The Effects Of Ghrelin On Hippocampal Neuronal Tau Phosporylation And Glucose Metabolism Induced By High Glucose And Its Mechanism

Ghrelin is a 28-amino acid peptide secreted predominantly from X/A-like cells of the gastric fundus as the endogenous ligand for the growth hormone (GH) secretagogue-receptor la (GHS-Rla). Circulating hormone has two forms:unacylated ghrelin (UAG) and acylated ghrelin (AG), which is essential for ghrelin bioactivity. Ghrelin has been on its role in neuroendocrine regulation in the central nervous system, for instance modulating appetite and food take, energy and glucose homeostasis, learning and memory performance. The data confirmed that ghrelin is a key mediator of hippocampal-dependent learning processes.Diabetes mellitus is an endocrine disorder of carbohydrate metabolism resulting from inadequate insulin release or insulin insensitivity, then causing sugar, protein, water abnormal metabolism. Hyperglycemia, as diabetic fundamental biochemistry character, has an important role in diabetes. Hyperglycemia in type 2 diabetes is not only a secondary manifestation of insulin resistance, but could also be responsible for directly inducing insulin resistance in the target tissue. Studies have been shown that hyperglycemia is a primary reason for nervous system impaired. Diabetic demented danger increased significantly, generally accompany with cognition dysfunction, such as learning and memory. Epidemiology studies have shown that diabetic patients with insulin resistant have a 30-65%increased risk of developing AD. Recent data indicate that brain insulin deficiency and the insulin-resistant brain state are related to Alzheimer's disease (AD), de la suggests that AD could be "Type 3 diabetes". Therefore, the common pathomechanism of diabetic encephalopathy and AD was insulin resistance, which led to metabolic disturbance and caused cognitive dysfunction. Ghrelin improved the cognitive action of AD model rats. Recent study suggests that ghrelin may modulate insulin sensitivity and offer a potentially novel therapeutic approach for the cognitive dysfunction seen in Type 2 DM. However, the underlying mechanisms have not been fully elucidated.Glucose is the primary energy source for mammalian brain and a continuous supply of this substrate is essential to maintain normal cerebral function. Currently,14 members of this sugar/polyol transporter protein family have been described. In the brain, although GLUT1 is expressed at the blood-brain barrier and in astrocytes, whereas GLUT3 is responsible for the neuronal glucose uptake independent insulin, GLUT4 translocation and consequently cellular glucose uptake in peripheral insulin-sensitive tissue as well as in some brain regions, such as in hippocampal neurons.AD has two pathological hallmarks:P-amyloid (Aβ) deposition and neurofibrillary tangles, which is composed of hyperphosphorylation tau protein and positive relation with cognitive dysfunction. In the process of tau phosphorylation, GSK-3βplays an important role. Schubert demonstrated that insulin signaling system disturbance resulted in Phosphatidylinositol-3 kinase (PI3-K)/Akt signaling pathway dysfunction, which caused GSK-3βactivity increase and led to tau hyperphosphorylation.PI3-K/Akt is the classical insulin signaling transduction pathway. AS 160 is one of Akt substrate, a GTP enzymes activation protein with Rab protein specificity action, which has an importance to GLUT4 traslocation. The last study showed that AS 160 was an signaling molecule the most closed to GLUT4 translocation. Grillol et al found that neuronal insulin utilized similar signal transduction even as peripheral tissues to stimulate GLUT4 trafficking to the plasma membrane.Evidences indicate that ghrelin may modulate insulin sensitivity, stimulate insulin-induced glucose uptake, moreover it also modulates memory. Therefore, we hypothesized that ghrelin may promote hippocampal neurons glucose uptake and improving insulin resistance exposure to high glucose, furthermore ghrelin may attenuate tau abnormal phosphorylation. In addition, examined potential mechanisms, including PI3-K/Akt-GSK-3βand GLUT4 expression. Further understand the function of ghrelin, and supply the theory for the relation of DM and AD.Materials and MethodsMaterials1. The agent of primary rat hippocampal neurons2. The agent of MTT3. The agent of GOD-POD of neurons4; The agent of [3H]-2-deoxy-D-glucose ([3H]-DG) of neurons5. The agent of immunofluorescence stain6. The agent of demi-quantitate RT-PCR to detect gene7. The agent of western blotting cross hybridizationMethods1. Rat hippocampal neurons isolate, culture and differentiation.