Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation

Abstract Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21217964 ◽

2020 ◽

Vol 21 (21) ◽

pp. 7964

Author(s):

Verena Stadlbauer ◽

Peter Lanzerstorfer ◽

Cathrina Neuhauser ◽

Florian Weber ◽

Flora Stübl ◽

...

Keyword(s):

Plasma Membrane ◽

Fluorescence Microscopy ◽

High Throughput ◽

Glucose Transporter ◽

Large Cell ◽

Live Cells ◽

Glut4 Translocation ◽

Plasma Membrane Vesicles ◽

Innovative Strategy ◽

Insulin Mimetic

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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Impairment of insulin-stimulated GLUT4 translocation in skeletal muscle and adipose tissue in the Tsumura Suzuki obese diabetic mouse: a new genetic animal model of type 2 diabetes

Acta Endocrinologica ◽

10.1530/eje.0.1450785 ◽

2001 ◽

pp. 785-790 ◽

Cited By ~ 45

Author(s):

T Miura ◽

W Suzuki ◽

E Ishihara ◽

I Arai ◽

H Ishida ◽

...

Keyword(s):

Type 2 Diabetes ◽

Skeletal Muscle ◽

Animal Model ◽

Adipose Tissue ◽

Plasma Membrane ◽

Blood Glucose ◽

Low Density ◽

Glut4 Translocation ◽

Genetic Animal Model

BACKGROUND: In skeletal muscle and adipocytes, insulin-stimulated glucose transport has been known to occur through the translocation of glucose transporter (GLUT) 4 from the intracellular pool to the plasma membrane. The Tsumura Suzuki obese diabetic (TSOD) mouse, a new genetic animal model of type 2 diabetes, develops moderate degrees of obesity and diabetes that are especially apparent in animals more than 11 weeks old. A defect in insulin stimulation of GLUT4 translocation also contributes to the characteristics of type 2 diabetes. OBJECTIVE: To characterize this mouse further, we examined the alteration in insulin-stimulated GLUT4 translocation in the skeletal muscle and adipose tissue. METHODS: For glucose and insulin tolerance tests, the mice were given glucose or insulin and blood samples were collected. After isolation of low-density microsomal membrane and plasma membrane from skeletal muscle and adipose tissue, insulin-stimulated translocation of GLUT4 in these TSOD mice was examined by Western blot. RESULTS AND CONCLUSIONS: TSOD mice showed a significant increase in blood glucose after the glucose load, and exhibited a significantly attenuated decrease in blood glucose concentrations after administration of insulin, compared with that in control Tsumura Suzuki non-obese (TSNO) mice. The insulin-stimulated translocation of GLUT4 from low-density microsomal membranes to plasma membrane was significantly reduced in both skeletal muscle and adipose tissue of TSOD mice. These results indicate that the reduced insulin sensitivity in diabetic TSOD mice is presumably due, at least in part, to the impaired GLUT4 translocation by insulin in both skeletal muscle and adipocytes.

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Calpain facilitates GLUT4 vesicle translocation during insulin-stimulated glucose uptake in adipocytes

Biochemical Journal ◽

10.1042/bj20030681 ◽

2003 ◽

Vol 376 (3) ◽

pp. 625-632 ◽

Cited By ~ 61

Author(s):

David S. PAUL ◽

Anne W. HARMON ◽

Courtney P. WINSTON ◽

Yashomati M. PATEL

Keyword(s):

Type 2 Diabetes ◽

Plasma Membrane ◽

Glucose Uptake ◽

Susceptibility Gene ◽

Cysteine Proteases ◽

Glut4 Translocation ◽

Calpain Activity ◽

Actin Reorganization ◽

Calpain 10

Calpains are a family of non-lysosomal cysteine proteases. Recent studies have identified a member of the calpain family of proteases, calpain 10, as a putative diabetes-susceptibility gene that may be involved in the development of type 2 diabetes. Inhibition of calpain activity has been shown to reduce insulinstimulated glucose uptake in isolated rat-muscle strips and adipocytes. In this report, we examine the mechanism by which calpain affects insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Inhibition of calpain activity resulted in approx. a 60% decrease in insulin-stimulated glucose uptake. Furthermore, inhibition of calpain activity prevented the translocation of insulin-responsive glucose transporter 4 (GLUT4) vesicles to the plasma membrane, as demonstrated by fluorescent microscopy of whole cells and isolated plasma membranes; it did not, however, alter the total GLUT4 protein content. While inhibition of calpain did not affect the insulin-mediated proximal steps of the phosphoinositide 3-kinase pathway, it did prevent the insulin-stimulated cortical actin reorganization required for GLUT4 translocation. Specific inhibition of calpain 10 by antisense expression reduced insulin-stimulated GLUT4 translocation and actin reorganization. Based on these findings, we propose a role for calpain in the actin reorganization required for insulin-stimulated GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes. These studies identify calpain as a novel factor involved in GLUT4 vesicle trafficking and suggest a link between calpain activity and the development of type 2 diabetes.

