In this paper, GLUT4 vesicles are observed in real-time under TIRF microscopy and a new three-dimensional single particle tracking algorithm according to the unique features of TIRF is put forward. Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time and mobile vesicles were segmented by an adaptive background subtraction method. Kalman filtering was then introduced to track the granules so as to reduce the searching range and to avoid the disturbance of background noise and false targets. In the experiments the algorithm was applied in analyzing the long-distance movement of GLUT4 vesicles. The experimental results indicate that the algorithm has achieved robust tracking of the vesicles in the imaging plane and has effectively calculated the position in the direction orthogonal to the imaging plane.
Glucose Transporter Type 4 ; metabolism ; Imaging, Three-Dimensional ; instrumentation ; methods ; Ion Transport ; Microscopy, Fluorescence ; methods

Previous studies proposed that the synergistic effect of fibroblast growth factor-21 (FGF-21) and insulin may be due to the improvement of insulin sensitivity by FGF-21. However, there is no experimental evidence to support this. This study was designed to elucidate the mechanism of synergistic effect of FGF-21 and insulin in the regulation of glucose metabolism. The synergistic effect of FGF-21 and insulin on regulating glucose metabolism was demonstrated by investigating the glucose absorption rate by insulin resistance HepG2 cell model and the blood glucose chances in type 2 diabetic db/db mice after treatments with different concentrations of FGF-21 or/and insulin; The synergistic metabolism was revealed through detecting GLUT1 and GLUT4 transcription levels in the liver by real-time PCR method. The experimental results showed that FGF-21 and insulin have a synergistic effect on the regulation of glucose metabolism. The results of real-time PCR showed that the effective dose of FGF-21 could up-regulate the transcription level of GLUT1 in a dose-dependent manner, but had no effect on the transcription level of GLUT4. Insulin (4 u) alone could up-regulate the transcription level of GLUT4, yet had no effect on that of GLUT1. Ineffective dose 0.1 mg kg(-1) FGF-21 alone could not change the transcription level of GLUT1 or GLUT4. However, when the ineffective dose 0.1 mg x kg(-1) FGF-21 was used in combination with insulin (4 u) significantly increased the transcription levels of both GLUT1 and GLUT4, the transcription level of GLUT1 was similar to that treated with 5 time concentration of FGF-21 alone; the transcription level of GLUT4 is higher than that treated with insulin (4 u) alone. In summary, in the presence of FGF-21, insulin increases the sensitivity of FGF-21 through enhancing GLUT1 transcription. Vice versa, FGF-21 increases the sensitivity of insulin by stimulating GLUT4 transcription in the presence of insulin. FGF-21 and insulin exert a synergistic effect on glucose metabolism through mutual sensitization.
Animals ; Blood Glucose ; Diabetes Mellitus, Experimental ; metabolism ; Drug Synergism ; Fibroblast Growth Factors ; pharmacology ; Glucose ; metabolism ; Glucose Transporter Type 1 ; metabolism ; Glucose Transporter Type 4 ; metabolism ; Hep G2 Cells ; Humans ; Insulin ; pharmacology ; Insulin Resistance ; Liver ; metabolism ; Mice

Animals ; Brain ; metabolism ; physiology ; Diet ; Glucose Transporter Type 1 ; genetics ; metabolism ; Glucose Transporter Type 4 ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Physical Conditioning, Animal ; physiology ; RNA, Messenger ; genetics ; metabolism ; Random Allocation

OBJECTIVE: To explore the effect of sodium tungstate on glucose metabolism in adipocytes and its mechanism. METHODS: After 3T3-L1 preadipocytes were differentiated into adipocytes, these adipocytes were incubated with sodium tungstate (0, 150, 300, 500, and 700 micromol/L) for 48 h, and then glucose consumption of the adipocytes was detected by glucose-oxidase assay. Glucose transport was determined by the uptake of 2-deoxy-[3H]-D-glucose, and the expression of glucose transport-4 (GLUT-4) mRNA was identified by semi-quantitative RT-PCR. RESULTS: Sodium tungstate (150 approximately 700 micromol/L) could significantly increase the glucose consumption and glucose transport with a concentration dependent-effect. Sodium tungstate could increase GLUT-4 mRNA expression. CONCLUSION Sodium tungstate can enhance the glucose metabolism of adipocytes by up-regulating the expression of GLUT-4 mRNA.
3T3-L1 Cells ; Adipocytes ; metabolism ; Animals ; Glucose ; metabolism ; Glucose Transporter Type 4 ; biosynthesis ; genetics ; Hypoglycemic Agents ; pharmacology ; Mice ; RNA, Messenger ; biosynthesis ; genetics ; Tungsten Compounds ; pharmacology ; Up-Regulation

