| Huntington's disease is an autosomal-dominant neurodegenerative disorder. The patients have involuntary dance movement, mental and emotional abnormalities, and progressive cognitive impairment. As a middle-aged disease, there is no effective treatment at present. Genetic feature is that CAG repeats expand(>35) in the code area of 4th chromosome, which produces mutant huntingtin(m Htt) with amplified polyglutamine(Poly-Q) amino-terminal. m Htt, especially, its amino-terminal fragments can accumulate in the cytoplasm or nucleus of neurons to form insoluble aggregates. m Htt and its aggregates have more cytotoxicity, such as the interference of intracellular gene expression, induction of oxidative stress, causing energy metabolic disorders and excitotoxic injury. These cytotoxicities play a key role in the pathogenesis of HD.m Htt amino terminal fragment can also form aggregates in endocrine cells such as islets and adrenal medulla cells, as well as in neurons. The aggregates could lead to cell dysfunction. Studies show that 50% ~ 60% HD patients have abnormal glucose tolerance test. Compared with the normal population, the incidence of diabetic HD patients is significantly increased, seven times as much as the normal populationincidence. However, the mechanism remains unclear. In this study,we transfected eukaryotic expression plasmid expressing normal or mutant Htt amino terminal fragment into NIT-1 mouse insulinoma ? cells, and detected the effect of m Htt on the cell viability of NIT-1 cells and its function. The study will provide clues for revealing pathogenesis of diabetes mellitus in HD patients.1. m Htt increased NIT-1 cell apoptosisIn transiently transfected NIT-1 cells, the normal Htt fragment which contained the amino-terminal 20 glutamine repeated sequences(20QHtt) was dispersed in the cytoplasm; the m Htt amino-terminal fragment which contained 160 glutamine repeated sequences(160QHtt) was also dispersed in the cytoplasm, but aggregates were observed in the cytoplasm of most cells.In order to examine the effect ofm Htton NIT-1 cell viability,At 48 hours after transfection, MTT analysis showed that there was no significant difference in the proliferation ability between 160QNIT-1 cells group and transfected empty vector(control group)/ 20QNIT-1 cells group. The results suggest that m Htt has no significant effect on NIT-1 cell proliferation. Then, we analyzed whether m Htt promoted NIT-1 cells apoptosis. Immunoblot analysis showed that caspase-3 expression was significantly higher in 160 Q cells than that in 20 Q cells after stimulation of apoptosis inducer, staurosporine. However, there was no significant difference in caspase-3 expression between 20 Q NIT-1 cells and the control group. These results suggest that m Htt causes that NIT-1 cells are more sensitive to apoptosis inducer, more vulnerable to apoptosis.2. m Htt impaired glucose-induced insulin secretion in NIT-1 cellsIn order to clarify the effection of m Htt on insulin secretion stimulated by glucose in NIT-1 cells, at 48 hours after cells transfected by 20 Q or 160 Q Htt,cells were stimulated with different concentrations of glucose(5.6mmol / L, 11.2mmol / L and 24.2mmol / L) for half an hour, insulin levels in the culture medium were analyzed by using mouse insulin ELISA assay kit. The results show that, with a higher concentrations of glucose(24.2mmol / L) stimulation, insulin levels in the culture medium of 20 Q and control cells were significantly increased. However, 160 Q cells displayed a modestly rise of insulin levels in culture medium. The increased rate was significantly lower than 20 Q and control cells. The results suggest that m Htt inhibits glucose-induced insulin secretion in NIT-1 cells.In order to clarify whether m Htt inhibits insulin secretion in NIT-1 cells by affecting its expression, at 48 hours after cells being transfected,cells were stimulated by three concentrations of glucose for half an hour. The expression of intracellular insulin m RNA and protein was detected by RT-PCR and immunoblot assay respectively. The results showed that, insulin m RNA expression level increased with high glucose compared with low glucose, and at the same concentrations of glucose, the 160 Q cells has no significant difference with 20 Q and control cells in insulin m RNA expression level. However, insulin protein levels of 160 Q cells were significantly reduced in high glucose stimulation group than that in low glucose stimulation group. Compared with the control cells, both insulin protein and m RNA expression levels in 20 Q NIT-1 cells have no significant changes. These results suggest that m Htt has no affect intracellular insulin transcription, the reason why insulin secretion reduced may have relationship with the decreased insulin content in cells.3. m Htt inhibited the insulin signaling pathway activated by glucoseInsulin signaling pathway plays an important role in ? cell growth, insulin synthesis and secretion, which can be activated by insulin and glucose. To make clear whether m Htt damages ? cell by affecting insulin signaling pathway, at 48 h after cells being transfected, the cells were incubated with KRBH buffer to inhibit endogenous insulin secreting, then cells were stimulated by exogenous insulin or glucose. The expression and activity of key signaling molecules in insulin signaling pathway were detected. The results show that, after 160 Q cells, 20 Q cells and control group cells were treated with exogenous insulin stimulation, PI3 K activity, Akt phosphorylation and Foxo1 phosphorylation levels were increased more than non-stimulated group, and there was no significant difference in three groups of cells. After high glucose stimulation, insulin signaling molecules including IRS-2 level, Akt phosphorylation and Foxo1 phosphorylation levels were elevated dramatically in 20 Q NIT-1 cells and control group. Whereas, there was a modestly rise of these proteins including IRS-2 level, Akt phosphorylation and Foxo1 phosphorylation levels in 160 Q NIT-1 cells stimulated by high glucose, which were lower than that in 20 Q NIT-1 cells stimulated by high glucose. The results suggest that, m Htt does not affect the activation of insulin-induced insulin signaling pathway in NIT-1 cells, but impairs the reactivity of glucose-regulated insulin signaling pathways, which leads to reduced insulin secretion induced by glucose in ? cells.Conclusion: m Htt can decrease the insulin secretion by damaging the activation of glucose-stimulated signaling pathway in ? cells. At the same time, the disorder of insulin signaling pathway also makes the ? cells apoptosis and decreases the number of ? cells. Further, the total amount of insulin secretion was reduced. Therefore, glucose homeostasis disorders and diabetes mellitus in HD patients may be related to the disorder of glucose-induce insulin signaling pathway. |
PDF Full Text Request