OBJECTIVETo observe the changes in the expression of glucose transporter-3 (GLUT3) in the cerebral cortex of rats during aging and investigate the role of GLUT3 in the aging process of the nervous system.

METHODSThe cerebral tissues were collected from rats of 3, 18, 24, and 30 months old (10 in each age group), and the expression of GLUT3 in the cerebral cortex was detected by immunohistochemistry.

RESULTSUnder optical microscope, GLUT3-positive cells were found in every group. Within the age range of 3 to 8 months, GLUT3-positive cells increased significantly with age (P<0.01), but at 24-30 months of age, the number of GLUT3-positive cells reduced significant with age (P<0.01).

CONCLUSIONThe expression changes of GLUT3 ir the cerebral cortex of rats during aging indicate that GLUT3 plays an important role in the maturation and aging of the nervous system.

Aging ; Animals ; Brain ; metabolism ; Cerebral Cortex ; metabolism ; Glucose Transporter Type 3 ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo examine the distribution of glucose transport 3 (GLUT 3) in different brain regions of aged rats and to investigate its role in ageing process of the nervous system.

METHODSThe GLUT 3 expression in different brain regions was examined with immunohistochemical method in rats aged 3, 18 and 30 months, respectively.

RESULTSThe number of GLUT 3-positive cells varied in the different brain regions in rats of all age groups (P<0.01); the CA1 region contained the greatest number of positive cells,and fewer in the motor cortex and cerebellum. The number of GLUT 3-positive cells was reduced in the brain of aged rats (P<0.01); and the neural cells in 4 different brain regions presented with large cell body and loose alignment.

CONCLUSIONThe expression of GLUT 3 decreased in aged rats, which suggests that GLUT 3 may be involved in the ageing process of nervous system.

Aging ; metabolism ; Animals ; Brain ; metabolism ; Glucose Transporter Type 3 ; metabolism ; Hippocampus ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley

BACKGROUNDThe delivery of glucose from the blood to the brain involves its passage across the endothelial cells of the blood-brain barrier (BBB), which is mediated by the facilitative glucose transporter protein 1 (GLUT(1)), and then across the neural cell membranes, which is mediated by GLUT(3). This study aimed to evaluate the dynamic influence of hyperglycemia on the expression of these GLUTs by measuring their expression in the brain at different blood glucose levels in a rat model of diabetes. This might help to determine the proper blood glucose threshold level in the treatment of diabetic apoplexy.

METHODSDiabetes mellitus was induced with streptozotocin (STZ) in 30 rats. The rats were randomly divided into 3 groups: diabetic group without blood glucose control (group DM1), diabetic rats treated with low dose insulin (group DM2), and diabetic rats treated with high dose insulin (group DM3). The mRNA and protein levels of GLUT(1) and GLUT(3) were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively.

RESULTSCompared with normal control rats, the GLUT(1) mRNA was reduced by 46.08%, 29.80%, 19.22% (P < 0.01) in DM1, DM2, and DM3 group, respectively; and the GLUT(3) mRNA was reduced by 75.00%, 46.75%, and 17.89% (P < 0.01) in DM1, DM2, and DM3 group, respectively. The abundance of GLUT(1) and GLUT(3) proteins had negative correlation with the blood glucose level (P < 0.01). The density of microvessels in the brain of diabetic rats did not change significantly compared with normal rats.

CONCLUSIONSChronic hyperglycemia downregulates GLUT(1) and GLUT(3) expression at both mRNA and protein levels in the rat brain, which is not due to the decrease of the density of microvessels. The downregulation of GLUT(1) and GLUT(3) expression might be the adaptive reaction of the body to prevent excessive glucose entering the cell that may lead to cell damage.

Animals ; Blood Glucose ; analysis ; Brain ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; Glucose Transporter Type 1 ; analysis ; genetics ; Glucose Transporter Type 3 ; analysis ; genetics ; Glycated Hemoglobin A ; analysis ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Streptozocin

