Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina

Summary

Multiple cell types in the retina, including endothelial cells, neurons, and glial cells, express glucose transporters (GLUTs) to enable glucose uptake into cells. Using ex vivo mouse neural retina and the fluorescent glucose analog 6-NBDG, we describe a relatively quick and inexpensive method to measure glucose uptake in the whole mouse retina.

Abstract

The retina is a highly metabolic tissue with multiple cell types requiring glucose and its derivatives to produce energy in the form of ATP. Retinal cells, including endothelial cells, neurons, photoreceptors, and glial cells, express glucose transporters (GLUTs; e.g., GLUT1-4) to enable the uptake of glucose for energy production. GLUT1 is the most abundantly expressed glucose transporter in the retina. This protocol enables researchers to measure the uptake of glucose in the neural murine retina in ex vivo conditions using the fluorescent glucose analog 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG). After retinal dissection, total retinal 6-NBDG levels can be easily determined via fluorescence endpoint measurement using a plate reader. For consistency, we recommend normalizing results to total protein levels. Although 6-NBDG is highly specific for GLUT1, uptake of this analog is detected in the presence of GLUT1 inhibitor BAY-876. As such, this assay provides a relatively quick and inexpensive method to measure glucose uptake ex vivo in whole mouse neural retina, which is partially mediated by GLUT1.

Introduction

Glucose is an essential metabolite for the neural retina, where it is used to fuel high rates of glycolysis and mitochondrial respiration to produce energy in the form of adenosine triphosphate (ATP)¹. Since glucose is the preferred energy substrate, many retinal cells express glucose transporters (GLUTs) to facilitate the uptake of glucose from the vasculature and surrounding tissue². GLUTs comprise a family of intrinsic membrane glycoproteins that are responsible for glucose transport into mammalian cells³. GLUT transporter-1 (GLUT1) is the primary glucose transporter in the retina, expressed th....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Vanderbilt University Medical Center.

1. Preparation for assay

NOTE: Preparation should be carried out on the day of the assay immediately prior to running the assay. This is necessary due to the time-sensitive nature of the protocol.

1. General preparation ahead of the experiment
   NOTE: 6-NBDG is light-sensitive and should be protected from light. Cover all 6-NBDG solutions with aluminum foil.
   1. A stock solution of 5 mM 6-NBDG in 50% DMSO/1x PBS is stable at -20 &#....

Results

Figure 4 shows representative glucose fluorescence measurements from WT mouse retina incubated with 6-NBDG for different periods of time. After 30 min incubation, 6-NBDG levels were an average of 336 ± 27.91 AU, whereas after 60 min, levels of 6-NBDG increased to an average of 616.3 ± 8.38 AU. A further incubation of 30 min led to a reduced level of 6-NBDG (506.4 ± 5.3 AU). At 60 min, variability in the results was at a minimum and thus was chosen as the optimal time for .......

Discussion

In summary, the method described enables basic science researchers to measure uptake of the fluorescent glucose analog, 6-NBDG, in ex vivo murine neural retina. Glucose is an essential metabolite for the neural retina, it's uptake supports the high rates of glycolysis and mitochondrial respiration needed to produce energy in the form of adenosine triphosphate (ATP)¹. Since glucose is the preferred energy substrate, many retinal cells express glucose transporters (GLUTs) to facilitate the .......

Materials

Name	Company	Catalog Number	Comments
# 5 forceps	Katena	K5-6550	Used for retina dissection
1.5 mL microcentrifuge tubes	Thermo Fisher Scientific	05-408-129
26 G x 5/8" needle	sol-M	112658	Used to puncture cornea during dissection
5 mL tubes	MTC bio	c2540
50 mL tubes	Avantor by VWR	89039-656
6-NBDG	Invitrogen	N23106	Fluorescent gucose analog
96 well plates black with clear bottom	Thermo Fisher Scientific	265301
Anesthetic Charcoal Filter Cannister	ReFresh	EZ-258	Used in anesthesia set up
BAY-876	Millipore Sigma	SML1774	For inhibition of GLUT1.
Centrifuge at 4 °C	Eppendorf	EPP-5424
Compressed gas (5% carbon dioxide, 95% oxygen)	Airgas	UN3156	Used in anesthesia set up
curved forceps	Roboz surgical instrument	RS-5137	Used for retina dissection
DDH2O	Elga LabWater	Elga PureLab Ultra	Used after ethanol to clean sonicator in between samples
Dissecting microscope	Olympus	szX12	Used for retina dissection
Ethanol 200 proof	Decon laboratories	2701	To be used to clean sonicator in between samples
Foam floating tube rack	Thermo Fisher Scientific	36-099-2328	For tubes during incubation in water bath steps
General scissors	Roboz surgical instrument	RS-680	Used for retina dissection
Isoflurane 250 mL bottle	Piramal critical care	NDC  6679401725	Anesthesia
Isoflurane equipment	Vetequip sold by VWR	89012-492	Used to anesthetize prior to euthanasia
Kim wipes	VWR	82003-820
Microplate reader	Molecular devices	SpectraMax M2 microplate reader	Used to read sample
Neurobasal- A media	Gibco	12349-015
Nose cone (low profile anesthesia mask)	Kent Scientific	SOMNO-0801	Used to deliver ansethesia
Objective on dissecting microscope	Olympus	DF plapo 1x pf	Used for retina dissection
Petri dish	VWR	25384-088	Used during retina dissection
Pierce assay reagent	Thermo Fisher Scientific	1861426
Pipette tips P20	Olympus Plastics	26-404
Pipette tips P200, P1000, P10 XL	VWR	76322-150, 76322-154, 76322-132
Pipetteman pipettes P200, P1000, P20, P10	VWR	F144055M, F144056M, F144058M, F144059M
SoftMax Pro software on computer	Molecular devices	SoftMax Pro 7 software	Software used to read sample
Sonic dismembrator	Thermo Fisher Scientific	FB50110	Sonicate sample (retina)
Transfer pipettes	Fisherbrand	13-711-9AM	Used to transfer retina from one tube to another
Vannas spring scissors	Katena	K4-5000	Used for retina dissection
Water bath set to 37 °C	N/A	N/A	Used for incubation