The Relationship Between The Expression Of GLUT1 And GLUT3 And Cerebral Ischemic Damage In Diabetic Rats

Objectives:Glucose is the primary metabolic fuel for the central nervous system, and a constant supply is essential to maintain normal cerebral development and function. The delivery of glucose from the blood to the brain involves its passage across the endothelial cells of the blood-brain barrier (BBB) and then across the neural cell membranes. These processes are mediated by the facilitative glucose transporter proteins GLUT₁ and GLUT₃. Studies have suggested that chronic hypoglycemia could enhance the GLUT₁ and GLUT₃ gene expression and induce the upregulation of the GLUT₁ and GLUT₃ proteins. However, whether the expressions of GLUT₁ and GLUT₃ were reduced in diabetes is unknown. Our study aims to use diabetic rats model to investigate the mRNA and protein expression of GLUT in brain under different blood glucose levels in diabetes, and further to evaluate the dynamic effect of hyperglycemia on the expression of GLUT₁ and GLUT₃.Materials and Methods:A total of 60 healthy male Wistar rats weighting 180-220g were used as study objects. 10 rats were randomly chosen as normal control group (group NC), the rest 50 rats were induced to be diabetes mellitus by STZ which was injected into the abdominal cavity. When the diabetic model was successfully established, the 40 diabetic rats were chosen as trial objects and were randomly divided into 4 groups: the DM1 group (the blood glucose was not controlled), the DM2 group (the blood glucose was bad controlled), the DM3 group (the blood glucose was general controlled), and the DM4 group (the blood glucose was good controlled), each group had 10 rats. Another 10 normal rats served as controls (the group NC). The four DM groups were treated with protamine zinc insulin(PZI) to control their blood glucose levels to>16.7 mmol/L, 14-16.7 mmol/L, 10-14 mmol/L and<10 mmol/L, respectively. After three months the HbA1c of five groups were measured, then all rats were decapitated and their whole brains were dissected. The reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry methods were used to measure the mRNA and protein expression of GLUT₁ and GLUT₃, respectively.Data were analyzed with the Statistical Package SPSS 11.0. Descriptive data were given as means±standard deviation (SD). The data were evaluated by a one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test as a post hoc test, and the relationships between the variables were analyzed by linear correlation. A P value of less than 0.05 was considered statistically significant.Results:1. During the experiment the weights of DM1, DM2 and DM3 groups were lower than the control group, the blood glucose and HbA1c were much higher than the control group. The rate of the weight growth of DM4 group was more rapid than DM1, DM2 and DM3 groups, and the control of blood glucose was better.2. Compared with NC group, the GLUT1 mRNA expression of DM1, DM2, DM3 and DM4 groups was decreased by 46.14%, 37.42%, 29.67%, 19.33% (P<0.001) respectively,3. Compared with NC group, the GLUT3 mRNA expression of DM1, DM2, DM3 and DM4 groups was decreased by 66.83%, 56.64%, 47.16%, 21.95% (P<0.001) respectively.4. Immunohistochemistry detection showed that the positive stained granules of GLUT₁ protein in BBB and GLUT₃ protein in the brain cortex and hippocampal CA region were abundant in NC group. In DM1, DM2 and DM3 group the positive stained granules of GLUT₁ and GLUT₃ protein reduced markedly, especially in DM1 group. The difference between DM4 and NC group was also significant (p<0.01).5. The density of micro vessels in the brain of diabetic rats had no significant change compared with normal rats.Conclusions:1. Chronic hyperglycemia could induce the "downregulation" of the GLUT₁ and GLUT₃ mRNA expression in the rat brain.2. Chronic hyperglycemia could induce the "downregulation" of the GLUT₁ and GLUT₃ protein expression in the rat brain.3. The decrease of the BBB GLUT₁ was not due to the decrease of the density of microvessels.4. When we controlled the blood glucose level using insulin, the expressions of GLUT₁ and GLUT₃ mRNA and protein increased, and there was negative correlation between GLUT expression and blood glucose level.5. The downregulation of GLUT₁ and GLUT₃ expression was the adaptive reaction of the body to avoid excessive glucose entering the cell and leading to the damnification of the cell. BackgroundDiabetes mellitus complicated with stroke is one of diabetic vascular complications. It is also the 2^nd major causes of death of patients with diabetes mellitus. And cerebral infarction is the principal causes of stroke. As the primary metabolic fuel for the central nervous system, a constant supply of glucose is essential to maintain normal cerebral development and function. The delivery of glucose from the blood to the brain cell must be mediated by the facilitative glucose transport proteins GLUT1 and GLUT3. Diabetes mellitus is often combined with ischemic cerebrovascular disease. Many researches have proved that acute hyperglycemia could