The Effects Of Central Insulin Resistance On Neuron-astrocyte Metabolic Coupling And Neuronal Functions

Aims:With the rapid development of social economy and changes in diet patterns,diabetes mellitus has become the leading disease threatening human health.Emerging evidence has demonstrated the existence of diabetes-induced brain dysfunctions,which is called diabetes-related cognitive decline.Insulin resistance,as one of the most important mechanisms of diabetes,has been reported to play a critical role in the development of diabetes-related complications.Studies have shown that central insulin resistance is intamately related to diabetes-induced cognitive decline.However,available studies in this field is quite limited,and the underlying mechanism of diabetes-related cognitive decline is unclear.Studies have shown that,in normal condition,neuron-astrocyte metabolic coupling is essential for learning,cognition and memory formation.However,it remains unknown whether the neuron-astrocyte metabolic coupling is affected under insulin resistance condition and its relationship with diabetes-related cognitive decline.Based on the above,we aim to establish an insulin resistant model in the neuron-astrocyte coculture system,to discover the impacts of central insulin resistance on neuronal morphology and functions,and to explore the role of neuron-astrocyte metabolic coupling in insulin resistance-induced neuronal dysfunctions.Furthermore,we will select some hypoglycemic agents(Metformin,Exendin-4,Linagliptin,Pioglitazone),which can improve insulin resistance and explore the effects of above hypoglycemic agents on neuronal functions and neuron-astrocyte metabolic coupling.Methods: ? The modified “sandwich type” method was used to establish the neuron-astrocyte coculture system with the aim to mimic the microenvironment between neural cells in brain.Neuronal morphology and cell viability between neurons in coculture system and neurons in non-coculture system were detected.Insulin,glucose and palmitic acid were used to induce neuronal insulin resistance by detecting the neuronal cell viability.Western blot was used to examine the alterations of protein expression in insulin signaling pathway.? HE staining,Nissl staining were used to detect the impact of insulin resistance on neuronal morphology.Fluorescence and flow cytometry were used to examine the neuronal apoptosis.Western blot was used to detect expression of proteins related to neuronal synaptic plasticity.?-galactosidase staining,telomerase activity assay and western blot were used to detect the neuron aging.? Western blot was used to detect the expressions of monocarboxylic transporters.The concentrations of energy substrates,including lactate,pyruvate,glutamate-glutamine cycle and the protein expressions of key enzymes as well as their activities in glycolysis were detected.? Hypoglycemic agents were used to detect their improvements in neuronal morphology,apoptosis,synaptic plasticity and neuronal aging.Protein expression and energy substrates were also detected after the hypoglycemic agents intervention.Results:? After establishing insulin resistant neuron-astrocyte coculture system,we found that neuron morphology and cell viability were improved.Comparing with insulin and glucose,palmitic acid intervention presented most significant cytotoxicity effect on cell viability.And 0.1mM palmitic acid could induced neuronal resistance,reflected by decreased protein expression of PI3 K,pIRS/IRS ratio and pAkt/Akt ratio.? 0.1mM palmitic acid-induced insulin resistance impaired the neuron morphology and sysnaptic plasticity,accelerated neuron apoptosis and neuron aging.? Central insulin resistance induced decreased expression of transporters in neuron-astrocyte metabolic coupling,along with significant alterations in energy substrates such as lactate and pyruvate.Moreover,neuronal glycolysis was enhanced significantly,with increased kinase activities and expressions of hexokinase,pyruvate kinase and lactate dehydrogenase.? Hypoglycemic agents improved neuronal morphology,synaptic plasticity,neuron apoptosis and neuron aging.Besides this,hypoglycemic agents presented positive effects on neuron-astrocyte metabolic coupling and glycolysis.Conclusions:Using “sandwich” coculture method and palmitic acid to establish the insulin resistant model in neuron-astrocyte coculture system,we concluded that:? Central insulin resistance induced significant neuron dysfunctions by impairing neuronal morphology,synaptic plasticity,neuronal apoptosis and neuronal aging;? Disrupted neuron-astrocyte metabolic coupling and enhanced glycolysis played a critical role in insulin resistance-induced neuronal dysfunctions;? Hypoglycemic agents improved insulin resistance-induced neuronal dysfunctions in the aspects of neuronal morphology,synaptic plasticity,apoptosis and aging,which were mediated by the improvements in the neuron-astrocyte metabolic coupling and cell glycolysis.