(1) Rat hippocampal neurons isolateHippocampal neurons were isolated from Sprague-Dawley neonate rats within 24 h of birth. The rats were decapitated and the heads were put in a 90 mm Petri dish placed on an ice-filled bag. The brains were removed from the skull and the bilateral hippocampi were derived. The hippocampi were carefully separated along with ablated vascellum and meninges, which had an extreme affect on neuronal purity. When all the hippocampi were isolated and washed twice in other Petri dishes using ice-cold D-Hank's, they were then chopped into approximately 1 mm3 pieces. The pieces were collected into a 50 ml conical tube, incubated in an equal volume of 0.25% trypsin/EDTA for approximately 30 min. To counteract the trypsin, an equal volume of a neuronal plating medium was added containing DMEM,10%heat-inactivated horse serum,10%heat-inactivated fetal bovine serum,1%glutamine,100 U/ml penicillin, and 100μg/ml streptomycin. This was centrifuged at 1200rmp for 10 min. The trypsinized hippocampi were triturated slowly and gently using a compatible plastic pipette until the tissue was completely dispersed. Then, neurons were slowly and gently dispersed with a plastic pipette and at 1.5×105, 5×105 or 2.5×106/well on polyethylenimine-coated 96,24 or 6 tissue culture plate and incubated in a humidified 5%CO2 incubator at 37℃.(2) Rat hippocampal neurons primary cultureNeurons were incubated in DMEM, including 10%heat-inactivated horse serum, 10%heat-inactivated fetal bovine serum,1%glutamine,100 U/ml penicillin, and 100μg/ml streptomycin and incubated in a humidified 5%CO2 incubator at 37℃. A neurobasal medium with B27 was used instead of the culture medium after the first 24h. After 7 days, neurons will be used. Neurobasal medium contains 25mM glucose, therefore,3 times of the concentration (75mM) will be as high glucose concentration.(3) Rat neurons immunohistochemical identificationNeurons were identified by NSE immunocytochemistry staining. Rat neurons were cleaned by PBS; and fixed by 4%paraformaldehyde for 20 min; 3%H2O2 blocked endogenous peroxydase 10min. Nonimmune animal serum for 15 min; The cells were then incubated overnight at 4℃the polyclonal anti-NSE-antibody (1:250); Immunoreactivity was detected using rabbit anti-rat IgG for 15min. Streptomycete avidin labeled HRP for 15 min. DAB colored and campeachy re-colored. Color status were recorded via inverted phase contrast microscope. Neurons accounted for about 90%of total cells and the experiments were executed on the 7th day of neurons being cultured.2. Cell viability testThe viability of neuronal cells was determined via colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT).0.5 mg/ml of MTT was added to each well for 4 h at 37℃. Medium with MTT was removed and 200μl of dimethylsulfoxide was added to each well to dissolve the prunosuscolored formazan particles. Absorbance was read at 490 nm with a microplate reader. 3. Neuronal glucose consumption via GOD-POD assayGlucose consumption was detected with a glucose GOD-POD assay kit according to the manufacturer's protocol. Cells plated in 96-well plates at a concentration of 1.5×105 cells/well. At the cultured the 7 day, preliminary medium was instead of different treating factors. Detected the remnant glucose concentration of medium via GOD-POD assay, the glucose concentration of uncultured cells as basal data, then counting the cells'glucose consumption.4. [3H]-2-deoxy-D-glucose uptake assayCultured neuronal cells were washed twice with Locke's buffer and incubated for 2 h at 37℃After incubation with or without insulin (100nM) for 30 min, [3H]-2-deoxy-D-glucose were added to the culture, and incubation was continued for 10 min. [3H]-2-deoxy-D-glucose was added to each culture well to give a final concentration of 600 nM (0.2μCi/ml). The cells were washed three times with ice-cold 0.1M PBS buffer and digested with 0.1 N NaOH. Cell lysates were used for determining radioactivity by means of a beta liquid scintillation counter and for an assay of total cell.protein. The experiment was carried out in triplicate with 5 wells per condition per replication.5. Demi-quantitate RT-PCR assayAfter the cells were washed twice with asepsis D-Hanks and added pre-cold TRIzol 1ml and to get out cell total RNA. To gather total RNA; To detect the putity and level of RNA; reverse transcription reaction; primer sequence and react condition; to prepare 3%agarose gel; electrophoresis; to detect electrophoresis density; to assay scanning flame. The OD value of