Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

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Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals

Molecules ◽

10.3390/molecules26144346 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4346

Author(s):

Verena Stadlbauer ◽

Cathrina Neuhauser ◽

Tobias Aumiller ◽

Alexander Stallinger ◽

Marcus Iken ◽

...

Keyword(s):

Plasma Membrane ◽

Blood Glucose ◽

Plant Extracts ◽

Glucose Transporter ◽

Glut4 Translocation ◽

In Ovo ◽

Insulin Mimetic ◽

Glucose Levels ◽

Intracellular Translocation

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.

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Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

Molecular Biology of the Cell ◽

10.1091/mbc.e13-02-0103 ◽

2013 ◽

Vol 24 (16) ◽

pp. 2544-2557 ◽

Cited By ~ 51

Author(s):

L. Amanda Sadacca ◽

Joanne Bruno ◽

Jennifer Wen ◽

Wenyong Xiong ◽

Timothy E. McGraw

Keyword(s):

Plasma Membrane ◽

Glucose Uptake ◽

Glucose Transporter ◽

Receptor Activation ◽

Glut4 Translocation ◽

Insulin Stimulation ◽

Transport Vesicles ◽

Glucose Transporter Glut4

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.

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Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes

Biochemical Journal ◽

10.1042/bj3210233 ◽

1997 ◽

Vol 321 (1) ◽

pp. 233-238 ◽

Cited By ~ 14

Author(s):

Eric HAJDUCH ◽

J. Carlos ALEDO ◽

Colin WATTS ◽

Harinder S. HUNDAL

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Glucose Uptake ◽

Glucose Transport ◽

Tetanus Toxin ◽

Glucose Transporter ◽

Detectable Effect ◽

Glut4 Translocation ◽

Rat Adipocytes ◽

Isolated Rat

Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusion, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocytes respectively. In this study we show that proteolysis of both cellubrevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transport or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate glucose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electroporation of adipocytes with 600 nM tetanus toxin resulted in a complete loss of both cellubrevin and VAMP expression within 60 min. However, toxin-mediated proteolysis of both these proteins had no effect on the ability of insulin to stimulate glucose transport which was elevated approx. 5-fold, an activation of comparable magnitude to that observed in cells electroporated without tetanus toxin. The lack of any significant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and VAMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and VAMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to the plasma membrane.

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Separation of Insulin Signaling into Distinct GLUT4 Translocation and Activation Steps

Molecular and Cellular Biology ◽

10.1128/mcb.24.17.7567-7577.2004 ◽

2004 ◽

Vol 24 (17) ◽

pp. 7567-7577 ◽

Cited By ~ 42

Author(s):

Makoto Funaki ◽

Paramjeet Randhawa ◽

Paul A. Janmey

Keyword(s):

Plasma Membrane ◽

Glucose Uptake ◽

Glucose Transporter ◽

Time Lag ◽

Inhibitory Effect ◽

Glut4 Translocation ◽

Glucose Levels ◽

Increase Glucose Uptake ◽

A Cell ◽

Peptide Treatment

ABSTRACT GLUT4 (glucose transporter 4) plays a pivotal role in insulin-induced glucose uptake to maintain normal blood glucose levels. Here, we report that a cell-permeable phosphoinositide-binding peptide induced GLUT4 translocation to the plasma membrane without inhibiting IRAP (insulin-responsive aminopeptidase) endocytosis. However, unlike insulin treatment, the peptide treatment did not increase glucose uptake in 3T3-L1 adipocytes, indicating that GLUT4 translocation and activation are separate events. GLUT4 activation can occur at the plasma membrane, since insulin was able to increase glucose uptake with a shorter time lag when inactive GLUT4 was first translocated to the plasma membrane by pretreating the cells with this peptide. Inhibition of phosphatidylinositol (PI) 3-kinase activity failed to inhibit GLUT4 translocation by the peptide but did inhibit glucose uptake when insulin was added following peptide treatment. Insulin, but not the peptide, stimulated GLUT1 translocation. Surprisingly, the peptide pretreatment inhibited insulin-induced GLUT1 translocation, suggesting that the peptide treatment has both a stimulatory effect on GLUT4 translocation and an inhibitory effect on insulin-induced GLUT1 translocation. These results suggest that GLUT4 requires translocation to the plasma membrane, as well as activation at the plasma membrane, to initiate glucose uptake, and both of these steps normally require PI 3-kinase activation.

