Visualization and quantitation of GLUT4 translocation in human skeletal muscle following glucose ingestion and exercise.

Insulin- and contraction-stimulated increases in glucose uptake into skeletal muscle occur in part as a result of the translocation of glucose transporter 4 (GLUT4) from intracellular stores to the plasma membrane (PM). This study aimed to use immunofluorescence microscopy in human skeletal muscle to quantify GLUT4 redistribution from intracellular stores to the PM in response to glucose feeding and exercise. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of ten insulin-sensitive men in the basal state and following 30 min of cycling exercise (65% VO2 max). Muscle biopsy samples were also taken from a second cohort of ten age-, BMI- and VO2 max-matched insulin-sensitive men in the basal state and 30 and 60 min following glucose feeding (75 g glucose). GLUT4 and dystrophin colocalization, measured using the Pearson's correlation coefficient, was increased following 30 min of cycling exercise (baseline r = 0.47 ± 0.01; post exercise r = 0.58 ± 0.02; P < 0.001) and 30 min after glucose ingestion (baseline r = 0.42 ± 0.02; 30 min r = 0.46 ± 0.02; P < 0.05). Large and small GLUT4 clusters were partially depleted following 30 min cycling exercise, but not 30 min after glucose feeding. This study has, for the first time, used immunofluorescence microscopy in human skeletal muscle to quantify increases in GLUT4 and dystrophin colocalization and depletion of GLUT4 from large and smaller clusters as evidence of net GLUT4 translocation to the PM.

Quantitative immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance and sprint interval training.

Increases in insulin-mediated glucose uptake following endurance training (ET) and sprint interval training (SIT) have in part been attributed to concomitant increases in glucose transporter 4 (GLUT4) protein content in skeletal muscle. This study used an immunofluorescence microscopy method to investigate changes in subcellular GLUT4 distribution and content following ET and SIT. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of 16 sedentary males in the overnight fasted state before and after 6 weeks of ET and SIT. An antibody was fully validated and used to show large (> 1 µm) and smaller (<1 µm) GLUT4-containing clusters. The large clusters likely represent trans-Golgi network stores and the smaller clusters endosomal stores and GLUT4 storage vesicles (GSVs). Density of GLUT4 clusters was higher at the fibre periphery especially in perinuclear regions. A less dense punctate distribution was seen in the rest of the muscle fibre. Total GLUT4 fluorescence intensity increased in type I and type II fibres following both ET and SIT. Large GLUT4 clusters increased in number and size in both type I and type II fibres, while the smaller clusters increased in size. The greatest increases in GLUT4 fluorescence intensity occurred within the 1 µm layer immediately adjacent to the PM. The increase in peripheral localisation and protein content of GLUT4 following ET and SIT is likely to contribute to the improvements in glucose homeostasis observed after both training modes.

Correlation of MRI biomarkers with tumor necrosis in Hras5 tumor xenograft in athymic rats.

Magnetic resonance imaging (MRI) can measure the effects of therapies targeting the tumor vasculature and has demonstrated that vascular-damaging agents (VDA) induce acute vascular shutdown in tumors in human and animal models. However, at subtherapeutic doses, blood flow may recover before the induction of significant levels of necrosis. We present the relationship between changes in MRI biomarkers and tumor necrosis. Multiple MRI measurements were taken at 4.7 T in athymic rats (n = 24) bearing 1.94 +/- 0.2-cm3 subcutaneous Hras5 tumors (ATCC 41000) before and 24 hours after clinically relevant doses of the VDA, ZD6126 (0-10 mg/kg, i.v.). We measured effective transverse relaxation rate (R2*), initial area under the gadolinium concentration-time curve (IAUGC(60/150)), equivalent enhancing fractions (EHF(60/150)), time constant (K(trans)), proportion of hypoperfused voxels as estimated from fit failures in K(trans) analysis, and signal intensity (SI) in T2-weighted MRI (T(2)W). ZD6126 treatment induced > 90% dose-dependent tumor necrosis at 10 mg/kg; correspondingly, SI changes were evident from T2W MRI. Although R2* did not correlate, other MRI biomarkers significantly correlated with necrosis at doses of > or = 5 mg/kg ZD6126. These data on Hras5 tumors suggest that the quantification of hypoperfused voxels might provide a useful biomarker of tumor necrosis.

Misregistration artifacts in image-derived arterial input function in non-echo-planar imaging-based dynamic contrast-enhanced MRI.

PURPOSE: To characterize misregistration artifact in arterial input function (AIF) pixels in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using a two-dimensional non-echo-planar imaging (EPI)-based gradient-recalled echo (GRE) sequence. MATERIALS AND METHODS: Dynamic gadopentetate-enhanced MRI was acquired in the rat using a semikeyhole acquisition scheme. The AIF was obtained from abdominal aorta pixels. Different sliding-window reconstruction techniques were applied to determine which lines in a series of the semikeyhole acquisition were associated with the misregistration artifacts. RESULTS: The misregistration along the phase-encoding direction arose when k-space lines were acquired during the rise-time of the aortic gadolinium concentration. The maximum blood concentration of gadolinium estimated from the phase shift calculation agreed with that estimated from dosage. CONCLUSION: AIF misregistration results from a phase shift due to increasing gadolinium concentration in the aorta, and may need to be considered in small animal DCE-MRI studies with a high rate of rise in the AIF in high-field MR applications.

Averaging keyhole pulse sequence with presaturation pulses and EXORCYCLE phase cycling for dynamic contrast-enhanced MRI.

The purpose of this study was to design a keyhole pulse sequence for quantitative 2D dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) based on a spoiled gradient echo T1-weighted acquisition. Saturation recovery was applied to achieve a linear correlation between signal intensity and contrast agent concentration in an arterial input function (AIF) while simultaneously removing time-of-flight effect. To remove ghosting artifacts arising from incomplete presaturation, EXORCYCLE phase cycling with averaging was applied to the pulse sequence. RF spoiling by radiofrequency switching with the synthesizer can be combined with EXORCYCLE phase cycling. Images affected by the large difference in signal intensity before and after contrast agent administration with the keyhole technique were improved by interleaving of peripheral lines of k-space with groups of central lines. Both peripheral and central lines were renewed during the dynamic scan. AIFs were obtained from the rat abdominal aorta with this keyhole sequence.