aggravate ischemic brain damage and increase the cerebral infarction volumes. However, hypoglycemia decreases energy supply for brain, which may exacerbate the injury of the brain tissue and affect the prognosis. Our previous study showed that chronic hyperglycemia could induce the downregulation of glucose transporters in the brain of diabetic rats. It could mean that there is a downregulation-compensation self-defending mechanism to avoid hyperglycemic lesion in diabetic hyperglycemic state. And we named the mechanism as hyperglycemia pre-accommodation (HGPA).As a compensatory mechanism for the protection . HGPA could help to maintain the normal metabolism of brain cells and to protect brain functions. However, basing the existence of HGPA, when a stroke happened, a relatively high blood glucose levels are needed to maintain the energy metabolism of brain for diabetic patients in the acute period of cerebral infarct. If blood glucose levels are too lower (even relatively low), HGPA may aggravate brain injury due to shortage of glucose transport in brain cells. Especially, it is not conducive to the functional recovery of the brain cells in ischemic penumbra region (ischemia penumbra, IP). Now, it is still controversial about controlling level of blood glucose ( glucose controlling threshold, GCT ) in diabetic patients with stroke. It is unclear that what blood glucose level controlled in the acute period is ideal for the treatment of diabetic patients with stroke. However, It has important clinical value and major scientific significance to explore the ideal GCT.ObjectivesWe aimed to investigate the relationship between the blood glucose levels, the GLUT gene expressions and the cerebral infarction volumes, further to explore the correlated mechanism of blood glucose levels and cerebral ischemia/reperfusion damage in diabetic rats. And to evaluate the ideal glucose controlling threshold in the acute cerebral infarction period for the treatment of diabetes with stoke. To provide a theoretical basis to identify suitable blood glucose threshold as a blood glucose control target for the clinical treatment of diabetes cerebral infarction.Materials and MethodsA total of 150 healthy male Wistar rats weighting 180-220g were used, 30 rats were randomly chosen to be normal control group (group NC),the rest 120 rats were induced to be diabetes mellitus by STZ injected into abdominal cavity. After the diabetic model was successfully established, the 90 diabetic rats were chosen to be experimental subjects , and were randomly divided into 3 groups: the group DM1 (the blood glucose was bad controlled), the group DM2 (the blood glucose was general controlled), and the group DM3( the blood glucose was good controlled), each group had 30 rats. The three DM groups were treated with neutral protamine hagedorn's insulin (NPH) to control their blood glucose levels to>16.7mmol/L, 10-14.0mmol/L, 6-8mmol/L, respectively. After 4-6 weeks , the model of cerebral ischemia-reperfusion injury (MCAO/R) in rats (four groups) was established with modified sutured-occluded method invented by Zea Longa. According to the sugar levels, each group was divided into 3 sub-groups. The Group NC was divided into NC_mcao1,NC_mcao2,NC_mcao3, and were injected 50% glucose solution into abdominal cavity, treated with NPH or isotonic Na chloride to control their blood glucose levels to the high sugar level( > 8mmol/L), normal sugar level, and low sugar level(2-3mmol/L), respectively. The Group DM1 was divided into DM1_Mcao1, DM1_mcao2 and DM1_mcao3, the sugar levels were controlled to > 16.7mmol/L, 10.0-14.0mmol/L and 6.0-8.0mmol/L by injecting NPH with different dosage, respectively. The group DM2 and DM3 were also divided into three subgroups: DM2_Mcao1, DM2_Mcao2, DM2_Mcao3 and DM3_Mcao1, DM3_Mcao 2 and DM3_Mcao3 with the same glucose level controlled. The rats were sacrificed by decapitation at 72 h after reperfusion, and the volume of cerebral infarction was assessed by tetrazolium chloride (TTC ) staining.Statistical analysis: Data was analyzed with the Statistical Package of the SPSS 13.0. Discriptive data were given as means±s. The comparison of multiple mean with analysis of the One-Way ANOVA, and the two-two comparisons among the means were done by Student-Newman-Keuls method. The relations between the variables were tested by linear correlation. A p-value of less than 0.05 were considered to be significant.Results1. Intra-groups comparison of changes in brain infarct volume(1) Intra-groups comparison of the three sub-groups of Group NC and comparison between each two sub-group showed significantly differences ( p=0.000). The result showed that the order of the changes in brain infarct volume was NC_mcao3 > NC_mcao1 >NC_Mcao2.(2) Intra-groups comparison of the three sub-groups of Group DM1 and comparison between each two sub-group showed significantly differences (p=0.000). The order of the changes in brain infarct volume was DM1_mcao1 > DM1_Mcao3 > DM1_Mcao2.