| Part I The correlational research on IDE, and insulin resistance, and Mild Cognitive Impairment of type 2 diabetes mellitusBackground:Insulin Degrading Enzyme (IDE) contributes to the degradation processes of insulin and Aβ, and is involved in chronic complications of type 2 diabetes mellitus (T2DM) and Alzheimer’s disease(AD). Insulin resistance (IR) is one of the initial factors for T2DM. Previous studies have shown that the IR could increase the risk of cognitive dysfunction and even AD, and is also a risk factor of conversion from Mild Cognitive Impairment (MCI) to AD.Objectives:We aimed to investigate the correlation among IDE, insulin resistance, and MCI of T2DM, and to assess the predict value of IDE for MCI in diabetic patients.Methods:A total of 239 individuals with type 2 diabetes were enrolled and divided into two groups according to the Montreal Cognitive Assessment (MoCA) score. Blood samples were obtained at 7 A.M. for fasting blood glucose (FBG), fasting C-peptide (FCP), glycosylated hemoglobin(HbAlc), liver and kidney function, blood lipids, and indexes of insulin resistance (HOMA-IR). A series of neuropsychological batteries were used to assess the subjects’ cognitive function. The polymerase chain reaction-restriction fragment length polymorphism method was used to detect the gene polymorphism of IDE rs4646958. The serum level of IDE was measured by enzyme-linked immunosorbent assay (ELISA) kits.Results:There were 132 patients with MCI and 107 patients without MCI. 1. Diabetic patients with MCI had a lower serum level of IDE (P < 0.001), and a higher level of HOMA-IR (P < 0.001),compared with the control group. 2. Among patients with MCI, serum IDE level was positively correlated with the MoCA score (r = 0.827; P < 0.001), and HOMA-IR was negatively correlated with the MoCA score (r = -0.644; P < 0.001). 3. Correlation analysis demonstrated that IDE was positively correlated with MoCA score (r = 0.798; P < 0.001) but negatively correlated with HOMA-IR (r = -0.586; P < 0.001), the Trail Making Test - A (r = -0.464; P < 0.001), the Trail Making Test - B (r = -0.399; P < 0.001), FBG (r = -0.409; P < 0.001), HbA1c (r = -0.237; P =0.015), and the mean amplitude of glycemic excursions (r = -0.502; P < 0.001) in all subjects. 4.In logistic regression analysis for MCI, IDE (P = 0.001), HbA1c (P = 0.024), and FCP (P < 0.001)was an independent variable, after adjusting for age, sex, education, liver function, kidney function, and lipid levels. 5. Meanwhile, no significant differences were found between IDE genotype and allele distributions of the cases and the controls.Conclusions:The results suggest that IDE is involved in IR-related cognitive dysfunction (especially executive function), and that it could be a useful biochemical marker of early cognitive impairment in T2DM. No significant differences were found between IDE genotype and allele distributions of the cases and the controls. Further studies with larger sample size or longitudinal studies are needed to verify these initial observations and the role of IDE rs4646958 in cognitive impairment of type 2 diabetes mellitus.Part Ⅱ The mechanism of IDE involved in cognitive impairment of KKAy mice and the intervention effect of PPARy agonistBackground:IR is the most characteristic feature of T2DM and appears much earlier than overt T2DM. IR involved in diabetic MCI and the conversion of AD. The expression of PPARy is positively correlated with insulin sensitivity. It could affect the expression of IDE by regulating insulin signaling pathway. PPARy agonist may improve insulin resistance, and reduce the occurrence of cognitive impairment and AD. Our previous study indicated that diabetic patients with MCI had a lower serum level of IDE, which was significantly correlated with cognitive function caused by IR, and that it could be a useful biochemical marker of early cognitive impairment in T2DM. On the basis of clinical research, we further used animal experiments to verify the mechanism of IDE involved in IR-related cognitive impairment of KKAy mice, and the prevention and intervention effect of PPARy agonist.Objectives:We aimed to discuss the possible protection mechanism of IDE on cognitive function of KKAy mice and its correlation with the level of Ap. We would like to investigate the effect of PPARy agonist on preventing and improving IR-related cognitive function in KKAy mice.Methods:This study used high-fat feeding KKAy mice as the model of MCI in T2DM, rosiglitazone as the PPARy agonist. Male spontaneous type 2 diabetes KKAy mice and C57BL/6j (C57) mice were selected. Animals were randomly divided into prevention, intervention and control group. The prevention group received daily administration of rosiglitazone at dose of 1, 3.3, 10 (mg/kg/day)at 8 weeks. The intervention group received daily administration of rosiglitazone at dose of 1,3.3,10 (mg/kg/day) at 15 weeks. The control group received 0.9% NaCl every day. 1. The weight,FBG, serum insulin were measured and HOMA-IR was calculated. 2. Morris water maze and Avoid dark experiment were completed to compare learning and memory changes in different groups. 3.The methods of Western and RT-PCR were used to measure the mRNA and protein levels of PPARy and IDE. 4. Hippocampus tissue was performed HE and Nissl’s to observe morphological changes. 