Mechanism Of Exenatide Regulating Mitochondrial Homeostasis To Improve Insulin Resistance-induced Neuronal Damage

Aims:Research suggests that people with obesity and type 2 diabetes have a significantly increased risk of cognitive decline and dementia,and that central insulin resistance may play an important role.However,the mechanism of cognitive impairment caused by central insulin resistance remains unclear.As a new type of hypoglycemic agent,GLP-1 receptor agonists’benefits on cognitive function in patients with type 2 diabetes have attracted increasing attention.However,the specific mechanism of their neuroprotective effect still needs to be further explored.The mitochondrial quality control system is an important part of mitochondrial homeostasis,and it is also crucial for the maintenance of neuron function.Therefore,in the current study,central insulin resistance models were established in vivo and in vitro,and GLP-1 receptor agonist exenatide was applied to intervene,to observe the effects of central insulin resistance on cognitive impairment and neuronal function,as well as the protective effect and potential mechanism of exenatide in this process,and to explore the role of mitochondrial quality control system.Methods:（1）Establishment of central insulin resistance mouse model and exenatide intervention:Male C57BL/6J mice aged 6 weeks were randomly divided into the Control group,the High-fat diet（HFD）group,and the exenatide（EXE）group.The Control group was given a low-fat diet,and the other two groups were given a high-fat diet.An oral glucose tolerance test（OGTT）was performed every 4 weeks.After 16 weeks of feeding,the EXE group was given exenatide by abdominal subcutaneous injection,and the other two groups were given normal saline as a control.After feeding for 4 weeks,the mice were sacrificed,and the levels of blood glucose,blood lipid,and insulin were detected respectively;Western blot was used to detect the expression of insulin signaling pathway-related proteins in the cortex and hippocampus after insulin stimulation in vitro.（2）Evaluation of spatial memory impairment and brain tissue injury in mice:The spatial memory ability of mice was evaluated by the Morris Water Maze test;HE staining and Nissl staining were used to observe the structural changes of the cortex and hippocampus;Western blot and immunofluorescence were used to detect the expression of synaptic plasticity-related proteins in the cortex and hippocampus to evaluate the synaptic function.（3）Mitochondrial structure,oxidative stress,and mitochondrial quality control system in mouse brain tissue were detected:The structure of mitochondria in the cortex and hippocampus of mice was observed by transmission electron microscopy;The content of ROS production in brain slices was detected by fluorescent probe;The Kits were used to detect the oxidative indexes H₂O₂,MDA,and antioxidant indexes CAT and SOD in cortex and hippocampus;Western blot and immunohistochemistry were used to detect the expression of mitochondrial quality control system-related proteins in the cortex and hippocampus;Immunofluorescence was used to observe mitophagy in brain slices.（4）In vitro Validation:A co-culture system of neurons and astrocytes was established to mimic the in vivo microenvironment;The insulin resistance model was established by the administration of 0.1m M palmitic acid and exenatide was used as an intervention.Western blot was used to detect the expression of insulin signaling pathway-related proteins to determine whether central insulin resistance occurred;The morphology of neurons was observed by immunofluorescence;Western blot was used to detect the expression of neuronal synaptic plasticity-related proteins;The oxidative stress level was detected by the kits;Western blot was used to detect the expression of mitochondrial quality control system-related proteins;Mitochondrial morphology was observed by fluorescence staining.（5）SIRT1/PGC-1αpathway was regulated to observe the effect on mitochondrial homeostasis imbalance and neuronal damage caused by central insulin resistance:After the central insulin resistance model was successfully established in vivo and in vitro,the expression of SIRT1 and PGC-1αprotein was detected by Western blot;Co-immunoprecipitation was used to detect PGC-1αacetylation levels;Immunofluorescence was used to detect the nuclear translocation of PGC-1αin vitro.SIRT1 was overexpressed in neurons in vitro,and the expression of mitochondrial quality control system-related proteins was detected by Western blot;The oxidative stress level was detected by kit;Immunofluorescence was used to detect the morphology of neurons;Western blot was used to detect the expression of synaptic plasticity-related proteins.