(3) Intra-groups comparison of the three sub-groups of Group DM2 showed significantly differences( p < 0.05). DM2_Mcao 1 VS DM2_Mcao 2 (p=0.005) and DM2_Mcao 1 VS DM2_mcao3 (p=0.000) showed significantly differences. The order of the changes in brain infarct volume was DM2_mcao1 > DM2_mcao2, DM2_mcao3.(4) Intra-groups comparison of the three sub-groups of Group DM3 showed significantly differences (p=0.000). DM3_Mcao 1 VS DM3_Mcao 2 and DM3_MCao1 VS DM3_mcao3 showed significantly differences (p=0.001). The order of the changes in brain infarct volume was DM3_Mcao1 > DM3_mcao2 , DM3_mcao3.2. Interclass comparison of changes in brain infarct volume(1) Comparison among the sub-groups with high sugar level in acute stage of diabetic cerebral infarction: Comparison among the three sub-groups of DMI_mcao1 , DM2 _mcao1 and DM3 _mcao1 showed no significant differences (P=0.255) .(2) Comparison among the sub-groups with medium sugar level in acute stage of diabetic cerebral infarction: Comparison among the three sub-groups of DM1 _mcao2, DM2 _mcao2 and DM3 _mcao2 showed significant differences (p=0.011) . DM1 _mcao 3 VS DM2 _mcao3 and DM1 _mcao3 VS DM3 _mcao 3 showed significantly differences (p < 0.05). The order of the changes in brain infarct volume was DM2 _mcao2, DM3 _mcao2 > DM1 _mcao 2.(3) Comparison among the sub-groups with low sugar level in acute stage of diabetic cerebral infarction: Comparison among the three sub-groups of DM1 _mcao3, DM2 _mcao3 and DM3 _mcao3 showed significant differences(P=0.000). DMI _mcao3 VS DM2_mcao3 and DM1_mcao3 VS DM3_mcao3 shown significantly differences (p=0.000). DM2 _mcao3 VS DM3_Mcao3 showed no significantly differences (P=0.447). The order of the changes in brain infarct volume was DM1_mcao3 > DM2_mcao3, DM3 _mcao3.(4) Comparison among the sub-groups with no changes of sugar level in acute stage of diabetic cerebral infarction: Comparison among the four sub-groups of NC_mcao2, DM1_mcao1, DM2_MCao2 showed significant differences (P=0.000). NC_MCao2 VS DM1_Mcao1 and NC_Mcao2 VS DM2_Mcao2 shown significantly differences ( p=0.000); DM3_MCAO3 VS DMI_mcao1 and DM3_Mcao3 VS DM2_MCAO2 showed significantly differences( p=0.000). NC_McaO2 VS DM3_Mcao3 showed no significantly differences(p=0.078). The order of the changes in brain infarct volume was DM1_MCAO1,DM2_MCAO2 > DM3_MCAO3, NC_Mcao2.3. Comparison of change range in brain infarct volume: In the acute stage of diabetic cerebral infarction, the blood glucose level controlled was correlated to cerebral infarct volume.4. The relation between change range in brain infarct volume and the blood sugar levels: The basic level of blood glucose was correlated to brain infarct volume negatively.Conclusion1. In the acute period of cerebral infarction, high or low blood glucose could aggravate infarction damage.2. Both basic blood glucose level before infarction and blood glucose levels controlled in the acute period of cerebral infarction were closely related to the volume of cerebral infarction in diabetic rats. There were positive correlation between basic blood glucose level before infarction and the injury of cerebral infarction. The higher the basic blood glucose level is, the bigger the injury of cerebral infarction is.3. If the basic blood glucose level before infarction was not well controlled, it was beneficial to degrade blood glucose level appropriately, it was likely to increase the volume of cerebral infarct injury to increase or maintain blood glucose levels.4. However, it could reduce infarct volume to control blood glucose appropriately. But it could not be suggested that the lower blood glucose controlled was, the smaller the volume of cerebral infarction was. The cerebral infarct injury might be aggravated while blood glucose level controlled was too lower. The mechanism might be related to the down regulation of brain glucose transporter protein, that was HGPA.5. While the basic blood glucose level before infarction was not well controlled, it was reasonable to degrade blood glucose to an extent of 4 - 6mmol / L. A blood glucose controlling threshold of 10.0 to 14.0mmol / L (GCT) was safe in the common situation. If the basic blood glucose level before infarction is well controlled, it is better to maintain the blood glucose level.6. There is a downregulation-compensation self-defending mechanism to avoid hyperglycemic lesion in diabetic hyperglycemic state. And we named the mechanism as hyperglycemia pre-accommodation (HGPA) in brain. When cerebral infarctions happened, the normalization of blood glucose level controlled might be not conducive to injury recovery of cerebral infarction. As the existence of HGPA, and the HGPA mechanism could not be relief timely when blood glucose level was degraded suddenly and sharply, a relatively high blood glucose level was needed to maintain the energy metabolism of brain cells. So when diabetic cerebral infarctions happened, the blood glucose level controlled should not be too lower to protect injury brain tissue, and to improve the prognosis of cerebral infarction.7. Our findings could elucidate the clinical phenomenon of hypoglycemia without hypoglycemia. And we will carry out a clinical experiment to test our experimental findings.