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Akt Activation Is Required at a Late Stage of Insulin-Induced GLUT4 Translocation to the Plasma Membrane

Molecular Endocrinology ◽

10.1210/me.2004-0413 ◽

2005 ◽

Vol 19 (4) ◽

pp. 1067-1077 ◽

Cited By ~ 64

Author(s):

Ellen M. van Dam ◽

Roland Govers ◽

David E. James

Keyword(s):

Plasma Membrane ◽

Glucose Transporter ◽

Cell Cortex ◽

Glut4 Translocation ◽

Akt Activation ◽

Multistep Process ◽

Intracellular Compartments ◽

Insulin Dependent ◽

Glucose Transporter Glut4 ◽

Golgi Network

Abstract Insulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortex—this process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PM—this process may be the major Akt-dependent step in the insulin regulation of glucose transport.

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GLUT4 Is Sorted to Vesicles Whose Accumulation Beneath and Insertion into the Plasma Membrane Are Differentially Regulated by Insulin and Selectively Affected by Insulin Resistance

Molecular Biology of the Cell ◽

10.1091/mbc.e09-08-0751 ◽

2010 ◽

Vol 21 (8) ◽

pp. 1375-1386 ◽

Cited By ~ 42

Author(s):

Wenyong Xiong ◽

Ingrid Jordens ◽

Eva Gonzalez ◽

Timothy E. McGraw

Keyword(s):

Insulin Resistance ◽

Plasma Membrane ◽

Fluorescence Microscopy ◽

Glucose Transport ◽

Total Internal Reflection ◽

Glucose Transporter ◽

Internal Reflection ◽

General Mechanism ◽

Recycling Endosomes ◽

Degree Of Redistribution

Insulin stimulates glucose transport by recruiting the GLUT4 glucose transporter to the plasma membrane. Here we use total internal reflection fluorescence microscopy to show that two trafficking motifs of GLUT4, a FQQI motif and a TELE-based motif, target GLUT4 to specialized vesicles that accumulate adjacent to the plasma membrane of unstimulated adipocytes. Mutations of these motifs redistributed GLUT4 to transferrin-containing recycling vesicles adjacent to the plasma membrane, and the degree of redistribution correlated with the increases of the GLUT4 mutants in the plasma membrane of basal adipocytes. These results establish that GLUT4 defaults to recycling endosomes when trafficking to specialized vesicles is disrupted, supporting the hypothesis that the specialized vesicles are derived from an endosomal compartment. Insulin stimulates both the accumulation of GLUT4 in the evanescent field and the fraction of this GLUT4 that is inserted into the plasma membrane. Unexpectedly, these two steps are differentially affected by the development of insulin resistance. We ascribe this selective insulin resistance to inherent differences in the sensitivities of GLUT4 vesicle accumulation and insertion into the plasma membrane to insulin. Differences in insulin sensitivities of various processes may be a general mechanism for the development of the physiologically important phenomenon of selective insulin resistance.

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Skeletal muscle plasma membrane glucose transport and glucose transporters after exercise

Journal of Applied Physiology ◽

10.1152/jappl.1990.68.1.193 ◽

1990 ◽

Vol 68 (1) ◽

pp. 193-198 ◽

Cited By ~ 96

Author(s):

L. J. Goodyear ◽

M. F. Hirshman ◽

P. A. King ◽

E. D. Horton ◽

C. M. Thompson ◽

...

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Glucose Uptake ◽

Glucose Transport ◽

Time Course ◽

Plasma Membranes ◽

Glucose Transporter ◽

Male Rats ◽

Intrinsic Activity ◽

Plasma Membrane Vesicles

Recent reports have shown that immediately after an acute bout of exercise the glucose transport system of rat skeletal muscle plasma membranes is characterized by an increase in both glucose transporter number and intrinsic activity. To determine the duration of the exercise response we examined the time course of these changes after completion of a single bout of exercise. Male rats were exercised on a treadmill for 1 h (20 m/min, 10% grade) or allowed to remain sedentary. Rats were killed either immediately or 0.5 or 2 h after exercise, and red gastrocnemius muscle was used for the preparation of plasma membranes. Plasma membrane glucose transporter number was elevated 1.8- and 1.6-fold immediately and 30 min after exercise, although facilitated D-glucose transport in plasma membrane vesicles was elevated 4- and 1.8-fold immediately and 30 min after exercise, respectively. By 2 h after exercise both glucose transporter number and transport activity had returned to nonexercised control values. Additional experiments measuring glucose uptake in perfused hindquarter muscle produced similar results. We conclude that the reversal of the increase in glucose uptake by hindquarter skeletal muscle after exercise is correlated with a reversal of the increase in the glucose transporter number and activity in the plasma membrane. The time course of the transport-to-transporter ratio suggests that the intrinsic activity response reverses more rapidly than that involving transporter number.