（6）Effects of regulation of the SIRT1/PGC-1αpathway on the restoration of mitochondrial homeostasis and improvement of neuronal injury by exenatide:The central insulin resistance model was established in vivo and in vitro and exenatide was used as an intervention.Western blot and co-immunoprecipitation were used to detect the expression of SIRT1/PGC-1αpathway-related proteins;The nuclear translocation of PGC-1αwas detected by immunofluorescence in vitro.SIRT1 was knocked down in neurons in vitro,and the expression of mitochondrial quality control system-related proteins was detected by Western blot;The oxidative stress level was detected by the kits;Immunofluorescence was used to detect the morphology of neurons;Western blot was used to detect the expression of synaptic plasticity-related proteins.Results:（1）The mice in the HFD group showed phenotypes of obesity,increased fasting blood glucose,dyslipidemia,and peripheral insulin resistance;In addition,the decreased expression of insulin signaling pathway-related proteins in the cortex and hippocampus suggested that the high-fat diet can induce central insulin resistance.Exenatide intervention reduced obesity,and improved blood glucose,blood lipid,and peripheral insulin resistance.Exenatide intervention also increased the expression of insulin pathway-related proteins in the cortex and hippocampus to improve central insulin resistance in mice.（2）Central insulin resistance caused spatial memory impairment in mice;In the cortex and hippocampus,the morphology of neurons was abnormal,the number of neurons was small,and the content of Nissl bodies in neurons was decreased.The decreased expression of synaptic plasticity-related proteins in tissues suggested that synaptic function is impaired.Exenatide intervention could significantly improve the spatial memory impairment of mice,and recover the morphology and synaptic function of nerve cells.（3）Central insulin resistance caused mitochondrial structure damage in the cortex and hippocampus of mice;The level of oxidative stress was increased,as indicated by increased ROS content,increased oxidative indexes H₂O₂ and MDA,and decreased antioxidant indexes CAT and SOD;The expression of mitochondrial fusion-related protein MFN2 was decreased,and the expression of mitochondrial division-related protein DRP1（Ser616）was increased;The expression of mitochondrial biosynthesis-related protein NRF1 was decreased;The protein expressions of Pink1 and Parkin were decreased,and the protein expressions of P62and LC3 were increased;Immunofluorescence showed that the co-localization coefficient between mitochondria and autophagosomes was increased,and the co-localization coefficient between autophagosomes and lysosomes was decreased.Exenatide intervention significantly alleviated the mitochondrial structural damage and dysfunction in the cortex and hippocampus of mice.（4）Insulin resistance was induced after 0.1m M palmitic acid was added to the in vitro co-culture system for 24 hours,which was manifested as abnormal neuronal morphology,impaired synaptic function,increased oxidative stress level,disordered mitochondrial quality control system,and fragmentation of mitochondrial morphology.Exenatide intervention alleviated insulin resistance,improved neuronal morphology and synaptic function,reduced oxidative stress,and improved the mitochondrial quality control system to restore mitochondrial homeostasis.（5）Central insulin resistance could inhibit the SIRT1/PGC-1αpathway and cause mitochondrial homeostasis imbalance and neuronal damage;Overexpression of SIRT1 in vitro could improve the inhibition of the SIRT1/PGC-1αpathway,restore mitochondrial homeostasis,inhibit oxidative stress,and improve neuronal morphology and synaptic function.Exenatide intervention could alleviate the inhibitory effect of central insulin resistance on the SIRT1/PGC-1αpathway,thereby improving neuronal mitochondrial homeostasis and synaptic function.Conclusions:（1）Central insulin resistance can cause the decline of learning and memory ability and cognitive dysfunction.GLP-1 receptor agonist exenatide can alleviate insulin resistance and improve cognitive function;（2）The imbalance of mitochondrial homeostasis and the damage to the mitochondrial quality control system play an important role in the process of mitochondrial dysfunction and accelerated neuronal damage caused by central insulin resistance;（3）Central insulin resistance mediates mitochondrial homeostasis imbalance by inhibiting SIRT1/PGC-1αpathway,which eventually leads to neuronal damage and synaptic dysfunction;（4）Exenatide ameliorates neuronal injury by regulating mitochondrial homeostasis through SIRT1/PGC-1αpathway.