OBJECTIVETo study the effects of conjugated linoleic acid (CLA) on expression of glucose transporter 4 (GLUT4) protein in skeletal muscle of insulin resistant rat, and explore the mechanism of resisting diabetes by CLA.

METHODSMale Wistar rats were randomly separated into control group, high-fat group and high fat plus CLA group (0.75 g%, 1.50 g%, 3.00 g% by deit weight), and the effects of CLA on blood glucose and insulin levels of insulin resistant rat were observed , by using Western blot technique to measure the expression level of GLUT4 protein in skeletal muscle of insulin resistant rat.

RESULTSThe serum insulin and glucose levels of obese rats were (11.11 +/- 2.73) microU/ml, and (5.09 +/- 0.66) mmol/L, the supplement of CLA might decrease the hyperinsulinemia and hyperglycemia, and in CLA groups (0.75 g%, 1.50 g%, 3.00 g% by deit weight) the serum insulin was (6.99 +/- 1.77) microU/ml, (7.36 +/- 1.48) microU/ml and (7.85 +/- 1.60) microU/ml (P < 0.05), and the glucose levels were (4.28 +/- 0.72) mmol/L, (4.18 +/- 0.55) mmol/L (P < 0.05), (4.06 +/- 0.63) mmol/L (P < 0.05) respectively. The expression of GLUT4 protein in skeletal muscle of rat fed with high fat diet were decreased as compared with those fed with basic deit, and CLA might increase the expression of GLUT4 protein in skeletal muscle fed with high fat diet.

CONCLUSIONSCLA improve the insulin resistance of obese rat, possibly acting through increasing the expression of GLUT4 protein in skeletal muscle of rat fed with high fat diet.

Animals ; Blood Glucose ; metabolism ; Glucose Transporter Type 4 ; metabolism ; Insulin ; blood ; Insulin Resistance ; Linoleic Acid ; pharmacology ; Male ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo study the effect of chronic intermittent hypoxia-induced inflammatory cytokines and reoxygenation on glucose transporter 4 (GLUT-4) expression in rat skeletal muscles.

METHODSTwenty-four male Sprague-Dawley rats were randomly assigned to blank control group, chronic intermittent hypoxia (CIH) group, and reoxygenation group. At the end of the experiment, fasting blood glucose (FBG), fasting blood insulin (FINS) and serum inflammatory cytokine levels were measured with glucose oxidase-peroxidase, insulin radioimmunoassay and ELISA, respectively. Homeostasis model assessment (IRI) was used to evaluate insulin resistance in the rats, and GLUT-4 protein expression in the skeletal muscles was measured with Western blotting.

RESULTSCompared with the blank control group, CIH resulted in significantly increased fasting blood glucose, blood insulin levels and insulin resistance index (IRI) (P<0.05); fasting blood glucose was significantly elevated in reoxygenation group (P<0.05). Inflammatory cytokines levels (IL-6 and TNF-α) were significantly higher in CIH group than in the blank control and reoxygenation groups (P<0.05), and were higher in reoxygenation group than in the blank control group. GLUT-4 expression in the skeletal muscles was significantly reduced after CIH (P<0.05) but increased after subsequent reoxygenation (P<0.05).

CONCLUSIONCIH can cause increased release of inflammatory cytokines to lower GLUT-4 protein expression in the skeletal muscles, which contributes to insulin resistance in adult rats.

Animals ; Blood Glucose ; Glucose Transporter Type 4 ; metabolism ; Hypoxia ; Insulin ; blood ; Insulin Resistance ; Interleukin-6 ; Male ; Muscle, Skeletal ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo evaluate the expression of glucose transporter-4 (GLUT4) mRNA in skeletal muscle and subcutaneous adipose tissues and investigate the mechanism of posttraumatic insulin resistance.

