Effects And Mechanisms Of Progesterone On Glucose Uptake In Neurons Of Alzheimer’s Disease Animals And Cell Models

The Alzheimer’s disease(AD),the most common type of dementia,is a degenerative disease of the central nervous system with progressive cognitive impairment and memory impairment.The current research shows that there is a close relationship between AD and glucose metabolism.All kinds of Alzheimer’s disease are accompanied by decreased glucose utilization,abnormal insulin pathway and energy utilization disorder,which lead to insufficient neuron function,decreased neuron number,apoptosis and necrosis,and aggravate the progress of AD.Therefore,in recent years,the research on the relationship between abnormal glucose metabolism and ad has attracted much attention.The glucose needed by the brain is completely from the circulating blood,which is transported to the central nervous system by glucose transporters(GLUTs)across the blood-brain barrier.The two most important subtypes of GLUTs are GLUT 3 and GLUT 4.In the brain of AD patients,the expression level of GLUT 3 and GLUT 4 decreased significantly,which led to the decrease of glucose transport rate,abnormal glucose metabolism of neurons,the disorder of energy metabolism of neurons,resulting in neuron degeneration and the corresponding symptoms of AD.It has been proved that the decrease of GLUT 3 and GLUT 4 can cause neuron degeneration,but not the decrease of GLUT 3 and GLUT 4.Therefore,up regulating the expression of GLUT3 and GLUT4,increasing glucose transport to neurons and improving the energy metabolism of neurons may be an important way to prevent the progression of AD.Progesterone(PROG),as an important neurosteroid,is an endogenous neuromodulator produced by the central nervous system and widely participates in many physiological and pathological processes of human body.Progesterone has a significant neuroprotective effect,improving learning and memory and the survival rate of newborn hippocampal neurons in rats.More importantly,progesterone is closely associated with the development of AD.Epidemiological and clinical investigations of AD have confirmed that the level of progesterone in the brain of AD patients is significantly lower than that of their normal peers.The above results suggest that progesterone may be a new target to delay the progression of AD.Previous studies in our laboratory have confirmed that progesterone can inhibit neuronal apoptosis,reduce oxidative stress and induce neural stem cells to differentiate into neurons in an AD cell model.However,whether progesterone can promote the glucose uptake ability of neurons in animal and cell models of AD has not been reported.Therefore,this study used the amyloid precursor protein(APP)/presenilin 1(PS1)transgenic mouse AD animal model and the Aβ25-35-induced primary cultured rat cortical neurons AD cell model to investigate the effect of progesterone on the glucose uptake capacity of AD neurons and the specific molecular mechanism.Part I Effects of progesterone on the expression of GLUT 3 and GLUT 4 in APP/PS1 double transgenic mice.Objective:To study the effect of progesterone on the expression of GLUT3 and GLUT4 in the cerebral cortex of APP/PS1 mice.Methods:APP/PS 1 mice were randomly divided into control group,APP/PS1 group and progesterone intervention group.The Morris water maze behavioral test was performed 40 days after oral administration of progesterone in the progesterone intervention group,and the mice were killed at the end of the behavioral test.The expression levels of GLUT 3 and GLUT 4 in the cerebral cortex of APP/PS1 mice were measured by immunohistochemistry and Western blot.Results:1.The effect of progesterone on the learning and memory ability of APP/PS1 transgenic mice.The results of Morris water maze navigation showed that there was no significant difference between the three groups.Compared with APP/PS1 group,the escape latency of APP/PS1+progesterone group was significantly shorter(P<0.05).The results of space exploration experiment showed that compared with APP/PS 1 group,the percentage of Ⅳ quadrant stay time and times of crossing platform area in progesterone intervention group were significantly increased(P<0.01).2.The effect of progesterone on the expression of GLUT 3 and GLUT 4 in the parietal cortex of APP/PS1 transgenic mice.The results of immunohistochemistry showed that the expression level of GLUT 3 and GLUT 4 in the parietal cortex of normal control group was higher than that of APP/PS1 group.After progesterone intervention,the expression level of GLUT 3 and GLUT 4 in the parietal cortex of APP/PS1+progesterone group was significantly higher than that of APP/PS1 group(P<0.01).After Western blot,the results showed