Live images of GLUT4 protein trafficking in mouse primary hypothalamic neurons using deconvolution microscopy

Chun Austin Changou, Szu Yi Chou, Reni Ajoy

Research output: Contribution to journalArticle

Abstract

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNFa, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear. In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

Original languageEnglish
Article numbere56409
JournalJournal of Visualized Experiments
Volume2017
Issue number130
DOIs
Publication statusPublished - Dec 7 2017

Fingerprint

Glucose Transporter Type 4
Insulin
Deconvolution
Protein Transport
Neurons
Microscopy
Microscopic examination
Proteins
Medical problems
Chemokines
Tissue
Inflammation
Membranes
Neurology
Image resolution
Knockout Mice
Type 2 Diabetes Mellitus
Hypothalamus
Interleukin-6
Central Nervous System

Keywords

  • Deconvolution microscopy
  • GLUT4
  • Issue 130
  • Liposome based transfection
  • Live image recording
  • Neurobiology
  • Primary mouse hypothalamic neuron culture

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

Live images of GLUT4 protein trafficking in mouse primary hypothalamic neurons using deconvolution microscopy. / Changou, Chun Austin; Chou, Szu Yi; Ajoy, Reni.

In: Journal of Visualized Experiments, Vol. 2017, No. 130, e56409, 07.12.2017.

Research output: Contribution to journalArticle

@article{2365434e4f47475fb7a6baa56e019b63,
title = "Live images of GLUT4 protein trafficking in mouse primary hypothalamic neurons using deconvolution microscopy",
abstract = "Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNFa, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear. In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.",
keywords = "Deconvolution microscopy, GLUT4, Issue 130, Liposome based transfection, Live image recording, Neurobiology, Primary mouse hypothalamic neuron culture",
author = "Changou, {Chun Austin} and Chou, {Szu Yi} and Reni Ajoy",
year = "2017",
month = "12",
day = "7",
doi = "10.3791/56409",
language = "English",
volume = "2017",
journal = "Journal of Visualized Experiments",
issn = "1940-087X",
publisher = "MYJoVE Corporation",
number = "130",

}

TY - JOUR

T1 - Live images of GLUT4 protein trafficking in mouse primary hypothalamic neurons using deconvolution microscopy

AU - Changou, Chun Austin

AU - Chou, Szu Yi

AU - Ajoy, Reni

PY - 2017/12/7

Y1 - 2017/12/7

N2 - Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNFa, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear. In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

AB - Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNFa, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear. In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

KW - Deconvolution microscopy

KW - GLUT4

KW - Issue 130

KW - Liposome based transfection

KW - Live image recording

KW - Neurobiology

KW - Primary mouse hypothalamic neuron culture

UR - http://www.scopus.com/inward/record.url?scp=85037702362&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85037702362&partnerID=8YFLogxK

U2 - 10.3791/56409

DO - 10.3791/56409

M3 - Article

AN - SCOPUS:85037702362

VL - 2017

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 130

M1 - e56409

ER -