5. The expression of PPARy, IDE and Aβ was detected by immunohistochemical staining.Results:1. Body mass, fasting blood glucose, fasting insulin and insulin resistance index: the KKAy mice had markedly higher body mass, FBG, insulin and HOMA-IR compared with the C57 mice (P< 0.05). No significant differences in body mass were observed between treated diabetic mice and untreated diabetic mice (P > 0.05). Moreover, FBG, insulin and HOMA-IR in prevention and intervention group exhibited lower than untreated diabetic group (P < 0.05). And the highest dose showed the best effect in all groups.2. Behavioral tests: in Morris water maze test, the model group had longer incubation period and lower platform-crossing activities than the control group (P < 0.05); prevention and intervention group had shorter incubation period and higher platform-crossing activities than the model group (P < 0.05). In avoid dark test, the model group had shorter avoid dark incubation period and increased number of errors than the model group (P < 0.05);prevention and intervention group had significantly improved the results (P < 0.05), especially the highest dose of prevention group.3. The protein and mRNA levels of PPARy and IDE: the model group had declined protein and mRNA levels of PPARy and IDE than the control group (P < 0.05); prevention and intervention group had higher protein and mRNA levels of PPARy and IDE than the model group (P < 0.05), especially the highest dose of prevention group.4. Morphologies of neurons in hippocampus HE staining: the structure of hippocampus in the control group was normal, and the nuclei are round and the cell morphology was normal. Neurons of hippocampus in the model group decreased and arranged disorderly. Nuclear atrophied and nucleolus was not clear. In the prevention and intervention group, the cell lines were comparatively clear and the number of cells significantly increased. The pathologic changes eased off.Nissl staining: neurons of hippocampus in the control group were morphological regularly,dense and rich. The cytoplasm and nuclear membrane were clear. While the neurons of hippocampus in the model group were relatively sparse, disorder, with less Nissl bodies. In the prevention and intervention group, the neuron number of hippocampus and Nissl bodies increased.5. Immunohistochemical staining: in the control group, the staining of Aβ was shallow and weak.In the model group, the expression of Aβ was notable positive and the IOD value of PPARy and IDE was decreased (P < 0.05). In the prevention and intervention group, the expression levels of PPARγ and IDE were increseaed obviously, and the expression of Aβ was significantly decreased (P < 0.05).Conclusion:The results suggest that IDE is involved in IR-related cognitive dysfunction and could protect cognitive function. PPARy agonist might prevente and improve the cognitive impairment of KKAy mice by up-regulating the levels of IDE,thus decreasing the deposition of Aβ.Part Ⅲ The effect of IDE-mediated PPARy agonist on expression of APP and Aβ in PC 12 cellsBackground:Our previous experiments have established that PPARy agonist could prevent and improve diabetic cognitive impairment. However, at a cellular level, the protective mechanism of the PPARy agonist on nerve cells is not that clear.Objectives:In present study, we aimed to investigate the effect of PPARy agonist on IDE in PC 12 cells, and the potential mechanism why PPARy agonist could protect the function of nerve cells.Methods:Undifferentiated PC 12 cell line was induced into neuronal cells by nerve growth factor. Compared to the control group, the treatment group had a significant statistical difference in the preliminary experiments, after the treatment with lOμmol/L rosiglitazone, 20pμmol/L chloroquine, and 12.5μmol/L T0070907, separately. Therefore, the subsequent experiments were completed with the concentration as the ideal concentration. After steady growth for 24 hours, the cell was randomly grouped into six groups, including the normal control group,rosiglitazone-intervened group, T0070907-intervened group, T0070907 pretreatment +rosiglitazone-intervened group, chloroquine-intervened group, chloroquine pretreatment +rosiglitazone- intervened group. Each group set up six parallel holes. After the intervention, the cell of each corresponding time points was collected. The methods of Western blotting and RT -PCR were used to measure the protein and mRNA levels of IDE, PPARy, and Amyloid Precursor Protein (APP). The Aβ level of cell culture supernatant was detected by ELISA.Results:The results showed that: 1. Compared to the normal control group, the rosiglitazone-intervened group had higher mRNA and protein levels of IDE and PPARy (P < 0.05), and a lower mRNA and protein level of APP (P < 0.05). 2. No significant differences in mRNA and protein levels of IDE, PPARy, and APP were observed between T0070907-intervened group and T0070907 pretreatment + rosiglitazone- intervened group (P > 0.05). 3. Chloroquine pretreatment+ rosiglitazone-intervened group had higher mRNA and protein levels of PPARy than chloroquine-intervened group (P < 0.05), but no significant differences in mRNA and protein levels of IDE and APP were observed between the two groups (P > 0.05). 4. Compared to the normal control group, the rosiglitazone-intervened group had decreased Ap level of cell culture supernatant (P < 0.05),but no significant differences in Aβ level of cell culture supernatant were observed among other groups (P > 0.05).Conclusion:The results showed that PPARy agonist can induce the expression of IDE,thus promote the degradation of APP and AβP. However, the protective effect of nerve cell damage induced by IDE,could be blocked by PPARγ or IDE inhibitors. It indicated that PPARy agonist may protect the function of nerve cell through IDE pathway, which is activated by PPARy. |
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