the same trend.3.The effect of progesterone on the expression of GLUT 1 and GLUT 8 in the parietal cortex of APP/PS1 transgenic mice.The results of Western blotting showed that the expression level of GLUT1 in the cortex of mice in each group was low,and progesterone had no significant effect on the expression of GLUT 1.Although the expression level of GLUT 8 in the cortex of APP/PS1 mice was significantly lower than that in the normal control group,progesterone also had no significant effect on the expression of GLUT 8.Conclusion:progesterone can improve the learning and memory ability of APP/PS1 mice.One of the mechanisms is that progesterone can improve the expression of GLUT3 and GLUT4 in parietal cortex.Part Ⅱ:progesterone up regulates GLUT 3 and GLUT 4 expression through PGRMC1.Objective:To study the specific molecular mechanism of progesterone promoting glucose uptake of cortical neurons.Methods:the primary cultured cortical neurons induced by a β 25-35 were selected as the cell model of AD,and AG205(PGRMC1 specific antagonist)and RU486(classic progesterone receptor selective antagonist)were used to study the specific molecular mechanism of progesterone on the glucose uptake of primary cultured cortical neurons.The effect of progesterone on glucose uptake of primary cultured cortical neurons was measured by glucose uptake experiment.Western blot and immunofluorescence were used to determine the effects of progesterone on the expression of GLUT3 and GLUT4 in primary cultured cortical neurons.Results:1.The effect of progesterone on glucose uptake of primary cultured cortical neurons.The results of glucose uptake experiment showed that Aβ significantly reduced the glucose uptake of cortical neurons compared with the normal control group.Progesterone significantly increased the glucose uptake of cortical neurons(P<0.01).AG205 can inhibit the increase of glucose uptake of cortical neurons induced by progesterone(P<0.01),but RU486 has no such effect.2.Effects of progesterone on the expression of GLUT3 and GLUT4 in primary cultured cortical neuronsWestern blot showed that Aβ significantly inhibited the expression of GLUT 3,GLUT 4 and membrane protein in primary cultured cortical neurons.Compared with Aβ group,the expression of GLUT 3,GLUT 4 and membrane protein in Aβ+progesterone group was significantly higher(P<0.01).AG205 could inhibit the expression of GLUT 3,GLUT 4 and membrane protein induced by progesterone(P<0.01).The results of immunofluorescence showed the same trend.Conclusion:progesterone can significantly enhance the glucose uptake of primary cultured cortical neurons.One of the mechanisms is that progesterone can enhance the expression of GLUT3 and GLUT4 by activating PGRMC1.Part Ⅲ Effects of progesterone on the expression of CREB and PPAR γin APP/PS1 double transgenic mice and Aβ induced ad cell model.Objective:To study the specific molecular mechanism of progesterone promoting the expression of GLUT 3 and GLUT 4 in cortical neurons.Methods:APP/PS1 double transgenic mice were selected as animal models,and the expression levels of CREB and PPAR y in the cerebral cortex of APP/PS1 mice were measured by immunohistochemistry.After extracting the protein from the cerebral cortex of APP/PS1 mice,Western blot was used to detect the expression level of CREB and PPAR γ.The primary cultured cortical neurons induced by Aβ were selected as cell models.AG205 was used to study the effect of progesterone on the expression of CREB and PPAR γ.Western blot was used to detect the expression of CREB and PPAR γ in primary cultured cortical neurons.After 666-15(CREB inhibitor)was used in the cell model,Western blot was used to measure the expression level of GLUT 3 in primary cultured cortical neurons.The expression of GLUT 4 in primary cultured cortical neurons was measured by Western blot after GW9662(PPAR γ inhibitor)was used in the cell model.Results:1.The effect of progesterone on the expression of CREB and PPAR γ in parietal cortex of APP/PS1 transgenic mice.The results of immunohistochemistry showed that the expression level of CREB and PPAR γ in the parietal cortex of normal control group was higher than that of APP/PS1 group.After progesterone intervention,the expression level of CREB and PPAR γ in the parietal cortex of APP/PS1+progesterone group was significantly higher than that of APP/PS1 group(P<0.01).The results of Western blot showed the same trend.2.The effect of progesterone on the expression of CREB and PPARγprotein in Aβ 25-35-induced ad cell model neurons.Western blot showed that Aβ significantly inhibited the expression of CREB and PPAR γ in primary cultured cortical neurons.Compared with Aβgroup,the protein expression of CREB and PPAR γ in Aβ+progesterone