Insulin Tolerance Test - Revision history

Davebrid at 16:43, 10 February 2025

2025-02-10T16:43:10Z

← Older revision		Revision as of 16:43, 10 February 2025
Line 37:		Line 37:
	===Taking into Account Differences in Fat Mass===		===Taking into Account Differences in Fat Mass===
	Since most glucose is taken up into non-fat tissues, it can be confounding if you dose based on total body weight, and the two groups differ greatly with respect to the amount of fat mass. A fat mouse and a lean mouse are likely to have very similar fat-free masses, and by giving more insulin to the fatter animal (because body weight is higher) you might make the fatter mouse seem more insulin sensitive, but really its just responding to a larger amount of insulin. In these cases the preferred practice is to '''measure fat free mass by echoMRI''', and then dose animals at approximately 1 U/kg of lean mass for a lean animal or 2.5 U/kg of lean mass for an obese animal.		Since most glucose is taken up into non-fat tissues, it can be confounding if you dose based on total body weight, and the two groups differ greatly with respect to the amount of fat mass. A fat mouse and a lean mouse are likely to have very similar fat-free masses, and by giving more insulin to the fatter animal (because body weight is higher) you might make the fatter mouse seem more insulin sensitive, but really its just responding to a larger amount of insulin. In these cases the preferred practice is to '''measure fat free mass by echoMRI''', and then dose animals at approximately 1 U/kg of lean mass for a lean animal or 2.5 U/kg of lean mass for an obese animal.

			[[Category:Animal Work]]
			[[Category:Mouse Work]]
			[[Category: Glucose]]

Davebrid: added abstract

2022-03-07T15:19:04Z

added abstract

← Older revision		Revision as of 15:19, 7 March 2022
Line 1:		Line 1:
			Insulin tolerance tests are a standard and common method for evaluating the insulin sensitivity of an animal. In this assay, animals are fasted to normalize blood glucose and then challenged by an intraperitoneal injection of insulin. As glucose is transported into tissues and glucose production is suppressed, glucose levels drop. A more insulin-sensitive animal will have a larger decrease in blood glucose. This has advantages over a glucose tolerance test which measures both insulin sensitivity and insulin responsiveness.

	==SOP==		==SOP==
	*[[Safety and Animal Training]]		*[[Safety and Animal Training]]

Reddj: added SOPS

2020-06-08T15:25:26Z

added SOPS

← Older revision		Revision as of 15:25, 8 June 2020
Line 1:		Line 1:
			==SOP==
			*[[Safety and Animal Training]]
			*[[SOP - Needle Capping]]

	==Materials (Bring to Room)==		==Materials (Bring to Room)==
	*Glucometer - AccuChek Advantage		*Glucometer - AccuChek Advantage

Davebrid: /* Materials (Bring Downstairs) */ removed link

2016-06-24T13:22:43Z

Materials (Bring Downstairs): removed link

← Older revision		Revision as of 13:22, 24 June 2016
Line 1:		Line 1:
	==Materials (Bring ~~Downstairs~~)==		==Materials (Bring to Room)==
	*Glucometer - AccuChek Advantage		*Glucometer - AccuChek Advantage
	*Glucose Test Strips - AccuChek Comfort Curve or equivalent		*Glucose Test Strips - AccuChek Comfort Curve or equivalent
	*Scale		*Scale
	*Beaker for weighing mice		*Beaker for weighing mice
	*echoMRI if the mice differ in body fat levels (see ~~[[#Taking_into_Account_Differences_in _Fat_Mass]]~~)		*echoMRI if the mice differ in body fat levels (see below)
	*Syringes		*Syringes
	*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of insulin, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass.		*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of insulin, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass.

Davebrid: changed section link

2016-06-24T13:22:04Z

changed section link

← Older revision		Revision as of 13:22, 24 June 2016
Line 4:		Line 4:
	*Scale		*Scale
	*Beaker for weighing mice		*Beaker for weighing mice
	*echoMRI if the mice differ in body fat levels (see [[~~Taking into Account Differences in Fat Mass~~]])		*echoMRI if the mice differ in body fat levels (see [[#Taking_into_Account_Differences_in _Fat_Mass]])
	*Syringes		*Syringes
	*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of insulin, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass.		*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of insulin, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass.

