Apr 14, 2020

Public workspaceIP Glucose Tolerance Test in Mouse

DOI
dx.doi.org/10.17504/protocols.io.wxhffj6
  • 1University of Alberta
  • CIRTNR2FIC
Nancy Smith
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DOI: dx.doi.org/10.17504/protocols.io.wxhffj6
External link: http://www.bcell.org
Protocol Citation: Nancy Smith, Mourad Ferdaoussi, Haopeng Lin, Patrick E Macdonald 2020. IP Glucose Tolerance Test in Mouse. protocols.io https://dx.doi.org/10.17504/protocols.io.wxhffj6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 09, 2019
Last Modified: April 14, 2020
Protocol Integer ID: 19145
Keywords: glucose tolerance test, IP, mouse, insulin, high fat diet, GTT, fasting,
Abstract
This protocol details the IP glucose tolerance test in mice. This is performed on C57Bl6, transgenic SENP1 KO and ZMIZ1 KO mouse models. Typically GTT's are done at 12 weeks of age. Following the GTT on chow diet, some mice are put on high fat diet for the next 10 weeks. Subsequent glucose tolerance tests are performed following 2 days, 4 weeks and 8 weeks of high fat diet.
Insulin tolerance test can also be performed. Once the in vivo studies are complete, the pancreas or islets are isolated for further ex vivo experiments.
Materials
MATERIALS
ReagentMouse Diet High Fat (60%)VWR International (Avantor)Catalog #F3282
Reagent5LOD Irradiated Pico Lab Rodent DietCatalog #6954
ReagentSterile Glucose (50% dextrose) solution 25g/50mlCatalog #06648(13)
ReagentMicrovette 100 LHSarstedtCatalog #20-1282-100
ReagentSTELLUX Chemi Rodent Insulin ELISAAlpcoCatalog #80-INSMR-CH01
ReagentOneTouch Ultra Blue Blood Glucose StripsCatalog #L8041261
ReagentOne Touch Ultra 2 Blood Glucose monitoring systemCatalog #L1540947
Reagent1cc SyringesBecton Dickinson (BD)Catalog #B309659
Reagent26G needleBecton Dickinson (BD)Catalog #305111
Fasting
Fasting
Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before IPGTT begins. Transfer mice to clean cage and wire top. Keep water bottles in cages during the fasting period.
Note
  • If using High Fat diet (HFD), save food to give back at the end of the IPGTT.
  • Mark tails with a sharpie for easier identification during IPGTT.

IPGTT
IPGTT
Weigh mouse to obtain body weight for dose calculations.
Calculate bolus dose of glucose (1g/kg).
Note
Weight of mouse (g) x 2 = ul sterile glucose (50% dextrose) solution

*If using HFD for 4 weeks or more, use 0.5g/kg (weight of mouse (g) x 1 = ul sterile glucose (50% dextrose) solution)

Download GTT- template spreadsheet.xlsxGTT- template spreadsheet.xlsx

Prepare syringes using 1cc syringe and 26G needle. Load all syringes with calculated glucose dose and position in front of home cage.
Prior to the delivery of the sterile glucose (50% dextrose) solution, a time “zero” blood glucose level must be measured. Restrain mouse and clean tail with 70% ethanol/gauze. Extract a small amount of blood from the tail vein onto One Touch Ultra blood glucose strips used with the One Touch Ultra 2 blood glucose monitoring system. Blood can be collected into microvette tubes that can be used to extract plasma which can be assayed for plasma insulin (see step 9 for plasma collection).

Note
Microvette tubes are blood collection tubes with a capillary. Tap tube until all the blood is collected in the tube. Place on ice until plasma collection.

Administer the calculated dose of sterile glucose (50% dextrose) solution using an IP (intraperitoneal) injection method.
  1. Restrain the mouse. Then place the mouse is in dorsal recumbency with the head down allowing the viscera to move cranially.
  2. Using the bend of the knee and the ventral abdominal midline as landmarks, the needle is inserted half way between the midline and lateral side of the animal at the natural bend of the knee. Insert the needle at a 30 to 45 degree angle into either the left or right lower abdominal quadrant.
  3. Prior to injection, aspirate to make sure the needle has not penetrated a blood vessel, the intestines or the urinary bladder. When aspirating the syringe, you should see an air bubble in the hub of the needle, and not any form of fluid.
  4. Inject and return mouse to cage.
Begin timing from the point of successfully delivering the sterile glucose (50% dextrose) solution.
Test and record the blood glucose level (see step 4) at times 15, 30, 60, 90, and 120 minutes after the initial glucose delivery. Record each value on Glucose Tolerance Test record sheet (template found on Step 2). Record condition of animal throughout and after procedure on Glucose Tolerance Test record sheet.
Note
Extract a small amount of blood from the tail veil at 15, 30, 60 and 120 minutes as well as testing the blood glucose level.
*skip blood collection at 90 minutes to ensure not to exceed 0.007 ml blood/ g body weight.

Following experiment, return food and environmental enrichments to cages.
Plasma collection
Plasma collection
Place tubes into pre-chilled centrifuge. Centrifguge for 2 mins at 2000 rpm.
Duration00:02:00
Temperature4 °C


Remove the capillary-lid and dispose in biohazard bin. Place the base lid on and seal the microvette (click position).
Centrifuge at 4°C for 10 mins at 10 000 rpm.
Duration00:10:00
Temperature4 °C


Using a pipette, transfer the plasma (top transparent layer) to a clean and labelled tube.

Note
Store plasma samples at -20°C until assayed.

Assay for insulin plasma on a STELLUX Chemi Rodent Insulin ELISA kit by ALPCO. See protocol at:
https://s3.amazonaws.com/alpco-docs/80/80-INSMR-CH01.pdf