Insulin stimulates glucose transport in muscle and fat cells by promoting the translocation of glucose transporter (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3-kinase) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt and PKC- zeta, those are known as the downstream target of PI3-kinase in regulation of GLUT4 translocation, is not known yet. An interesting possibility is that these protein kinases phosphorylate GLUT4 directly in this process. In the present study, PKB- alpha and PKC- zeta were added exogenously to GLUT4-containing vesicles purified from low density microsome (LDM) of the rat adipocytes by immunoadsorption and immunoprecipitation for direct phosphorylation of GLUT4. Interestingly GLUT4 was phosphorylated by PKC- zeta and its phosphorylation was increased in insulin stimulated state but GLUT4 was not phosphorylated by PKB- alpha. However, the GST-fusion proteins, GLUT4 C-terminal cytoplasmic domain (GLUT4C) and the entire major GLUT4 cytoplasmic domain corresponding to N-terminus, central loop and C-terminus in tandem (GLUT4NLC) were phosphorylated by both PKB- alpha and PKC- zeta. The immunoblots of PKC- zeta and PKB- alpha antibodies with GLUT4-containing vesicles preparation showed that PKC- zeta was co-localized with the vesicles but not PKB- alpha. From the above results, it is clear that PKC- zeta interacts with GLUT4-containing vesicles and it phosphorylates GLUT4 protein directly but PKB- alpha does not interact with GLUT4, suggesting that insulin-elicited signals that pass through PI3-kinase subsequently diverge into two independent pathways, an Akt pathway and a PKC- zeta pathway, and that later pathway contributes, at least in part, insulin stimulation of GLUT4 translocation in adipocytes via a direct GLUT4 phosphorylation.
Adipocytes* ; Animals ; Antibodies ; Cytoplasm ; Glucose ; Glucose Transport Proteins, Facilitative ; Glucose Transporter Type 4 ; Immunoprecipitation ; Insulin ; Microsomes ; Phosphatidylinositol 3-Kinases ; Phosphorylation* ; Protein Kinases ; Proto-Oncogene Proteins c-akt ; Rats*

The effect of CoCl(2) pretreatment on glucose transport activity of cultured newborn rat hippocampal neurons and its role in neuronal hypoxic tolerance were observed. The results showed that the 2-deoxy-D-[1-(3)H ]glucose uptake rate and the mRNA expressions of glucose transporters (GLUT1 and GLUT3) in the hippocampal neurons were significantly increased after a 24-hour pretreatment with CoCl(2). The cell injury induced by 6-hour or 8-hour hypoxic exposure was also greatly reduced by CoCl(2) pretreatment. The protective effect of CoCl(2) on the neurons was largely abolished by cytochalasin B, a specific inhibitor of glucose transporters. The results suggest that CoCl(2) can increase mRNA expressions of GLUT1 and GLUT3 and glucose transporter activity of the neurons, which may be an important mechanism for the increased tolerance of the neurons to hypoxia.
Animals ; Animals, Newborn ; Cell Hypoxia ; Cell Survival ; drug effects ; Cells, Cultured ; Cobalt ; pharmacology ; Glucose Transporter Type 1 ; metabolism ; Glucose Transporter Type 3 ; metabolism ; Hippocampus ; cytology ; Hypoxia ; metabolism ; Neurons ; drug effects ; metabolism ; Organometallic Compounds ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo investigate the expression of glucose transporter (Glut)1, Glut3, and hypoxia inducible factor (HIF)-1 alpha in human non-small cell lung carcinoma (NSCLC), and its clinical significance.

METHODSSpecimens of cancer tissues and paracancerous lung tissues from 34 cases of NSCLC and 17 specimens of benign lung lesions were collected. The expressions of Glut1, Glut3, and HIF-1 alpha were detected with immunohistochemical staining, RT-PCR, and Western blot.

RESULTThe relative mRNA expressions of Glut1 and HIF-1 alpha were 0.689 +/-0.245, 0.693 +/-0.248 in cancer tissues; and 0.338 +/-0.157, 0.351 +/-0.184 in paracancerous lung tissues (P <0.001); while those of Glut3 were 0.506 +/-0.246 in cancer tissues and 0.482 +/-0.238 in paracancerous tissues (P >0.05). The relative protein expressions of Glut1 and HIF-1 alpha were 0.582 +/-0.247, 0.525 +/-0.246 in cancer tissues and 0.288 +/-0.151, 0.261 +/-0.135 in paracancerous lung tissues (P<0.001), but the protein expressions of Glut3 were 0.551 +/-0.251 and 0.436 +/-0.224 respectively (P>0.05). Glut1 and HIF-1 alpha expressions were higher in poor differentiation group and in stage III group, than those in medium and well differentiation group and stage I and II group. Moreover, there was a significant correlation between the expression of Glut1 and HIF-1 alpha (r=0.854, P<0.01).

CONCLUSIONGlut1 and HIF-1 alpha are highly expressed in NSCLC, and their expressions are associated with tumor differentiation and clinical stage.

Aged ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Glucose Transporter Type 1 ; metabolism ; Glucose Transporter Type 3 ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Male ; Middle Aged

BACKGROUNDBlood glucose control improves the outcome of diabetic patients with stroke, but the target range of blood glucose control remains controversial. The functional recruitment of ischemia penumbra is extremely important to the recovery after stroke. The present study aimed to explore the expression of brain-type glucose transporters (GLUT1 and GLUT3) in cerebral ischemic penumbra at different blood glucose levels and different ischemic-reperfusion time in diabetic hypoxia-ischemia rats. The results might provide an experimental basis for clinical treatment of diabetic patients with stroke.