group was significantly higher(P<0.01).AG205 could inhibit the expression of CREB and PPAR γ protein induced by progesterone(P<0.05).3.The effect of 666-15 on GLUT3 protein expression in Aβ25-35-induced AD cell model neurons.Western blot showed that the expression level of GLUT3 protein was higher in the normal control group,and Aβ25-35 could significantly inhibit the expression of GLUT 3.Progesterone significantly increased GLUT 3 protein expression in primary cortical neurons(P<0.01).666-15 significantly inhibited the expression of GLUT 3 induced by progesterone(P<0.01).4.The effect of GW9662 on GLUT4 protein expression in Aβ25-35-induced ad cell model neurons.Western blot showed that the expression level of GLUT4 protein was higher in the normal control group,and Aβ25-35 could significantly inhibit the expression of GLUT 4.Progesterone significantly increased GLUT 4 protein expression in primary cortical neurons(P<0.01).GW9662 significantly inhibited the expression of GLUT 4 induced by progesterone(P<0.01).Conclusion:Progesterone up regulates the expression of CREB,a transcription factor upstream of GLUT 3.One of the mechanisms of progesterone up regulating GLUT 4 expression is that progesterone up regulates PPAR γ,a transcription factor upstream of GLUT 4.Part Ⅳ Effects of progesterone on the translocation of GLUT 4 membrane in APP/PS1 double transgenic mice and Aβ induced ad cell model.Objective:To study the specific molecular mechanism of GLUT 4 membrane translocation induced by progesterone.Methods:APP/PS1 double transgenic mice were selected as animal models.After extracting the protein in the cerebral cortex of APP/PS1 mice,Western blot was used to detect the expression level of AS 160,P-AS160,AKT and P-AKT in the protein of the cortex.The primary cultured cortical neurons induced by Aβ25-35 were selected as the cell model of AD,and the specific molecular mechanism of GLUT4 membrane translocation induced by progesterone was studied by AG205.Western blot and immunofluorescence were used to determine the effects of progesterone on the expression of AS 160,P-AS160,AKT and P-AKT in primary cultured cortical neurons.Results:1.The effect of progesterone on the expression of AS 160,P-AS160,AKT and P-AKT and protein in parietal cortex of APP/PS1 transgenic mice.The results of Western blot showed that there was no significant difference in AKT and AS 160 expression in the cerebral cortex of each group.Compared with the control group,the expression levels of P-AKT and P-AS160 in the parietal cortex of APP/PS1 transgenic mice decreased significantly(P<0.01).Compared with APP/PS1 group,the expression level of P-AKT and P-AS160 in parietal cortex of APP/PS1+progesterone group was significantly higher(P<0.01).2.The effect of progesterone on the expression of AS 160,P-AS160,AKT and P-AKT protein in Aβ25-35-induced ad cell model neurons.The results of Western blot showed that there was no significant difference in AKT and AS 160 expression in the cerebral cortex of each group.Compared with the control group,the expression level of P-AKT and P-AS160 in primary cultured neurons induced by Aβ decreased significantly(P<0.01).Compared with Aβ group,progesterone significantly increased the expression of P-AKT and P-AS160 in primary cultured neurons(P<0.01).AG205 could significantly inhibit the expression of P-AKT and P-AS160 induced by progesterone(P<0.01).The results of immunofluorescence showed the same trend.Conclusion:One of the mechanisms of progesterone promoting GLUT 4 membrane translocation is that progesterone stimulates PGRMC1 and promotes AKT and AS 160 phosphorylation.In conclusion,APP/PS1 transgenic mice were used as the animal model of AD,primary cultured cortical neurons induced by Aβ25-35 were used as the ad cell model,and immunohistochemistry,Western blot,glucose uptake experiment,immunofluorescence and other experimental techniques were used to study the effect of progesterone on the glucose uptake ability of cortical neurons and the specific molecular mechanism.The results showed that progesterone could significantly promote the glucose uptake of cortical neurons in APP/PS 1 transgenic mice and Aβ25-35-induced ad cell model.Progesterone stimulates the expression of GLUT 3 and GLUT 4 in cortical neurons by activating PGRMC 1,thus improving the glucose uptake of cortical neurons.Further study showed that progesterone promoted the expression of CREB,a transcription factor upstream of GLUT 3,while progesterone promoted the expression of PPAR γ,a transcription factor upstream of GLUT 4.In addition,progesterone can promote the translocation of GLUT4 membrane.One of the molecular mechanisms is that progesterone promotes the phosphorylation of AKT and AS 160 by activating PGRMC1.