Davebrid: wrote section regarding fat mass considerations

2016-06-24T13:21:02Z

wrote section regarding fat mass considerations

← Older revision		Revision as of 13:21, 24 June 2016
Line 1:		Line 1:
	==Materials (Bring Downstairs)==		==Materials (Bring Downstairs)==
	*Glucometer - AccuChek Advantage		*Glucometer - AccuChek Advantage
	*Glucose Test Strips - AccuChek Comfort Curve~~. Order these through Materiels Service by calling 936-6077 and ordering product 2535. For information see [here]~~		*Glucose Test Strips - AccuChek Comfort Curve or equivalent
	*Scale		*Scale
	*Beaker for weighing mice		*Beaker for weighing mice
			*echoMRI if the mice differ in body fat levels (see [[Taking into Account Differences in Fat Mass]])
	*Syringes		*Syringes
	*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. This may need to be adjusted depending on the insulin sensitivity of the mice. ~~This~~ is diluted from Humulin R-100 and is purchased through the veterinary staff.		*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. If you are using a higher or lower dose of insulin, add more or less to the 10 mL of PBS, so that injections are 10 uL/g of mass.
			**This may need to be adjusted depending on the insulin sensitivity of the mice, and this is based on a normal C57BL/6J mouse on chow.
			**In general for insulin resistant mice, such as those >40g on a high fat diet or such, increase the dose to 2 or 2.5U/kg.
			** In general you want the insulin to decrease blood glucose by about 60-70% in the most responsive of your too group so if your response is <20% of >70% change in blood glucose you will probably have to change your dose and retry.
			**The insulin is diluted from Humulin R-100 and is purchased through the veterinary staff.
	*Timer		*Timer

	==Protocol==		==Protocol==
	*Remove food from mice for about 6h. Add do not feed tag to cages, or move cage to procedure room.		*Remove food from mice for about 6h by putting them in a fresh cage. Add do not feed tag to cages, or ideally move cage to procedure room. Try to make sure that the mice are in a quiet, undisturbed temperature controlled room with the lights on.
	*Typically starve the mice at ~~9AM~~ and aim to start injections at ~~3 PM~~		*Typically starve the mice at 8AM and aim to start injections at 2PM
	*Weigh mice, mark tails if necessary and take fasting glucose measurement via a tail clip.		*Prepare a 1 g/10mL solution of glucose in case some animals become hypoglyemic.
			*Weigh mice, mark tails if necessary with different colors for rapid identification and take fasting glucose measurement via a tail clip.
	*Prepare insulin syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).		*Prepare insulin syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).
	*At approximately 1 min intervals, inject appropriate amount of insulin into interperitoneal cavity of the mouse.		*At approximately 1 min intervals, inject appropriate amount of insulin into interperitoneal cavity of the mouse.
Line 18:		Line 24:
	**Insert syringe (do not inject) into cavity		**Insert syringe (do not inject) into cavity
	**Eject syringe.		**Eject syringe.
	*At desired intervals (normally 15, 30, 45, 60, 75, 90, 105 and 120 min), take blood glucose measurements from tail vein. If needed re-snip the tail vein. When measuring glucose just lift the tail of the mouse, while leaving it in the cage, rather than removing and restraining the mouse.		*At desired intervals (normally 15, 30, 45, 60, 75, 90, 105 and 120 min), take blood glucose measurements from tail vein. If needed re-snip the tail vein. When measuring glucose just lift the tail of the mouse, while leaving it in the cage, rather than removing and restraining the mouse, which can be more stressful.
	*~~Analyse~~ data by both % change from fasting glucose and absolute values		**If blood glucose drops below 10 mg/dL, or the animal appears catatonic, inject 300 uL of the glucose solution prepared above to rescue the mice and prevent hypoglycmic shock. Ignore the data from this mouse from that point on.
			*Analyze data by both % change from fasting glucose and absolute values. Our preferred outcome is to report fasting glucose levels and report percent change as a figure
			*The preferred statistical model is a mixed linear model using the time points as ordinate values and testing for a main effect or an interaction of the treatment/genotype. Use the R [https://cran.r-project.org/web/packages/lme4/lme4.pdf lme4] package for this.

			===Taking into Account Differences in Fat Mass===
			Since most glucose is taken up into non-fat tissues, it can be confounding if you dose based on total body weight, and the two groups differ greatly with respect to the amount of fat mass. A fat mouse and a lean mouse are likely to have very similar fat-free masses, and by giving more insulin to the fatter animal (because body weight is higher) you might make the fatter mouse seem more insulin sensitive, but really its just responding to a larger amount of insulin. In these cases the preferred practice is to '''measure fat free mass by echoMRI''', and then dose animals at approximately 1 U/kg of lean mass for a lean animal or 2.5 U/kg of lean mass for an obese animal.

Davebrid: fixed glucose typo

2014-01-17T16:39:35Z

fixed glucose typo

← Older revision		Revision as of 16:39, 17 January 2014
Line 12:		Line 12:
	*Typically starve the mice at 9AM and aim to start injections at 3 PM		*Typically starve the mice at 9AM and aim to start injections at 3 PM
	*Weigh mice, mark tails if necessary and take fasting glucose measurement via a tail clip.		*Weigh mice, mark tails if necessary and take fasting glucose measurement via a tail clip.
	*Prepare ~~glucose~~ syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).		*Prepare insulin syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).
	*At approximately 1 min intervals, inject appropriate amount of insulin into interperitoneal cavity of the mouse.		*At approximately 1 min intervals, inject appropriate amount of insulin into interperitoneal cavity of the mouse.
	**Immobilize mouse and restrain tail with one hand		**Immobilize mouse and restrain tail with one hand

Davebrid: added ordering details for humulin

2014-01-17T15:29:25Z

added ordering details for humulin

← Older revision		Revision as of 15:29, 17 January 2014
Line 5:		Line 5:
	*Beaker for weighing mice		*Beaker for weighing mice
	*Syringes		*Syringes
	*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. This may need to be adjusted depending on the insulin sensitivity of the mice		*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. This may need to be adjusted depending on the insulin sensitivity of the mice. This is diluted from Humulin R-100 and is purchased through the veterinary staff.
	*Timer		*Timer

