The Role Of Lactate-Mediated Metabolic Coupling And HCAR1-cAMP-PKA Signaling Cascades Between Neurons And Astrocytes On Neuron Dysfunction Induced By Hypoglycemia

Objectives:That brain damage induced by hypoglycemia has been widely concerned by researchers.But recent studies show that the effects of hypoglycemia on neuronal and cognitive function are controversial,varying from protective to detrimental.Energy demands of the brain account for at least 20% of the body's energy consumption,and glucose is a critical energy substrate in the brain,hence,hypoglycemia can induce energy metabolic alterations in brain.Astrocyte-derived lactate,as an energy substrate,can provide energy for neurons.Moreover,lactate can act as a signaling molecule to activate lactate receptor on the membrane of neurons and influence neuronal functions.Base on the above results,we aimed to establish a hypoglycemic neuron-astrocyte coculture system,to observe the impacts of hypoglycemia on neuronal morphology and functions,to reveal the role of lactate-mediated metabolic coupling and signaling cascades between neurons and astrocytes on neuron dysfunction induced by hypoglycemia.Furthermore,we used si RNA to downregulate the expressions of MCT2 and lactate receptor HCAR1 in neurons,to explore the role of lactate in neuronal dysfunction induced by hypoglycemia.Methods:(1)Duplicate the mature neuron-astrocyte coculture system established by our research group,and we established hypoglycemic coculture system by replacing the glucose-free medium.MTT was used to detect neuronal cell viability.(2)HE staining,Nissl staining were used to detect the impacts of hypoglycemia on neuronal morphology.Fluorescence and flow cytometry were used to detect cell cycle and the apoptosis.Western blot was used to examine protein expressions of cyclin A and synaptic plasticity related proteins.(3)Glutamate content,glycogen content,the activity of GPa in astrocytes,as well as the activity of key enzymes related to glycolysis in both neurons and astrocytes were detected.Lactate and pyruvate contents in astrocytes,medium,neurons were detected.Western blot was used to examine the protein expressions of MCTs and key enzymes related to glycolysis.si RNA was used to downregulate MCT2 expression,HE staining was used to detect neuronal morphology.Lactate content in medium and neurons were detected,ATP level in neurons was detected.MTT was used to detect neuronal cell viability;(4)Western blot was used to examine protein expressions of HCAR1,p PKA,PKA in neurons.ELISA kit was used to detect c AMP level.si RNA was used to downregulate HCAR1 expression,HE staining was used to detect neuronal morphology,MTT was used to detect neuronal cell viability,and western blot was used to examine expressions of proteins related to synaptic plasticity.(5)si RNA was used to downregulate both MCT2 and HCAR1 expressions,HE staining was used to detect neuronal morphology.Lactate content in medium and neurons were detected,ATP level in neurons was detected.MTT was used to detect neuronal cell viability;western blot was used to examine protein expressions of HCAR1,p PKA,PKA and synaptic plasticity related proteins in neurons.ELISA kit was used to detect c AMP level.Results:(1)We established hypoglycemic neuron-astrocyte coculture system successfully by using glucose deprivation treatment for 0.5h,1h,2h,6h,which can simulate the dynamic effects of hypoglycemia on the brain.(2)Hypoglycemia can alter the neuronal morphology,induce cell cycle arrest,prolonged hypoglycemia can cause neuronal apoptosis.Protein expressions of synaptic plasticity decreased significantly from GD 1h.(3)Glycolysis in astrocytes showed from strengthen to weaken during hypoglycemia;glutamate uptake,glycogenolysis,as well as the activity and expressions of key enzymes in glycolysis showed dynamic alterations in astrocytes.Lactate shuttle between neuron and astrocyte showed from strengthen to weaken characterized with the dynamic alterations of MCTs,and further resulted in the dynamic alterations of energy substrates as lactate and pyruvate.Moreover,glycolysis in neuron also presented from strengthen to weaken during hypoglycemia,characterized with the dynamic alterations of activity and expressions of key enzymes in glycolysis.Hypoglycemia caused ATP level reduced significantly from GD 2h.Inhibition of neuron-astrocyte metabolic coupling can accelerate neuronal impairment induced by hypoglycemia.(4)Lactate can act as a signaling molecule to active HCAR1 receptor during hypoglycemia,and then inhibit c AMP-PKA pathway,resulted in downstream CREB-BDNF pathway inhibited,which was related to the synaptic plasticity damage.HCAR1 knockdown can improve neuronal morphology,and cell viability and protein expressions of synaptic plasticity during hypoglycemia.(5)MCT2 knockdown activated neuronal HCAR1 and its downstream signaling cascades to accelerate the neuronal synaptic plasticity damage.Hypoglycemia induced synergistic dynamic alterations of lactate mediated metabolic coupling and signaling cascades between neuron and astrocyte.Lactate as a signal molecule mainly regulates synaptic plasticity.Conclusions:(1)Neurons cocultured with astrocytes are more resistant to hypoglycemia than those non-cocultured.Hypoglycemia impaired neuronal morphology,induced cell cycle arrest,neuronal apoptosis and synaptic plasticity damage.(2)Hypoglycemia induced synergistic dynamic alterations of lactate mediated metabolic coupling and signaling cascades between neuron and astrocyte,which induced adaptive mechanisms to defense neuronal dysfunction during the early stage of hypoglycemia,and may cause detrimental mechanisms to accelerate neuronal injury during the prolonged hypoglycemia.(3)Inhibition of neuron-astrocyte metabolic coupling can accelerate neuronal impairment induced by hypoglycemia,and the activation of HCAR1 was involved in this process.Inhibition of expressions of HCAR1 can improve neuronal synaptic plasticity,which may provide a new target for the therapy of hypoglycemia related cognitive dysfunction.