METHODSSixteen adult male Wistar rats were randomly divided into 2 group (n equal to 8 in each group), i.e., severe traumatic brain injury (TBI) group due to falls from a height and normal control group. Blood glucose and serum insulin were measured at 0.5 h before trauma and 3 h, 24 h, 72 h, 7 d after trauma, respectively. And insulin sensitivity was calculated by insulin activity index (IAI) formula. Skeletal muscle and subcutaneous adipose tissue samples were collected at the same time when blood was sampled. The changes of expression of GLUT4 mRNA were observed using reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSAccompanied by the decrease of insulin sensitivity, the expression of GLUT4 mRNA was significantly decreased in adipose tissues at 24 h and 72 h after trauma (P less than 0.01), however, such phenomena did not appear in skeletal muscle samples.

CONCLUSIONSTo some extent, the development of posttraumatic insulin resistance is related to the abnormality of transcription activity of GLUT4 gene. Adipose tissues show some difference in the transcriptional level of GLUT4 gene after trauma as compared with skeletal muscle tissues.

Adipose Tissue ; metabolism ; Animals ; Brain Injuries ; metabolism ; physiopathology ; Glucose Transporter Type 4 ; metabolism ; Insulin Resistance ; physiology ; Male ; Muscle, Skeletal ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar

BACKGROUNDThe change of glucose transporter 4 (GLUT4) expression could influence glucose uptake in the myocardial cells and then effect myocardial metabolism, which maybe one of the factor for the diabetes cardiovascular disease. This study aimed to explore the influence of glucose and insulin at different concentrations on H9c2 (2-1) cell proliferation and its GLUT4 expression in vitro, and evaluate the correlation between myocardial cells proliferation and GLUT4 expression. This might be helpful for understanding the relationship between glucose metabolism and cardiovascular disease.

METHODSAccording to glucose concentrations in culture medium, cultured H9c2 rat myocardial cells were divided into five groups: control group (NC, glucose concentration 5.0 mmol/L), low glucose group (LG, glucose concentration 0.1 mmol/L), high glucose group 1 (HG1, glucose concentration 10 mmol/L), high glucose group 2 (HG2, glucose concentration 15 mmol/L), high glucose group 3 (HG3, glucose concentration 20 mmol/L). Then according to different insulin concentrations in culture medium, each group was further divided into two subgroups: normal insulin subgroup (INSc, insulin concentration 3.8 mU/L), high insulin subgroup (INSh, insulin concentration 7.6 mU/L). H9c2 (2-1) cells were cultured for 1, 2, 3 days, the proliferation of cells were assayed by cell counting Kit-8 assay, the expressions of GLUT4 mRNA and protein were detected with RT-PCR and Western Blotting technique, and the relation between myocardial cells proliferation and GLUT4 expression was evaluated.

RESULTSCompared with NC group, cell proliferation (OD value) was lower in LG, HG2, HG3 group but higher in HG1 group on the second and the third day (P < 0.05). There was a negative correlation between OD value and the glucose level in HG1, HG2, HG3 groups (P < 0.05). OD value in INSc subgroups was lower than that in INSh subgroups (P < 0.05). GLUT4 mRNA was lower in LG, HG2, HG3 groups than that in NC group (P < 0.05). Compared with NC group, GLUT4 mRNA level in HG1 group was higher on the first day but lower on the second and third day (P < 0.05). In HG1, HG2 and HG3 groups, GLUT4 mRNA level had a negative correlation with the level of glucose (P < 0.05). GLUT4 mRNA in INSc subgroups was lower than that in INSh subgroups (P < 0.05). The expression of GLUT4 protein was similar to that of GLUT4 mRNA. There was a positive correlation between H9c2 cell proliferation and GLUT4 expression (P < 0.02).

CONCLUSIONSGlucose levels could regulate glucose uptake in myocardial cells through influencing GLUT4 expression, and thus affected the cell proliferation and cell function. Insulin levels could affect the myocardial cell function by regulating GLUT4 expression. Effects of glucose and insulin on the myocardial cells proliferation might be mediated through regulating GLUT4 expression. There may be a mechanism of hyperglycemia pre-accommodation (HGPA) in myocardial cells mediated through regulation of GLUT4 expression.