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Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation

Journal of Molecular Endocrinology ◽

10.1530/jme-17-0041 ◽

2017 ◽

Vol 59 (3) ◽

pp. 257-268 ◽

Cited By ~ 5

Author(s):

Raquel S Campello ◽

Luciana A Fátima ◽

João Nilton Barreto-Andrade ◽

Thais F Lucas ◽

Rosana C Mori ◽

...

Keyword(s):

Plasma Membrane ◽

Glucose Uptake ◽

Subcellular Distribution ◽

Glucose Transporter ◽

Biological Effects ◽

Glut4 Translocation ◽

Akt Phosphorylation ◽

Akt Activation ◽

Glut4 Expression ◽

Protein Kinase Akt

Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17β-estradiol (E2) modulates SLC2A4/GLUT4 expression, but the involved mechanisms are unclear. Although E2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4/GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4/GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4/GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.

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Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles

Biochemical Journal ◽

10.1042/bj20091283 ◽

2009 ◽

Vol 424 (2) ◽

pp. 163-167 ◽

Cited By ~ 105

Author(s):

Ilya Levental ◽

Fitzroy J. Byfield ◽

Pramit Chowdhury ◽

Feng Gai ◽

Tobias Baumgart ◽

...

Keyword(s):

Plasma Membrane ◽

Phase Separation ◽

Membrane Vesicles ◽

Correlation Spectroscopy ◽

Model Systems ◽

Live Cells ◽

Lipid Phase ◽

Plasma Membrane Vesicles ◽

Liquid Ordered ◽

Lipid Phase Separation

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

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Sphingomyelinase activates GLUT4 translocation via a cholesterol-dependent mechanism

AJP Cell Physiology ◽

10.1152/ajpcell.00073.2003 ◽

2004 ◽

Vol 286 (2) ◽

pp. C317-C329 ◽

Cited By ~ 33

Author(s):

Ping Liu ◽

Brian J. Leffler ◽

Lara K. Weeks ◽

Guoli Chen ◽

Christine M. Bouchard ◽

...

Keyword(s):

Plasma Membrane ◽

Glucose Transporter ◽

Degradation Products ◽

Membrane Integrity ◽

Time Dependent ◽

Membrane Cholesterol ◽

Glut4 Translocation ◽

Dose Dependent ◽

Plasma Membrane Cholesterol ◽

Dependent Mechanism

A basis for the insulin mimetic effect of sphingomyelinase on glucose transporter isoform GLUT4 translocation remains unclear. Because sphingomyelin serves as a major determinant of plasma membrane cholesterol and a relationship between plasma membrane cholesterol and GLUT4 levels has recently become apparent, we assessed whether GLUT4 translocation induced by sphingomyelinase resulted from changes in membrane cholesterol content. Exposure of 3T3-L1 adipocytes to sphingomyelinase resulted in a time-dependent loss of sphingomyelin from the plasma membrane and a concomitant time-dependent accumulation of plasma membrane GLUT4. Degradation products of sphingomyelin did not mimic this stimulatory action. Plasma membrane cholesterol amount was diminished in cells exposed to sphingomyelinase. Restoration of membrane cholesterol blocked the stimulatory effect of sphingomyelinase. Increasing concentrations of methyl-β-cyclodextrin, which resulted in a dose-dependent reversible decrease in membrane cholesterol, led to a dose-dependent reversible increase in GLUT4 incorporation into the plasma membrane. Although increased plasma membrane GLUT4 content by cholesterol extraction with concentrations of methyl-β-cyclodextrin above 5 mM most likely reflected decreased GLUT4 endocytosis, translocation stimulated by sphingomyelinase or concentrations of methyl-β-cyclodextrin below 2.5 mM occurred without any visible changes in the endocytic retrieval of GLUT4. Furthermore, moderate loss of cholesterol induced by sphingomyelinase or low concentrations of methyl-β-cyclodextrin did not alter membrane integrity or increase the abundance of other plasma membrane proteins such as the GLUT1 glucose transporter or the transferrin receptor. Regulation of GLUT4 translocation by moderate cholesterol loss did not involve known insulin-signaling proteins. These data reveal that sphingomyelinase enhances GLUT4 exocytosis via a novel cholesterol-dependent mechanism.

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