	==Protocol==		==Protocol==
	*Remove food from mice for about 6h. Add do not feed tag to cages, or move cage to procedure room.		*Remove food from mice for about 6h. Add do not feed tag to cages, or move cage to procedure room.
	*Separate mice into individual cages and mark with mouse ID. Put a water bottle on the cage.
	*Typically starve the mice at 9AM and aim to start injections at 3 PM		*Typically starve the mice at 9AM and aim to start injections at 3 PM
	*Weigh mice, mark tails as necessary and take fasting glucose measurement via a tail clip.		*Weigh mice, mark tails if necessary and take fasting glucose measurement via a tail clip.
	*Prepare glucose syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).		*Prepare glucose syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).
	*At approximately 1 min intervals, inject appropriate amount of ~~glucose~~ into interperitoneal cavity of the mouse.		*At approximately 1 min intervals, inject appropriate amount of insulin into interperitoneal cavity of the mouse.
	*Immobilize mouse and restrain tail with one hand		**Immobilize mouse and restrain tail with one hand
	*Aim needle between the midline and the hip bone		**Aim needle between the midline and the hip bone
	*Insert syringe (do not inject) into cavity		**Insert syringe (do not inject) into cavity
	*~~Pull up syringe and ensure that only air is aspirated. If urine or blood appears in the syringe, start over~~		**Eject syringe.
	*Eject syringe.		*At desired intervals (normally 15, 30, 45, 60, 75, 90, 105 and 120 min), take blood glucose measurements from tail vein. If needed re-snip the tail vein. When measuring glucose just lift the tail of the mouse, while leaving it in the cage, rather than removing and restraining the mouse.
	*At desired intervals (normally 15, 30, 45, 60, 75, 90 and 120 min), take blood glucose measurements from tail vein
	*Analyse data by both % change from fasting glucose and absolute values		*Analyse data by both % change from fasting glucose and absolute values

Davebrid: updated with new starvation and added additional information

2011-05-13T15:00:52Z

updated with new starvation and added additional information

← Older revision		Revision as of 15:00, 13 May 2011
Line 1:		Line 1:
	==Materials (Bring Downstairs==		==Materials (Bring Downstairs)==
	*Glucometer - AccuChek Advantage		*Glucometer - AccuChek Advantage
	*Glucose Test Strips - AccuChek Comfort Curve. Order these through Materiels Service by calling 936-6077 and ordering product 2535. For information see [here]		*Glucose Test Strips - AccuChek Comfort Curve. Order these through Materiels Service by calling 936-6077 and ordering product 2535. For information see [here]
Line 5:		Line 5:
	*Beaker for weighing mice		*Beaker for weighing mice
	*Syringes		*Syringes
	*0.~~07U~~/mL humulin in PBS (make as ~~7uL~~ of 100 U/mL in 10 mL, sterile filtered). This will correspond to ~~0.7U~~/kg injections.		*0.1 U/mL humulin in PBS (make as 10uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 1 U/kg injections. This may need to be adjusted depending on the insulin sensitivity of the mice
	Timer		*Timer

	==Protocol==		==Protocol==
	*Remove food from mice for about 6h. Add do not feed tag to cages, or move cage to procedure room.		*Remove food from mice for about 6h. Add do not feed tag to cages, or move cage to procedure room.
	*~~Bring~~ mice ~~to procedure room~~. Put water bottle on cage.		*Separate mice into individual cages and mark with mouse ID. Put a water bottle on the cage.
	*Weigh mice, mark tails as necessary and take fasting glucose measurement.		*Typically starve the mice at 9AM and aim to start injections at 3 PM
			*Weigh mice, mark tails as necessary and take fasting glucose measurement via a tail clip.
	*Prepare glucose syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).		*Prepare glucose syringes with 10 uL per g mouse weight (ie for a 30g mouse, 300 uL).
	*At 1 min intervals, inject appropriate amount of glucose into interperitoneal cavity of the mouse.		*At approximately 1 min intervals, inject appropriate amount of glucose into interperitoneal cavity of the mouse.
	*Immobilize mouse and restrain tail with one hand		*Immobilize mouse and restrain tail with one hand
	*Aim needle between the midline and the hip bone		*Aim needle between the midline and the hip bone

Davebrid: updated insulin concentration

2010-11-01T14:19:17Z

updated insulin concentration

← Older revision		Revision as of 14:19, 1 November 2010
Line 5:		Line 5:
	*Beaker for weighing mice		*Beaker for weighing mice
	*Syringes		*Syringes
	*0.07U/mL humulin in PBS (make as ~~70uL~~ of 100 U/mL in 10 mL, sterile filtered). This will correspond to 0.~~7mU~~/kg injections.		*0.07U/mL humulin in PBS (make as 7uL of 100 U/mL in 10 mL, sterile filtered). This will correspond to 0.7U/kg injections.
	Timer		Timer