Histone H3K9 Trimethylation(H3K9me3) Is Associated With The High Glucose Metabolic Memory In Nervous System

ObjectiveWe suppose that in neural cells the global H3K9me3 and the subsequent chromatin changes may be the main reason for the metabolic memory.so we want to detect the association between the H3K9me3 and the high glucose metabolic memory in the brain of the diabetic mice and in the cultured neural cells. Methods1. T1 DM mice were preparated with high-dose STZ. High glucose metabolism memory model were maked by insulin injection(i.p.) to control the blood glucose between normal range. The expression of H3K9me3 and methyltransferase SUV39H1 in cerebral cortex and hippocampus were detected by western blot.2. Three neural cell types were stimulated with high glucose(neural progenitor, neuron and HT22 cells) for different time. The expression of H3K9me3 and methyltransferase SUV39H1 were detected by western blot.3. After the stimulation with high glucose, the medium were changed into normal glucose level to preparated the metabolic memory model. The expression of H3K9me3 and methyltransferase SUV39H1 in these cells were tested by western blot.4. in order to test if the heterochromatin structure changes was happened accompany with the H3K9me3 during the high glucose metabolic memory, the following experiments were designed:(1) In HT22 cells, the expression of H3K9me3 and HP1 were tested by western blot, both in the high glucose stimulated cells and in the metabolic memory model cells.(2) In HT22 cells, the expression of major satellite DNA was tested by Real-Time PCR, both in the high glucose stimulated cells and in the metabolic memory model cells.(3) In HT22 cells, the relative occupancy of major satellite DNA with H3K9me3 was tested by qChIP, both in the high glucose stimulated cells and in the metabolic memory model cells.5. In HT22 cell, the cellular bioenergetics was tested by the seahourse XFe/XF 24 and Mito-stress-text kit, both in the high glucose stimulated cells and in the metabolic memory model cells. Results(1) H3K9me3 increased in diabetic mouse brain and kept high even after the hyperglycemia has been controlled by insulin;(2) with high glucose(HG) stimulation in three types of neural cells, H3K9me3 was increased and reached its peak for short time;(3) after the withdrawn of high glucose stimulation in three types of neural cells, the expression level of H3K9me3 remained high for a long time and displayed a hysteresis;(4) during the high glucose metabolic memory period in HT22 cells, the heterochromatin protein 1(HP1) remained high and displayed the similar hysteresis accompany with the high H3K9me3;(5) during the high glucose metabolic memory period in HT22 cells, as well as, the relative occupancy of the major-SAT region(heterochromatin condensation marker) by H3K9me3 remained high and a hysteresis phenomenon;(6) in HT22 cells, the mitochondria ATP production increased in response to the high glucose stimulation, but returned to the control level rapidly after the withdrawn of high glucose with a very short time hysteresis..