Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Changou, Chun A.; Ajoy, Reni; Chou, Szu Yi

doi:10.3791/56409

Cited by 6 publications

(4 citation statements)

References 17 publications

(22 reference statements)

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“…The housing of mice and the animal for primary neuronal cell isolation were performed in accordance with protocols approved by the Institutional Animal Care and Use Committees of the Taipei Medical University (Protocol numbers: LAC-2015-0397; LAC-2019-0020). Primary neurons were cultured from mice embryos at day 16.5 (E16.5–E17.5), as described previously . In short, dishes were coated with poly l -lysine (Sigma-Aldrich, USA) overnight.…”

Section: Methodsmentioning

confidence: 99%

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Characterization and Chromatographic Isolation of Platelet Extracellular Vesicles from Human Platelet Lysates for Applications in Neuroregenerative Medicine

Nyam-Erdene

Nebie

Delila

et al. 2021

ACS Biomater. Sci. Eng.

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Human platelet lysates (HPLs) made from clinical-grade platelet concentrates are currently evaluated in the preclinical models of Parkinson’s disease, Alzheimer’s disease, traumatic brain injury, and others, as a new polyvalent neuroprotective biotherapy of the central nervous system. However, the presence and content of extracellular vesicles (EVs) in HPLs and their potential contribution to the neuroprotective and neurorestorative activities of HPLs are still unknown. We, therefore, characterized the EVs present in four different HPL preparations and after purification by size-exclusion chromatography. We then tested the effect of the isolated EVs on neuronal cell repair. We identified that all four HPLs contained a high and similar amount of EVs (1011 to 1012/mL) with a mean size ranging from ca. 50 to 300 nm and a negative zeta potential as determined by nanoparticle tracking analysis and dynamic light scattering. Western blot analysis revealed that the EVs present in HPLs expressed the clusters of differentiation 41 (CD41) and 61 (CD61) characteristic of platelets. These EVs were efficiently isolated from HPL proteins by Sepharose CL-2B size-exclusion column chromatography as confirmed by total protein determination and protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with 73–85% recovery and maintenance of their size, negative zeta potential, and CD41 and CD61 expression. Interestingly, the EVs purified from the four HPLs exhibited a differential capacity to promote cell growth and migration in a wound-healing assay using SH-SY5Y neuronal cells, and one EV preparation stimulated network formation in primary neuronal cultures. These data indicated that the EVs present in HPLs have different neuroregenerative capacities and that some EV preparations may have interesting applications as a stand-alone therapy for usage in neuroregenerative medicine.

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Section: Methodsmentioning

confidence: 99%

“…Primary neurons were cultured from mice embryos at day 16.5 (E16.5−E17.5), as described previously. 38 In short, dishes were coated with poly L-lysine (Sigma-Aldrich, USA) overnight. Pregnant mice were sacrificed by CO 2 intoxication and the embryos were separated from the embryonic sac.…”

Section: Pcs and Preparation Of Hpls The Institutional Reviewmentioning

confidence: 99%

Characterization and Chromatographic Isolation of Platelet Extracellular Vesicles from Human Platelet Lysates for Applications in Neuroregenerative Medicine

Nyam-Erdene

Nebie

Delila

et al. 2021

ACS Biomater. Sci. Eng.

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“…Pregnant mice at embryonic day 16–17 (E16-E17) were euthanized after being anesthetized with high-dose Avertin (400 mg/kg). Embryonic hypothalamic regions were dissected from brains on ice-cold DPBS immediately as a previous protocol described [31]. Hypothalamic tissues were digested with EDTA Trypsin (0.125%, Gibco) at 37 °C for 15 min.…”

Section: Methodsmentioning

confidence: 99%

A subpopulation of Bdnf-e1–expressing glutamatergic neurons in the lateral hypothalamus critical for thermogenesis control

You

Chu

Guo

et al. 2020

Molecular Metabolism

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ObjectiveBrown adipose tissue (BAT)–mediated thermogenesis plays a key role in energy homeostasis and the maintenance of body temperature. Previous work suggests that brain-derived neurotrophic factor (BDNF) is involved in BAT thermogenesis, but the underlying neural circuits and molecular mechanism remain largely unknown. This is in part due to the difficulties in manipulating BDNF expression in different brain regions through different promoters and the lack of tools to identify neurons in the brain specifically involved in BAT thermogenesis.MethodsWe have created several lines of mutant mice in which BDNF transcription from a specific promoter was selectively disrupted by replacing Bdnf with green fluorescent protein (GFP; Bdnf-e1, -e4, and -e6−/− mice). As such, cells expressing Bdnf-e1, -e4, or -e6 were labeled with GFP. To identify BAT-connected thermogenesis neurons in brain, we applied the retrograde pseudorabies virus labeling method from BAT. We also used chemogenetic tools to manipulate specific neurons coupled with BAT temperature recording. Moreover, we developed a new TrkB agonist antibody to rescue the BAT thermogenesis deficits.ResultsWe show that selective disruption of Bdnf expression from promoter 1 (Bdnf-e1) resulted in severe obesity and deficits of BAT-mediated thermogenesis. Body temperature response to cold was impaired in Bdnf-e1−/− mice. BAT expression of Ucp1 and Pcg1a, genes known to regulate thermogenesis, was also reduced, accompanying a decrease in the sympathetic activity of BAT. Staining of cells expressing Bdnf-e1 transcript, combined with transsynaptic, retrograde-tracing labeling of BAT-connected neurons, identified a group of excitatory neurons in lateral hypothalamus (LH) critical for thermogenesis regulation. Moreover, an adaptive thermogenesis defect in Bdnf-e1−/− mice was rescued by injecting an agonistic antibody for TrkB, the BDNF receptor, into LH. Remarkably, activation of the excitatory neurons (VGLUT2+) in LH through chemogenetic tools resulted in a rise of BAT temperature.ConclusionsThese results reveal a specific role of BDNF promoter I in thermogenesis regulation and define a small subset of neurons in LH that contribute to such regulation.

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“…Changou et al [71] reported a detailed protocol for another cell type in GLUT4 insulin signal regulation research, primary hypothalamic neurons. They applied GFP-GLUT4 protein to track GLUT4 membrane translocation upon insulin stimulation.…”

Section: General Labeling Strategies For Glut4 Visualization By Tradi...mentioning

confidence: 99%

Fluorescence microscopy-based quantitation of GLUT4 translocation

Heckmann

Klanert²,

Sandner

et al. 2022

Methods Appl. Fluoresc.

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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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Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Cited by 6 publications

References 17 publications

Characterization and Chromatographic Isolation of Platelet Extracellular Vesicles from Human Platelet Lysates for Applications in Neuroregenerative Medicine

Characterization and Chromatographic Isolation of Platelet Extracellular Vesicles from Human Platelet Lysates for Applications in Neuroregenerative Medicine

A subpopulation of Bdnf-e1–expressing glutamatergic neurons in the lateral hypothalamus critical for thermogenesis control

Fluorescence microscopy-based quantitation of GLUT4 translocation

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