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Inflammatory Markers Associated With Diabetes Mellitus – Old and New Players

Current Pharmaceutical Design ◽

10.2174/1381612826666201125103047 ◽

2020 ◽

Vol 26 ◽

Author(s):

Emir Muzurović ◽

Zoja Stanković ◽

Zlata Kovačević ◽

Benida Šahmanović Škrijelj ◽

Dimitri P Mikhailidis

Keyword(s):

Diabetes Mellitus ◽

Inflammatory Markers ◽

High Specificity ◽

Future Directions ◽

Effective Strategies ◽

Advantages And Disadvantages ◽

Type 2 Dm ◽

Increased Risk ◽

Made In

: Diabetes mellitus (DM) is a chronic and complex metabolic disorder, and also an important cause of cardiovascular (CV) diseases (CVDs). Subclinical inflammation, observed in patients with type 2 DM (T2DM), cannot be considered the sole or primary cause of T2DM in the absence of classical risk factors, but it represents an important mechanism that serves as a bridge between primary causes of T2DM and its manifestation. Progress has been made in the identification of effective strategies to prevent or delay the onset of T2DM. It is important to identify those at increased risk for DM by using specific biomarkers. Inflammatory markers correlate with insulin resistance (IR) and glycoregulation in patients with DM. Also, several inflammatory markers have been shown to be useful in assessing the risk of developing DM and its complications. However, the intertwining of pathophysiological processes and the not-quite-specificity of inflammatory markers for certain clinical entities limits their practical use. In this review we consider the advantages and disadvantages of various inflammatory biomarkers of DM that have been investigated to date as well as possible future directions. Key features of such biomarkers should be high specificity, non-invasiveness and cost-effectiveness.

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Gundelia tournefortii: Fractionation, Chemical Composition and GLUT4 Translocation Enhancement in Muscle Cell Line

Molecules ◽

10.3390/molecules26133785 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3785

Author(s):

Sleman Kadan ◽

Sarit Melamed ◽

Shoshana Benvalid ◽

Zipora Tietel ◽

Yoel Sasson ◽

...

Keyword(s):

Glucose Transporter ◽

Rapid Development ◽

Muscle Cells ◽

Flash Chromatography ◽

Glucose Disposal ◽

Gas Chromatography Mass Spectrometry ◽

Glut4 Translocation ◽

Toxic Concentrations ◽

L6 Muscle Cells

Type 2 diabetes (T2D) is a chronic metabolic disease, which could affect the daily life of patients and increase their risk of developing other diseases. Synthetic anti-diabetic drugs usually show severe side effects. In the last few decades, plant-derived drugs have been intensively studied, particularly because of a rapid development of the instruments used in analytical chemistry. We tested the efficacy of Gundelia tournefortii L. (GT) in increasing the translocation of glucose transporter-4 (GLUT4) to the myocyte plasma membrane (PM), as a main strategy to manage T2D. In this study, GT methanol extract was sub-fractionated into 10 samples using flash chromatography. The toxicity of the fractions on L6 muscle cells, stably expressing GLUTmyc, was evaluated using the MTT assay. The efficacy with which GLUT4 was attached to the L6 PM was evaluated at non-toxic concentrations. Fraction 6 was the most effective, as it stimulated GLUT4 translocation in the absence and presence of insulin, 3.5 and 5.2 times (at 250 μg/mL), respectively. Fraction 1 and 3 showed no significant effects on GLUT4 translocation, while other fractions increased GLUT4 translocation up to 2.0 times. Gas chromatography–mass spectrometry of silylated fractions revealed 98 distinct compounds. Among those compounds, 25 were considered anti-diabetic and glucose disposal agents. These findings suggest that GT methanol sub-fractions exert an anti-diabetic effect by modulating GLUT4 translocation in L6 muscle cells, and indicate the potential of GT extracts as novel therapeutic agents for T2D.

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Secretory granule behaviour adjacent to the plasma membrane before and during exocytosis: total internal reflection fluorescence microscopy studies

Acta Physiologica ◽

10.1111/j.1748-1716.2007.01818.x ◽

2007 ◽

Vol 192 (2) ◽

pp. 303-307 ◽

Cited By ~ 25

Author(s):

R. W. Holz ◽

D. Axelrod

Keyword(s):

Plasma Membrane ◽

Fluorescence Microscopy ◽

Secretory Granule ◽

Total Internal Reflection ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence

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Spontaneous Type 2 Diabetic Rodent Models

Journal of Diabetes Research ◽

10.1155/2013/401723 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 22

Author(s):

Yang-wei Wang ◽

Guang-dong Sun ◽

Jing Sun ◽

Shu-jun Liu ◽

Ji Wang ◽

...

Keyword(s):

Diabetes Mellitus ◽

Insulin Resistance ◽

Type 2 Diabetes ◽

Health Care ◽

Animal Models ◽

Rodent Models ◽

Advantages And Disadvantages ◽

Characteristic Features ◽

Type 2 Diabetic

Diabetes mellitus, especially type 2 diabetes (T2DM), is one of the most common chronic diseases and continues to increase in numbers with large proportion of health care budget being used. Many animal models have been established in order to investigate the mechanisms and pathophysiologic progress of T2DM and find effective treatments for its complications. On the basis of their strains, features, advantages, and disadvantages, various types of animal models of T2DM can be divided into spontaneously diabetic models, artificially induced diabetic models, and transgenic/knockout diabetic models. Among these models, the spontaneous rodent models are used more frequently because many of them can closely describe the characteristic features of T2DM, especially obesity and insulin resistance. In this paper, we aim to investigate the current available spontaneous rodent models for T2DM with regard to their characteristic features, advantages, and disadvantages, and especially to describe appropriate selection and usefulness of different spontaneous rodent models in testing of various new antidiabetic drugs for the treatment of type 2 diabetes.

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