GAPDH production as basal data was count each gene ratio, and semiquantitative analysis.6. Immunocytochemical stain detected ptau[Ser 199] expressionHippocampal neurons were cultured on coverslips. Rat neurons were cleaned by PBS; and fixed by 4%paraformaldehyde for 20 min; 3%H2O2 blocked endogenous peroxydase 10min. Nonimmune animal serum for 15 min; The cells were then incubated overnight at 4℃the polyclonal ptau[Serl99](1:500), GLUT4(1:250); Immunoreactivity was detected using rabbit anti-rat IgG for 15min. Streptomycete avidin labeled HRP for 15 min. DAB colored. Water flushed 5min, neutro-resin coverd. Color status were recorded via inverted phase contrast microscope.7. Western blotting analysis pAkt [Ser473], ptau [Serl99], pGSK-3β[Ser9] and AS 160 phosphorylationThe cells were lysed using a RIPA lysis buffer. The protein underwent SDS-PAGE and was transferred to polyvinylidene fluoride membranes. After being washed three times with a 1×TBST (pH 7.5) buffer, the membranes were soaked in 3%BSA for 2 h at room temperature and incubated with the antibodies at 4℃overnight. Then the membranes were washed five times with the 1×TBST (pH 7.5) buffer again. After incubation with the HRP-conjugated secondary antibody for 2 h at room temperature, the immune complexes were visualized by enhanced chemiluminescence methods. The band intensity was measured and half quantitated. The resulting images were analyzed with Image Analysis Software V 7.0.8. Statistical analysisAll data were expressed as means±SD and analyzed by SPSS 13.0 software. Statistical analysis of the data for multiple comparisons was performed by an analysis of variance (ANOVA) followed by post-hoc analysis. For single comparison, the significance of differences between means was determined by a t-test. p<0.05 was considered significant.Results1. Neuronal glucose metabolism decreased significantly in high glucose compared to in normal glucose; in 1-100nM ghrelin administration, there is no great change at lnM ghrelin, however, there is significant change at lOnM and 100nM ghrelin, in a dose-depent fashion. After 100nM insulin stimulated, this change is much more significant.2. Immunocytochemical method was detected the change of phosphorylation. Tau hyperphosphorylation is colored deeper in high glucose group than in normal group, and treating with ghrelin made the color change light.3. Western blotting was detected the change of tau phosphorylation. Tau hyperphosphorylation increased in high glucose group, ghrelin made the change much more. However, total tau protein had no change.4. RT-PCR was detected the gene expression of GLUT3, GLUT4and PPARy. In primary cultured hippocampal neurons have the expression of GLUT4 besides GLUT3, however, the latter is much more than the former. With or without ghrelin and insulin, the expression of GLUT4 and PPARy have no change, which suggested that the effect of ghrelin improvement on insulin resistant was not via PPARy and on glucose uptake was not via the quantity of GLUT4 in short time. The change of glucose uptake may be linked with GLUT4 translocation.5. Western blotting was detected the change of Akt, AS 160 and GSK-3βphosphorylation. Ghrelin increased the level of Akt, AS 160 and GSK-3p phosphorylation in different glucose concentration.Under high glucose group, the level of Akt, AS 160 and GSK-3βphosphorylation decreased significantly compared to normal group.Treated with ghrelin, Akt, AS 160 and GSK-3βphosphorylation increased greatly, however, total protein had no significant change.6. Immunocytochemical stain detected GLUT4 translocation. GLUT4 was expressed in plasm and membrane. Under high glucose 24h, translocation was refrained, after insulin and/or ghrelin treatment,the action was improved.7. Additon PI3-K specific inhibitor wortmannin (50nM) attenuated the effect of ghrelin on increase glucose uptake and improvement tau abnormal phosphorylaiton.Conclusion1. Hippocampal neuronal tau hyperphosphorylation and glucose metabolism decreased, existed insulin resistance in high glucose concentration probably. 2. Ghrelin increased hippocampal neuronal glucose metabolism in high glucose concentration and improved insulin resistance. Additon insulin, the effect increased significantly. Ghrelin and insulin is synergistic action on glucose metabolism.3. Ghrelin decreased tau hyperphosphorylation resulted from high glucose.4. Ghrelin regulated hippocampal neuronal glucose uptake via GLUT4 translocation probably at last.5. Ghrelin ameliorated insulin resistance and tau hyperphosphorylation partly via PI3-K/Akt-GSK-3βsignaling pathway.