Animals ; Blotting, Western ; Cell Line ; Cell Proliferation ; drug effects ; Glucose ; pharmacology ; Glucose Transporter Type 4 ; genetics ; metabolism ; Insulin ; pharmacology ; Myocardium ; cytology ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

The aim of this project is to establish a fibroblast growth factor-21 (FGF-21) signaling pathway targeted cell model, for screening a class of FGF-21 receptor agonists as anti-diabetic candidates. FGF-21 requires beta klotho transmembrane proteins as co-receptor for the activation of tyrosine kinase FGF receptor (FGFR) signaling, thereby activating a series of intracellular signaling pathways and regulating gene transcription for glucose metabolism. Firstly a recombinant plasmid expressing co-receptor beta klotho and EGFP reporter genes was constructed. After introducing the recombinant plasmid into package cells, the cell culture supernatant was used to infect 3T3-L1 cells, which were then screened for stably expressing beta klotho gene. Administration of FGF-21 increased the expression of GLUT1 and stimulated GLUT1-mediated glucose uptake. This novel cell model can be conveniently used in high-throughput drug screening of FGF-21 or FGF-21 analogues.
3T3-L1 Cells ; Animals ; Drug Evaluation, Preclinical ; Fibroblast Growth Factors ; metabolism ; pharmacology ; Glucose ; metabolism ; Glucose Transporter Type 1 ; genetics ; metabolism ; Glucose Transporter Type 4 ; genetics ; metabolism ; HEK293 Cells ; Humans ; Hypoglycemic Agents ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; NIH 3T3 Cells ; Plasmids ; RNA, Messenger ; metabolism ; Receptors, Fibroblast Growth Factor ; agonists ; Recombinant Proteins ; genetics ; metabolism ; Retroviridae ; genetics ; Signal Transduction ; Transfection

BACKGROUNDThe persistence of sleep disordered breathing (SDB) symptoms after tonsil and/or adenoid (T&A) surgery are common in children with obstructive sleep apnea (OSA). We tested the hypothesis that disturbances of glucose transporters (GLUTs) in intraabdominal adipose tissue caused by chronic intermittent hypoxia (CIH) from the pedo-period could facilitate the appearance of periphery insulin resistance in Sprague-Dawley (SD) rats. We tested the hypothesis that the changes of GLUTs in adipose tissue may be one of the reasons for persistent SDB among clinical OSA children after T&A surgery.

METHODSThirty 21-day-old SD rats were randomly divided into a CIH group, a chronic continuous hypoxia (CCH) group, and a normal oxygen group (control group) and exposed for 40 days. The changes of weight, fasting blood glucose and fasting blood insulin levels were measured. Hyperinsulinemic-euglycemic clamp techniques were used to measure insulin resistance in each animal. Real-time quantitative PCR and Western blotting were used to measure GLUT mRNA and proteins in intraabdominal adipose tissue. Additional intraabdomial white adipose tissue (WAT) was also processed into paraffin sections and directly observed for GLUTs1-4 expression.

RESULTSWhen compared with control group, CIH increased blood fasting insulin levels, (245.07 +/- 53.89) pg/ml vs. (168.63 +/- 38.70) pg/ml, P = 0.038, and decreased the mean glucose infusion rate (GIR), (7.25 +/- 1.29) mg x kg(-1) x min(-1) vs. (13.34 +/- 1.54) mg x kg(-1) x min(-1), P < 0.001. GLUT-4 mRNA and protein expression was significantly reduced after CIH compared with CCH or normal oxygen rats, 0.002 +/- 0.002 vs. 0.039 +/- 0.009, P < 0.001; 0.642 +/- 0.073 vs. 1.000 +/- 0.103, P = 0.035.

CONCLUSIONSCIH in young rats could induce insulin resistance via adverse effects on glycometabolism. These findings emphasize the importance of early detection and treatment of insulin insensitivity in obese childhood OSA.

Adipose Tissue ; metabolism ; Animals ; Blood Glucose ; metabolism ; Blotting, Western ; Glucose Clamp Technique ; Glucose Transporter Type 4 ; metabolism ; Hypoxia ; physiopathology ; Immunohistochemistry ; Insulin ; blood ; Insulin Resistance ; physiology ; Male ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction