Visual Cell Based Real Time Assay Biology Essay

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Insulin-stimulated glucose transporter 4 (GLUT4) translocation leading to glucose uptake is vital to glucose homeostasis and is a defined target of antidiabetic drug research. Existing functional assays to detect the process of GLUT4 translocation are hampered by assay variability and low sensitivity, thus slowing down progress towards development of preferred alternative to insulin. This chapter describes a real time, visual, cell-based qualitative GLUT4 translocation assay suitable for screening insulin mimetics. The basic strategy consists of establishment of insulin sensitive CHO-HIRc-myc-GLUT4eGFP cells that stably express myc and eGFP tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by tracking the movement of GLUT4 associated GFP fluorescence from perinuclear space to plasma membrane by employing cooled charge-coupled device (CCD) camera attached to a simple fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provides rapid and foolproof visual evidence suitable for screening GLUT4 translocation modulators. This assay is further validated by complementary assays.

1. INTRODUCTION

It is well established that insulin action leading to glucose uptake by adipocytes and muscle cells through glucose transporter 4 (GLUT4) is a major mechanism for clearance of excess exogenous glucose in blood and hence is the rate limiting step in diabetes (Pessin and Saltiel, 2000). GLUT4 is one of 13 sugar transporter proteins (GLUT1- GLUT12, and HMIT) that facilitates hexose transport across cell membranes (Joost and Thorens, 2001). These transporters differ in respective substrate specificities and concentration. Skeletal muscle, adipose tissue and cardiac cells primarily express maximum of GLUT4, in addition to a distinct set of other transporters. GLUT4 has 12 transmembrane domains with unique sequences in NH2 and COOH terminal domains that navigates its membrane trafficking ability (Fig. 1A). It has a specific substrate binding domain as well as a domain for cytochalasin, which is a known inhibitor of receptor transport. Exo-facial loop of the GLUT4 is a site for insertion of epitope (either myc or HA) that would assist in tracking of insulin induced translocation. Under basal or nonstimulated conditions more than 95% GLUT4 is localized to perinuclear region or in the cytoplasm typically close to the plasma membrane. Dynamically GLUT4 is retained inside cells as it is swiftly internalized and slowly recycled. Insulin stimulation shifts the balance in favor of recycling of GLUT4 to the PM, resulting in an increased presence of GLUT4 on the surface and an associated increase in glucose transport (Suzuki and Kono, 1980). All these factors make GLUT4 a most promising target of antidiabetic drug research.

2. GLUT4 TRANSLOCATION ASSAY

2.1 Rational for development of real time GLUT4 translocation assay for drug discovery

An oral hypoglycemic agent that offers a needle less alternative to insulin therapy is yet to be fulfilled target of pharmaceutical research. With no limit of natural products and synthetic compounds or derived products for antidiabetic drug research, the need of the hour is efficient in vitro systems and methods for GLUT4 translocation assay to facilitate rapid screening. General GLUT4 translocation assays employs indirect methods such as western blot analysis of PM fractions, photoaffinity labeling, binding assay using cytochalasin B and qualitative assessment by immunofluorescence or immunoelectron microscopy (Kozka et al.,1991; Smith et al.,1991). All these assays are not only relatively less sensitive and but also time consuming. Direct visualization of GLUT4 trafficking by tagging of GLUT4 with GFP (green fluorescent protein) and its expression in cells has made detection of GLUT4 movement relatively easy by various microscopy techniques (Dobson et al., 1996). However, the resolution obtained especially with immunofluorescence microscopy may sometimes be inadequate to differentiate between GLUT4 molecules that are inserted into the PM and those residing close to the PM of certain cell lines. Therefore, addition of specific tags within first exofacial loop of GLUT4 is an added advantage for immunofluorescence detection or ELISA of the PM associated GLUT4 distribution in fixed and nonpermeabilized cells (Kanai et al., 1993; Dawson et al., 2001). Similar to the endogenous GLUT4 the overexpressed GLUT4 chimera is also localized to perinuclear region and shows similar response to insulin treatment (Fig 1B and C). Since epitope-tagged GLUT4 helps in detecting cell-surface expression with GFP chimeras revealing subcellular distribution and total expression of GLUT4, it is better to have fusion chimeras between GFP and GLUT4 with an HA or myc tag in their exofacial loops that will not alter its function (Fig 1D and E). Chimera is shown to remains intact, and that the junction between the two proteins is not susceptible to proteolysis which generates native GFP (Dobson et al. 1996). In addition, presence of an exofacial tag and GFP in a chimera provides the opportunity to normalize cell-surface expression levels to total expression levels in a single cell. Such a model system would be of immense use in screening antidiabetic drug or natural products for GLUT4 translocation modulators.

2.2 Rational for development of insulin sensitive cell line for drug discovery

One of the arduous tasks in the antidiabetic drug development is screening enormous numbers of lead compounds for an appropriate biochemical or cellular outcome. The ways of making this screening faster, more effective and less expensive which can revolutionize antidiabetic drug development are being relentlessly pursued and developed. Cell or tissue culture models of diabetes are being used to understand the cellular and molecular processes involved in the disease and also to characterize the cellular or molecular actions of a lead compound in advanced stages of drug development. 3T3-L1, L6 and C2C12 cells are the generally used in vitro models for GLUT4 translocation assays. Yet, they are far from insulin sensitive muscle and adipose cells as a reliable in vitro model. Prerequisite of differentiation and the presence of GLUT1 in these cell lines leave ambiguity in tedious pharmaceutical drugs screening (Mehra et al. 2007; Liu et al. 2009). In addition, traditionally, these cells are least competent for transfection and hence the establishment of stable cell line expressing GLUT4 chimera with tags will be difficult. We were also not successful in stably transfecting 3T3-L1 cells with GLUT4 chimera, as GFP displayed considerably lower levels of fluorescence in 3T3-L1 cells and GFP fluorescence was diminished over a period of time in culture and was eventually lost.

Due to high transfection efficiency, a number of laboratories use CHO cell line that allows the greatest intensity of GFP fluorescence. Differential levels of fluorescence intensity of GFP is attributed to the redox state of the cell, variations in intracellular pH, differences in oxygen tension or expression of heat shock proteins (Dobson et al., 1996). Since, insulin signal transduction machinery essential for GLUT4 translocation is incomplete in CHO cell line, it is essential to manipulate this cell line by overexpressing insulin receptor (IR) and GLUT4 to make them responsive to insulin (Kanai et al. 1993; Quon et al. 1994; Perfetti et al. 1997; Lampson et al. 2000). To establish suitable insulin sensitive CHO cells generally clones expressing IR and tagged GLUT4 chimera are selected. The most preferred chimera of GLUT4 with tag is mycGLUT4eGFP or HAGLUT4eGFP (Dawson 2001 and Jiang 2002). It will also be useful in exploring the intracellular sorting signals involved in the insulin-regulated trafficking pathways of GLUT4. Therefore, CHO cells expressing IR and mycGLUT4eGFP or HAGLUT4GFP is a strong in vitro model for the development of screening assays to test anti-diabetic drugs. Insulin-stimulated translocation of mycGLUT4eGFP vesicles to the PM can be visualized by live cell imaging based on the movement of GFP fluorescence (Oatey et al. 1997, Vijayakumar et al 2011).

3. METHODS AND PROCEDURES

In this part we provide the methods and procedure for the establishment of a stable insulin sensitive CHO cell line, its application in GLUT4 translocation assay and its validation by different approaches. The protocol involves sequential transfection of cells with insulin receptor and GLUT4 (Are you meaning Glut4 GFP fusion-write complete) expression vectors. This is followed by selection and screening of ideal clones for insulin sensitivity as well as by real time assay for GLUT4 translocation with further validation using complementary experiments.

Reagents

Hams F12 medium penicillin, streptomycin, LipofectAMINE 2000 (LF-2000), fetal bovine serum (US origin) and trypsin-EDTA are available from Invitrogen (Carlsbad, CA, USA). Tissue culture wares such as 35 mm and 60 mm petridishes 24 well plates and chambered slide with cover can be procured from Nunc, Rochester, NY, U.S.A. Fine chemicals and reagents 2-deoxy glucose (2-DG), cytochalasin, Paraformaldehyde, sodium dodicyl sulfate and bovine insulin are available from Sigma. 14C-2DG Radiolabelled glucose can be purchased from American radiochemicals (ARC……..). Monoclonal anti-myc antibody, polyclonal IR antibody and rhodamine conjugated got anti-mouse secondary antibody are available from Santacruz Biotechnology (Santa Cruz, CA, U.S.A.). Stock solution of insulin (100 M) is prepared by dissolving 5.7 mg insulin in 10 ml of acidified water.

3.1 Methodological Considerations

Generation of vectors and transfection into desired cell line by molecular biology techniques for the transient or stable expression of specific proteins tagged with a fluorescent moiety for visualization has revolutionized studies on cellular as well as receptor dynamics and analysis. CHO cells stably overexpressing IR (is this human) and GLUT4 (is this human)has several advantages in antidiabetic drug screening, as they avoid the need for expensive and repeated transient transfections and also provide a nearly homogenous expression. CHO cells are tolerant for exogenously expressed proteins well. In addition stable cell line will ensure perinuclear localization of GLUT4 and their response to insulin or insulin mimetics by translocation. To establish an insulin sensitive cell line, CHO cells are to be transfected with the Wild-type Human Insulin Receptor-A eukaryotic expression vector containing the B isoform (exon 11- ) of wild-type human IR (HIRc). Most of the established laboratories are generous to provide the construct and its map upon mutual understanding or after signing material transfer agreement for non-commercial, research and development. However, it is important to verify the construct by standard restriction digestion analysis taking clue from the map or by sequencing. The commonly used plasmid that carry human IR is pCVSVHIRc . This plasmid contains the origin of replication and the ampicillin-resistance gene of the Escherichia coli plasmid pBR322 and an insert containing the human IR coding sequence under the control of the SV40 early promoter (SVE) (Ref………).

3.2 Transfection procedure to establish CHO cells over expressing IR involves following steps

Twenty four hours before transfection, exponentially growing cells are split into 35 mm dish and should be 60-80% confluent for the transfection procedure. To avoid clumping of the cells, do not agitate the cells by tapping or shaking the culture dish during incubation with trypsin.

Cotransfect CHO cells with plasmid pCVSVHIRc (4 g) and a plasmid (pSVEneo) coding for neomycin resistance gene under control of SVE (1 g) by LF-2000 method as described by the manufacturer (Invitrogen). For this, dilute pCVSVHIRc and pSVEneo plasmids to 200 l with F12 media and incubate for 5 min. Dilute 5 l of Lipofectamine reagent to 200 l with F12 media separately and incubate for 5 min.

Mix the solutions of diluted plasmids and LF-2000 and incubate for 30-45 min at room temperature to allow DNA-liposome complex formation.

Add this complex to the cells in culture plates incubated in 1.6 ml of fresh F12 medium for 1 hour. Incubate the cells for further 24 h before the medium was changed to fresh F12 medium.

Twenty four to 48 h posttransfection, the cells are split 1:3 or 1:5 into separate, preferably 60 mm, dishes.

After a 24-h period of recovery, add 800 g/ml of the neomycin analog G418 to the medium to select cells expressing neomycin resistance gene contained in the pSVEneo plasmid. Selection media is replaced every second or third day. Selection is continued generally for 2 to 3 weeks.

Once the majority of the cells are dead, surviving cells tend to form colonies which is monitored and these are tracked by marking at the bottom of the dish.

When the colony size is approximately 1-3 mm in diameter, they are detached with 25 l of trypsin and placed directly into wells of a 48 or 24 well plate and cultured in presence of G418 to be propagated as cell line.

The IR expression levels in each clone should be verified by standard western blotting procedure. The cells that express IR 5-10 fold over control CHO cells should be selected and amplified in tissue culture. It is utmost important to cryopreserve cells in liquid nitrogen as soon as the expression levels are verified. We have observed that any kind of stress due to undesired pH of the media and low density (Culturing less than 2.5x105 cells in 35 mm plate or 5x105 cells in T25) causes decrease in IR expression. The level of IR expression can also be determined by insulin binding assay (White et al., 1987).

3.3 Development of CHO-HIRc-mycGLUT4eGFP cells

The back bone of the plasmid construct used to establish this stable cell line is modified pGreen Lantern Vector with EcoR1 and XbaI sites and the cDNA coding for mycGLUT4eGFP. DNA sequence for myc epitope is between the 66th and 67th amino acid of GLUT4 and GFP encoding sequences is at C-terminal of myc-GLUT4. The protein, mycGLUT4eGFP expressed constitutively under cytomegalovirus (CMV) promoter is shown to be translocated to membrane upon insulin stimulation.

Transfection procedure to establish CHO-HIRc-mycGLUT4eGFP cells involves following steps

Cotransfect CHO-HIRc cells with 4 µg of pGreen Lantern mycGLUT4eGFP and 1 µg pTk-Hyg plasmid using LF-2000 method as mentioned earlier except that selection was done using 200 g/ml hygromycin B. GFP clones should be picked up and maintained separately.

Before proceeding on to drug screens, the stably expressed GLUT4 chimera should be evaluated for its translocation efficiency in response to insulin. It is also important to establish the response of chimera to a broad range of insulin concentration (1-1000 nM) to measure the dose responsiveness of the translocation event. Specificity of insulin sensitivity can be further verified by pretreating the cells with pharmacological inhibitors of insulin action (wortmannin, cytochalasin, genestein etc.) followed by insulin treatment (Vijayakumar et al 2005).

3.3.1 Simple protocol for verification of insulin sensitivity

Plate 5000-10000 cells per well in a chambered slide with cover (Nunc, Rochester, NY, U.S.A) and allow cells to adhere and grow for a day.

Wash the cells with serum free media and then serum starved further in Ham's F-12 media (100 l) containing 1 mg/ml BSA for 3 h. It is recommended to have BSA present in the medium if experiments are performed for more than 5-10 minutes.

Remove 50 l media and add 2X concentration of 50 l insulin diluted in pre-warmed F12 containing 1 mg/ml BSA at 37oC so that final volume of 100 l and desired concentration is achieved. To verify the specificity of insulin induced GLUT4 translocation, pharmacological inhibitor of insulin action to be added to incubating media.

GLUT4 translocation based on visual increase in membrane GFP fluorescence upon stimulation with different concentrations of insulin (10-1000 nM) for 1-20 min can be visualized under a fluorescent microscope (Olympus, Shinjuku-ku, Tokyo, Japan). Alternatively, following insulin treatment, cells can be fixed with 3% paraformaldehyde in PBS pH 7.4, for 10 min at room temperature, rinsed with PBS three times and quenched with 1% glycine for 5 min, wash with PBS and observe under a microscope. We have observed that fixed cells give better resolution as far as images are concerned.

Clones in which GLUT4 chimera translocated to PM upon insulin stimulation and the level of membrane associated GFP fluorescence was distinct from that of cytoplasmic should be selected.

Verified clones may be designated as CHO-HIRc-mycGLUT4eGFP with appropriate numbering and can be cryopreserved and used for further experiments. During the selection process of transfected cells with hygromycin, we had obtained eight clones that expressed GLUT4 chimera and were designated as CHO-HIRc-mycGLUT4eGFP clone 1 to 8. However, only clone 4 and 6 exhibited concentration dependent sensitivity to insulin.. Details mentioned in this chapter are experiments done with clone 4.

3.3.2 Real time GLUT4 translocation assay

Major goal of a biologist is to understand the molecular process that happens in cell in real time. Live cell imaging (LCI) helps in non-invasively analyzing dynamic processes in living cells using microscope and computer vision techniques. LCI allow viewing live cellular processes in real time with resolution and has great benefit in basic as well as drug screening. Cellular processes take place so quickly that the motion of cellular machinery can be complex and spontaneous. Real time microscopy experiments using GFP fusion proteins and computational methods reveal many important properties of cells unachievable by traditional in vitro biochemical methods. Fluorescence microscopy is the most proficient technique for studying the dynamic behavior in LCI. It distinguishes GFP chimera with a high degree of specificity from non-fluorescing matters in a live cell, and hence the dynamics of individual cellular components can be analyzed in real time. By incorporating a monochrome or color chip video (CCD) camera on a microscope, time-lapse images of cells could be recorded onto magnetic storage media (Figure 2A). Video imaging and analysis is less complex and expensive, with the added advantage that the molecular process of interest can be analyzed in real time. Video cameras can detect contrast differences invisible to the human eye, and these differences can be electronically amplified. Since GLUT4 translocation is a very dynamic and visual process, LCI can reveal much information on the complex motions involving other molecules. It is advantageous to store videos for subsequent analysis as GLUT4 translocation involves many accompanying events. One of the limitation of these imaging technologies is accumulation of huge amount of digital image data and to extract reproducible as well as quantitative information, computer-based image analysis is required. In this article, we briefly outline the methods of live cell imaging and quantitative analysis of GLUT4 translocation. These assays are based on the principles of GLUT4 chimera movement monitored directly by GFP.

3.3.3 Simple protocol for real time GLUT4 translocation assay

Plate the cells in 35 mm petridishes and allow cells to attach and grow for a day

Wash the cells with serum free media and then serum starved further in Ham's F-12 media (1 ml) containing 1 mg/ml BSA for 3 h.

Remove 500 ul media and place the plate on a fluorescent microscope.

Focus cells under 40X objective as images at this magnification increases the area of the field that provides reliable and collective information about significantly large number of cells (n>25), a most desired parameter in any form of research.

Add 500 ul pre-warmed reagents at 2X concentration. With a quick final adjustment of focus, images were captured at room temperature at the rate of one frame per min for 10-30 min using CCD, 1.4 megapixel, 12 bit camera (Olympus) attached to fluorescent microscope.

Choose exposure times for GFP fluorescence such that more image pixel intensities are below camera saturation. Therefore, it is necessary to adjust exposure time to keep the background minimum to obtain maximum signal (2 B and C). Importantly, exposure times should be kept constant within each experiment to avoid variability in quantitation. Video files are saved in .avi format and can be played with windows media player, VLC media player, quick time movie player etc.

Analysis of images and conversion of videos to individual frames can be done (Fig 3) using Image-Pro Plus-AMS software (MediaCybernetics, Silver Spring, MD, USA.) or any compatible software (Metamorph image-processing software, Molecular Devices Corp). This aids to visualize kinetics of GLUT4 translocation under normal (supplementary video 1) as well as stimulated conditions (supplementary video 2).

Open the video files and define the area of the cell membrane for analysis. The software quantifies GLUT4 translocation based on increase in mean GFP fluorescence density on defined area of PM of the cells in the video frame (Fig 4). We have observed that monochrome images are sharp and would give better read out.

The mean density data obtained versus frame (one frame per minute) can be used to calculate fold GLUT4 translocation per minute. Repeat the analysis for 10-20 cells per experiment to obtain statistically significant values This provides the optimum time at which GLUT4 translocation was maximum. Standardization dose and of time point is advantageous in further molecular studies of suitable GLUT4 translocation modulators.

Alternatively, using the same method, measure the fluorescence background and subtract these background values from specific signals for each individual myc-GLUT4-GFP-expressing cell. Plot the intensity versus frame/time.

3.4 Immunofluorescence microscopy

Data obtained by LCI of GLUT4 translocation in CHO-HIRc-mycGLUT4eGFP cells can be further validated by immunofluorescence detection of GLUT4. Since direct immunological detection of GLUT4 on the cell surface with an anti-GLUT4 antibody being difficult due to conformational changes accompanying stimulation or un exposure of domains in the membrane, most of the investigators rely on detection of inserted epitope (myc or HA) on expressed GLUT4 GFP chimera by the binding of anti-myc antibody. This assay detect the proportion of mycGLUT4eGFP in the plasma membrane of non-permeabilized cells, and aid in comparing the amounts of GLUT4 in the PM in the basal and insulin-stimulated states In this assay, the amount of mycGLUT4eGFP in the PM is quantified by measuring the amount of myc epitope on the surface of cells. In a cell-by-cell analysis, the amount of anti- myc antibody bound to the plasma membrane (surface mycGLUT4eGFP) to the amount of GFP expressed in the cell (total mycGLUT4eGFP) is normalized. This normalization corrects for different levels of total expression of mycGLUT4eGFP among the cells.

3.4.1 Protocol for detection of myc epitope

Plate and treat the cells as mentioned in the section. Simple protocol for verification of insulin sensitivity (what do you mean).

Wash the cells quickly with ice cold PBS and fix by treating with 3% paraformaldehyde in PBS pH 7.4, for 10 min at room temperature. Rinse the cells with PBS three times and quench with PBS containing 1% glycine for 5 min followed by washing with PBS three times.

Block the cells with PBS containing 5% BSA and 5% FBS for 1 h at room temperature.

Incubate cells with anti-myc (9E10) antibody or respective isotype control for overnight at 4oC or 2h at room temperature at a dilution of 1: 50 in PBS containing 2.5% FBS.

Wash the cells 5 times at 2 min interval with PBS to eliminate unbound antibody.

Incubate the cells with a rhodamine conjugated goat-anti-mouse secondary antibody at a dilution of 1:100 for additional 1 h, at room temperature. Wash again five times with PBS at 2 min interval and mount using vectashield (Vector Laboratories, Burlingame, CA, USA) for visualization by immunofluorescence or confocal microscopy (LSM510, Carl Zeiss).

3.5 Glucose transport assay

Insulin stimulation of insulin sensitive cells expressing GLUT4 results in rapid recruitment of GLUT4 transporter proteins from internal compartment to the plasma membrane with resultant increase in the rate of glucose transport. Being the rate limiting step, this process is often impaired in patients with type 2 diabetes. It is assumed that therapies which augment insulin stimulated GLUT4 translocation should also increase glucose uptake in target tissues, thereby improve insulin sensitivity. Therefore, glucose uptake assay could be considered as another validation assay for real time GLUT4 translocation assay.

3.5.1 Procedure for glucose uptake assay

Wash the cells (1X105) grown in 24 well plates with serum free media and then further serum starved for 3 h in F12 medium containing 0.1% BSA.

Wash the cells twice in KRP buffer (137 mM NaCl, 4.7 mM KCl, 10 mM sodium phosphate pH 7.4, 0.5 mM MgCl2, 1 mM CaCl2, 2 mg/ml BSA), incubate at 37oC for 30 min and then treat with various concentration of insulin (0.1-1000 nM) or test compounds for additional time. Control cells are to be treated with respective vehicle control.

The glucose uptake reaction was initiated by adding cocktail of 0.1 mM 2-deoxy glucose (DG) and 0.5 Ci/ml 14C-2-DG in final volume of 250 l per well. After incubation at 37oC for 10 min, the reaction is terminated by keeping the cells on ice and washing three times with ice-cold PBS containing 20 mM D-glucose.

Add 50 l 0.1% SDS to solubilize cells. Following protein estimation, transfer the lysate to unifilter-96/GFB plates (PerkinElmer, Waltham, MA, USA) and allow to dry at 37oC for 6 h.

