A Detection Method for GLUT4 Exocytosis Based on Spontaneous Split Luciferase Complementation

Endo, Mizuki; Miyasaki, Masashi; Li, Qiaojing; Kawamura, Genki; Ozawa, Toshio

doi:10.2116/analsci.19c003

Cited by 7 publications

(4 citation statements)

References 14 publications

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“…It is particularly interesting that spontaneously interacting fragments of split NLuc were developed at the K D for 700 pM. The resultant 11 amino acid peptide was used for quantifying proteins and monitoring protein trafficking [ 64 , 65 ]. Further mutation generated a brighter NLuc mutant called teLuc (D19S, D85N, and C164H), which emits 2.6-fold brighter bioluminescence than NLuc with diphenylterazine ( Figure 3 c), or yeLuc (F1L, A14D, L18Q, D19A, S28T, V27L, Q69R, R112Q, L142R, and C164S) which emits 11.5-fold brighter bioluminescence with selenoterazine ( Figure 3 d) [ 24 ].…”

Section: Development Of a Brighter Bioluminescence Systemmentioning

confidence: 99%

Advanced Bioluminescence System for In Vivo Imaging with Brighter and Red-Shifted Light Emission

Endo

Ozawa

2020

IJMS

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In vivo bioluminescence imaging (BLI), which is based on luminescence emitted by the luciferase–luciferin reaction, has enabled continuous monitoring of various biochemical processes in living animals. Bright luminescence with a high signal-to-background ratio, ideally red or near-infrared light as the emission maximum, is necessary for in vivo animal experiments. Various attempts have been undertaken to achieve this goal, including genetic engineering of luciferase, chemical modulation of luciferin, and utilization of bioluminescence resonance energy transfer (BRET). In this review, we overview a recent advance in the development of a bioluminescence system for in vivo BLI. We also specifically examine the improvement in bioluminescence intensity by mutagenic or chemical modulation on several beetle and marine luciferase bioluminescence systems. We further describe that intramolecular BRET enhances luminescence emission, with recent attempts for the development of red-shifted bioluminescence system, showing great potency in in vivo BLI. Perspectives for future improvement of bioluminescence systems are discussed.

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Section: Development Of a Brighter Bioluminescence Systemmentioning

confidence: 99%

Advanced Bioluminescence System for In Vivo Imaging with Brighter and Red-Shifted Light Emission

Endo

Ozawa

2020

IJMS

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“…Therefore, additional phytochemicals that stimulate GLUT4 translocation and thereby reduce blood glucose levels need to be identified, and their mode of action needs to be studied in detail. For this purpose, different wet-lab chemistry [ 14 , 15 ], luminescence [ 16 ] and microscopy techniques [ 17 , 18 ] have been implemented, offering varying sensitivity and applicability as well as different throughput capabilities.…”

Section: Introductionmentioning

confidence: 99%

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

Schwarzinger

Neuhauser

Aumiller³

et al. 2021

Molecules

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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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“…Therefore, there is an urgent need for alternative approaches that are robust, high throughput, and sensitive. A•luminescence-based system described previously could be an appropriate solution in this regard [17]. However, the sensitivity and ease of implementation in comparison to fluorescence-based assays might be lower.…”

Section: Introductionmentioning

confidence: 99%

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

Schwarzinger

Lanzerstorfer

Neuhauser

et al. 2020

IJMS

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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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A Detection Method for GLUT4 Exocytosis Based on Spontaneous Split Luciferase Complementation

Cited by 7 publications

References 14 publications

Advanced Bioluminescence System for In Vivo Imaging with Brighter and Red-Shifted Light Emission

Advanced Bioluminescence System for In Vivo Imaging with Brighter and Red-Shifted Light Emission

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

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

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