Effect Of KLF4 In Leptin Regulation Of Feeding Behavior And Its Potential Mechanism

Background: Leptin is a hormonal factor secreted by white adipocytes that regulates energy metabolism.Previous evidence has shown that in the hypothalamus,leptin can regulate feeding behavior through the LR-JAK2-STAT3 signaling pathway and the PI3K-AKT signaling pathway.Leptin directly acts on LR-expressing presynaptic GABAergic neurons and reduces the inhibitory effect of GABAergic neurons on postsynaptic POMC neurons.Our previous study found that leptin inhibits energy restrictioninduced increases in food intake,an effect blocked by GABA-BR antagonists.The GABAergic neurotransmitter transmission system plays an important role in the regulation of central feeding behavior of leptin,but its specific mechanism is not fully understood.KLF4 is a member of the zinc-finger transcription factor family.KLF4 was originally discovered from the gastrointestinal tract and plays an important role in adipocyte differentiation.It was later found that KLF4 is widely involved in central nervous system development,neural differentiation,and repair of nerve damage.Our previous study reported that KLF4 cooperated with pentobarbital to regulate the hypothalamic GABAergic system,induce the expression of c-Fos in the hypothalamus,and then exert a sedative effect.However,there are no reports on the regulation of KLF4 on the feeding behavior of energy-restricted(16-hour fasting)mice,and there are no experiments to explore the mechanism of KLF4 in the regulation of feeding behavior by leptin.Based on the above background,this study will investigate the role of KLF4 in energy restriction and leptin regulation of feeding behavior in mice.We will use behavioral pharmacology,molecular biology and stereotaxic injection of mouse brain to interfere with virus sh KLF4 and other experimental methods,focusing on the LRJAK2-STAT3 signaling pathway,the PI3K-AKT signaling pathway,and the hypothalamic GABAergic system,explored the possible regulatory mechanisms,and further clarified the molecular mechanism of central regulation of feeding behavior.It provides a possible molecular target for metabolic diseases with abnormal feeding behavior such as obesity and diabetes.Objective: In this study,energy-restricted(16-hour fasting,the same below)mice were used as models,and experimental methods such as behavioral pharmacology,molecular biology,and immunohistochemistry were used to explore the molecular mechanism of leptin regulation of food intake in mice in the energy-restricted model.The role of KLF4 in the regulation of feeding behavior by leptin.This study provides a new theoretical basis for elucidating the mechanism of action of leptin in regulating feeding behavior.Methods: First,the effect of energy restriction on food intake in mice and its mechanism were investigated.Male mice were randomly divided into two groups: control group and fasting group.The food was taken according to the group,and after 16 hours,the food intake of the two groups of mice was measured,and the mouse brain tissue was collected.The changes of KLF4 levels and the expression levels of leptin signaling pathway proteins,POMC and TH were investigated by immunoblotting experiments.At the same time,in order to explore the effect of energy restriction on GABA neurons,the changes in the expression levels of glutamate decarboxylase: GAD65 and GAD67 were also investigated.Secondly,the effect of leptin on the food intake of mice was investigated.Fasting mice were randomly divided into three groups: fasting + saline group,fasting + 1 mg/kg leptin group,and fasting + 2 mg/kg leptin group.After intraperitoneal administration,behavioral pharmacology experiments were conducted to investigate the effect of different doses of leptin on food intake in mice.Using immunoblotting experiments,leptin doses that had significant effects on the expression levels of KLF4 and related proteins in the hypothalamus and brainstem were analyzed to determine the mechanism by which leptin affects feeding.Finally,based on the successfully constructed energy restriction model and the selected dose of leptin,the role and mechanism of KLF4 in regulating the feeding behavior of mice were investigated.The interference virus sh KLF4 was injected into the lateral ventricle through a brain stereotaxic instrument to reduce the expression of KLF4,and the control virus was injected into the lateral ventricle of the remaining mice as a control group.After successful virus transfection,control mice and sh KLF4 mice with similar body weight were selected and randomly divided into: control + saline group,control + leptin group,sh KLF4 + saline group,and sh KLF4 + leptin group.After fasting,leptin or saline was injected intraperitoneally according to the group.Mice were randomly selected for behavioral experiments.Brain tissue and blood were taken from the remaining mice.The expression levels of leptin signaling pathway proteins,GAD65,GAD67,POMC and TH were examined by western blotting,and the expression of POMC was detected by immunofluorescence experiments in relevant brain regions.The target site of KLF4 action was explored by immunohistochemical staining.The differences in blood glucose between different groups were detected,and the differences in plasma insulin content were detected by Elisa test.Results: The results of the study on the energy-restricted mouse model showed that the food intake of the mice increased significantly between 0-0.5 hours and 0.5-1 hours after the end of the fast.In the brainstem and hypothalamus,energy restriction led to a significant decrease in the expression levels of LR,p-JAK2,p-STAT3,PI3 K,and AKT,a significant decrease in POMC levels,a significant increase in TH expression,and an increase in KLF4 levels.At the same time,energy restriction resulted in a significant increase in brainstem and hypothalamic GAD67 levels and a significant increase in hypothalamic GAD65 levels.Exploring the effect of different doses of leptin on feeding behavior and its molecular mechanism showed that leptin could reduce the increase in food intake caused by energy restriction at 0-0.5 hours and 0.5-1 hours.1mg/kg leptin significantly changed brainstem p-STAT3,AKT and GAD65,GAD67 levels.The expression level of brainstem POMC was significantly increased under the action of leptin.However,leptin did not induce significant changes in brainstem KLF4.The experimental results for the hypothalamus showed that 1mg/kg leptin most significantly increased the levels of LR,p-JAK2,p-STAT3 and AKT in the hypothalamus leptin signaling pathway.Meanwhile,the 1mg/kg leptin group had significant effects on the levels of KLF4,GAD65 and GAD67,POMC and TH in the hypothalamus.The results of exploring the role and mechanism of KLF4 in the regulation of leptin’s feeding behavior in mice showed that,within 0.5-1 h after the end of fasting,the reduction of KLF4 level reversed the decrease in food intake caused by leptin.Brainstem and hypothalamus KLF4 levels were significantly decreased after sh KLF4 virus transfection relative to control virus.While KLF4 did not affect the effects of leptin on brainstem LR,p-JAK2,p-STAT3,PI3 K and AKT,GAD65 and GAD67,and POMC and TH.In the hypothalamus,the effects of leptin on LR,p-JAK2,PI3 K and AKT were not affected by sh KLF4.sh KLF4 had no effect on p-STAT3 expression,but the effect of leptin on p-STAT3 was blocked by sh KLF4.Meanwhile,sh KLF4 blocked the effect of leptin on GAD65 and POMC.Sustained low KLF4 levels blocked the inhibitory effect of leptin on neurons in hypothalamic DM and LH regions.In the arcuate nucleus of the hypothalamus and AHP,leptin affected c-fos expression,but KLF4 did not.In VMH,leptin and KLF4 had no effect on neurons.In the energy restriction model,serum insulin levels were only regulated by leptin,and KLF4 had no effect on insulin.Conclusion: 1.Energy restriction promotes feeding by inhibiting the leptin signaling pathway and activating the GABA system.Leptin administration reverses the promoting effect of energy restriction on feeding behavior.2.KLF4 affects leptin-regulated feeding behavior through p-STAT3-POMC pathway and glutamate decarboxylase of GABA system.3.KLF4 mediates the activation of leptin on hypothalamic DM and LH nuclei neurons and POMC neurons.