Add 20 l of scintillation fluid (PerkinElmer----other details) per well and radioactivity incorporated into cells is quantified with a top count microplate scintillation counter (Packard, Albertville, MN, USA). 6.Nonspecific uptake, measured in the presence of 10 M cytochalasin B, to be subtracted from all values. To examine the specificity of the signaling pre-treat cells with specific pharmacological inhibitor of insulin signaling activation. This assay aids in establishing a dose response curve (EC-50) of insulin or any GLUT4 modulator. EC-50 of insulin in CHO-HIRc-mycGLUT4eGFP cells is found to be 1 nM.

Conclusions and future applications

GLUT4 translocation in response to its activation can be monitored and measured by real time GLUT4 translocation assay described here. Being rapid and fool proof assay, it offers multiple methods for application in antidiabetic drug discovery. Presence of a fluorescent GFP tag aids in monitoring GLUT4 translocation from perinuclear space to plasma membrane by GLUT4 modulators automatically. Considering the fact that several lead compounds await screening and identification for GLUT4 modulation activity, the strategy presented here may augment the antidiabetic drug discovery process.

Acknowledgements

We thank Dr. G.C. Mishra, Director, NCCS for being very supportive and giving all the encouragement to carry out this work. We thank Department of Biotechnology, Government of India for providing financial support. We thank Dr M. P. Czech, University of Massachusetts Medical School, Worcester, MA, USA for generously gifting pGreen Lantern mycGLUT4eGFP construct and Dr. M.. Bernier, National Institute of Aging, Baltimore, MD for kindly providing CHO cells over expressing wild type insulin receptor (CHO-HIRc). We thank Dr. Amredra Kumar Ajay for image analysis and Mrs. Aswini Atre for confocal microscopy studies. We thank Journal of Bioscience allowing us to reproduce from our published article in this chapter.

Figure legends

Figure 1. A. Structural features of GLUT4 protein and schematic representation of GLUT4 translocation event. (B) In unstimulated cells GLUT4 chimera is localized primarily to perinuclear space. (C) Insulin stimulation lead to translocation of GLUT4 chimera to plasma membrane (D) GLUT4 translocation can be detected by tracking GFP fluorescence or (E) immunodetection by anti-myc-Ab and rhodamine conjugated secondary antibody.

Figure 2. A model set up for live cell imaging. (A) Microscope body (B) Colour chip CCD camera (C) DP-30 monochrome CCD camera (D) Mercury burner (E) Computer (F) Software to monitor GLUT4 translocation. (G and H) Software displays images at one frame per minute and aid to visualize translocation of mycGLUT4eGFP vesicles to the plasma membrane, up on insulin stimulation based on the movement of GLUT4 associated GFP fluorescence. Sequence tool bar of the software aid to select the frame number.

Figure 3. An example for video image analysis of insulin stimulated GLUT4 translocation. Video files are extracted to obtain frames using Image Proplus 5.0 software. Frames aid the qualitative analysis of GLUT4 translocation. (A) GLUT4 translocation in control cells (B) insulin stimulated GLUT4 translocation. Arrow mark indicates the localization of GLUT4 chimera in the plasma membrane. Legends on the figure indicate the time points at which images were captured following treatment.

Figure4. Quantitative analysis of GLUT4 translocation. Software aided quantitative analysis of GLUT4 translocation is based on increase in mean GFP fluorescence density at defined area of plasma membrane of the cells in the video using Image-Pro Plus-AMS software (MediaCybernetics, Silver Spring, MD, USA.). The mean density data obtained versus frame (one frame per minute) is used to calculate fold GLUT4 translocation per minute. It also provides the optimum time point at which maximum GLUT4 translocation was achieved.

Figure 5. Confocal microscopy analysis of GLUT4 translocation in CHO-HIRc-mycGLUT4eGFP cells. GLUT4 translocation was visualized based on GFP fluorescence or immunostaining of myc epitope present in non-permeabilized cells.

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