral vectors by tail vein, continued high-fat diet for2weeks.Green fluorescent expression was observed in liver underfluorescence microscopy. Western blotting was used to detect adiponectinprotein expression in liver. Elisa was used for detecting blood adiponectinlevels. Islet apoptosis was detected by TUNEL assay. Expression ofCleaved caspase3,8,9was detected by immunohistochemistry. Blood lipidlevels were detected by chemical methods. Blood glucose level wasmeasured by blood glucose meter.Results: Type2diabetic mice were induced successfully by multipleinjections of small doses of streptozotocin plus high-fat diet, with higherplasma blood lipids and glucose level than control group mice(p <0.01).14days after injection, obvious green fluorescent proteins wereobserved in liver frozen sections under a fluorescence microscope inadiponectin intervention group and vector injection group. No expressionwere observed in control group and saline injection group. Western blottingshowed that adiponectin protein expressed only in adiponectin interventiongroup. Elisa also showed that plasma level of adiponectin in adiponectinintervention group was significantly higher than in other three groups (p<0.01). The concentration was48.03±3.79ug/ml in adiponectin intervention group. Adiponectin levels did not differ between vectorinjection group and saline injection group, but were significantly lowerthan in control group (p <0.05).Compared with control group, low body weight and high bloodglucose/lipid level were found in saline injection group and vector injectiongroup. No difference was found between saline injection group and vectorinjection group,Compared with saline injection group and vector injection group,adiponectin intervention group have lower blood glucose level and TGlevel（p<0.05,p<0.01）. Apoptotic β cell’s number significantly reduced andthe expressions of Cleaved caspase3,8,9in islet were significantly lowerin adiponectin intervention group.Conclusion:Mutiply injection of small doses of STZ plus high fat dietcan successfully induce type2diabetes in mice. Lentiviral vectors for genetherapy can increase plasma adiponectin level. Improving bloodadiponectin level can be a way to inhibit pancreatic β cell apoptosis，toimprove glucose and lipid metabolisms，to intervene diabetes.