METHODSThe Wistar rats included in this study were randomly assigned to 4 groups (50 rats each): normal control group (NC), uncontrolled diabetic group (DM1), poorly-controlled diabetic group (DM2), and well-controlled diabetic group (DM3). Diabetic rats were induced by single intraperitoneal injection of streptozotocin, and the focal ischemic rat model of middle artery occlusion (MCAO) was made by insertion of fishing thread in 6 weeks after the establishment of the diabetic model. Each group was divided into 5 subgroups (10 rats each): four focal ischemic subgroups at different ischemic-reperfusion time (at 3,12, 24 and 72 hours after reperfusion, respectively) and one sham-operated subgroup. The mRNA and protein expression of GLUT1 and GLUT3 was assessed by RT-PCR and Western blotting, respectively.

RESULTSThere was significant difference in the mRNA expression of GLUT1 and GLUT3 between the four focal ischemic subgroups and the sham-operated subgroup at different reperfusion time in each group. The mRNA expression of GLUT1 and GLUT3 in the 4 ischemic groups began to increase at 3 hours, peaked at 24 hours after reperfusion and maintained at a higher level even at 72 hours compared with that of the sham-operated subgroup. The mRNA expression of GLUT1 increased more significantly than that of GLUT3. The mRNA expression of GLUT1 and GLUT3 was significantly different between the diabetic groups and normal control group. The mRNA expression of GLUT1 and GLUT3 was increased more significantly in the diabetic groups than that in the normal control group. There was a significant difference in the mRNA expression in the groups with different blood glucose levels. The mRNA expression tended to decrease with increased blood glucose levels. The expression trend of GLUT1 and GLUT3 protein was similar to that of GLUT1 and GLUT3 mRNA.

CONCLUSIONSGLUT1 and GLUT3 expression was notably up-regulated in the penumbra region after cerebral ischemia in this study. But the up-regulated amplitude of GLUT1 and GLUT3 in the diabetic rats with cerebral ischemic injury became smaller than that of the normal controls. In the treatment of diabetic patients with cerebral embolism, blood glucose control should not be too strict, otherwise the up-regulation of GLUT1 and GLUT3 induced by cerebral ischemic injury might not be able to meet the needs of energy metabolism in cells.

Animals ; Blotting, Western ; Brain ; metabolism ; Brain Ischemia ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; Glucose Transporter Type 1 ; genetics ; metabolism ; Glucose Transporter Type 3 ; genetics ; metabolism ; Male ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

AIMTo investigate the expression of GLUT1 and GLUT3 in the hippocampus after cerebral hypoxic-ischemia (HI) in newborn rats and the effect of progesterone (PROG) on them.

METHODSForty newborn SD rats were randomly divided into four groups: normal group, sham-operated group, hypoxic-ischemic group and progesterone group. Model of hypoxic-ischemia encephalopathy (HIE) was established in the 7-day-old newborn SD rats. Immunohistochemical method was applied to detect the expression of GLUT1 and GLUT3 in hippocampus.

RESULTSGLUT1 and GLUT3 were slightly seen in normal and sham operation group, there was no obviously difference between the two groups (P > 0.05). The expression of GLUT1 and GLUT3 in hypoxic-ischemia group were all higher than that in sham operated group (P < 0.05). Not only the expression of GLUT in progesterone group were significantly higher than that in sham operated group (P < 0.01), but also than that in hypoxic-ischemia group (P < 0.05).

CONCLUSIONPROG could increase the tolerance of neuron to hypoxic-ischemia with maintaining the energy supply in the brain by up-regulating GLUT expression.

Animals ; Animals, Newborn ; Glucose Transporter Type 1 ; genetics ; metabolism ; Glucose Transporter Type 3 ; genetics ; metabolism ; Hippocampus ; metabolism ; Hypoxia-Ischemia, Brain ; metabolism ; Progesterone ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Up-Regulation ; physiology

Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Glucose Transporter Type 1 ; metabolism ; Glucose Transporter Type 3 ; metabolism ; Head and Neck Neoplasms ; metabolism ; pathology ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged

OBJECTIVE: To construct an effective lentiviral vector for RNA interference (RNAi) with human glucose transporter 3 (GLUT3)gene.
 METHODS: Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1, pLV-shRNA-GLUT3-2, pLV-shRNA-GLUT3-3, and pLV-shRNA-GLUT3-4. The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing. One of effective vectors was selected and co-transfected into 293T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LV-GLUT3. After viral titer determination, U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection (MOI) of 10. Finally, the expression of GLUT3 protein was detected by Western blot. 
 RESULTS: DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors. In HeLa cells, the expression of GLUT3-mRNA was significantly down-regulated by the recombinant vectors compared with negative control. The recombinant lentivirus LV-GLUT3 harvested from 293T cells had a titer of 1.5×10(9) TU/mL. After infection with LV-GLUT3, the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated. 
 CONCLUSION An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.
Genetic Vectors ; Glucose Transporter Type 3 ; genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Lentivirus ; Plasmids ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection