Difference between revisions of "Pyruvate Tolerance Test"
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| − | ''' | + | Pyruvate tolerance tests are typically done to understand the rates of pyruvate conversion into glucose (as an indirect measure of gluconeogenesis). The assay is relatively straightforward, we fast mice for ~6h to normalize glycemia then perform intraperitoneal injections of pyruvate. That pyruvate is converted into glucose, primarily by the liver. The rate of glucose production from pyruvate is indicated by the peak, or area under the curve of the glucose-time curve. A couple of key caveats. Several substrates could be used for this including pyruvate, glycerol, lactate, alanine, or glutamine, but pyruvate appears to be the most common in the literature. Second, you should '''always''' perform a [[Glucose Tolerance Test|glucose tolerance test]] to interpret these results. This is because if you have impaired glucose disposal, you will also have elevated disposal of pyruvate-elicited glucose. That would mean that glucose intolerance could be interpreted as excessive pyruvate-glucose conversion. While this assay is relatively easy and inexpensive, the gold standard of glucose production ''in vivo'' is to monitor glucose production during a euglycemic clamp either in response to insulin (suppressive to gluconeogenesis) or glucagon (promoting it). |
| − | Glucometer - AccuChek Advantage | + | ==Materials== |
| − | Glucose Test Strips - AccuChek Comfort Curve. Order these through Materiels Service by calling 936-6077 and ordering product 2535. For information see [here] | + | * Glucometer - AccuChek Advantage |
| − | Scale | + | * Glucose Test Strips - AccuChek Comfort Curve. Order these through Materiels Service by calling 936-6077 and ordering product 2535. For information see [here] |
| − | Beaker for weighing mice | + | * Scale or echoMRI to determine mouse mass or mouse lean mass |
| − | Syringes | + | * Beaker for weighing mice |
| − | 2.5g pyruvate in 10 mL PBS- This will correspond to 2.5 g/kg injections. | + | * Syringes |
| − | Timer | + | * 2.5g pyruvate in 10 mL PBS, dissolve then adjust pH to 7.3-7.5 - This will correspond to 2.5 g/kg injections. |
| + | * Timer | ||
| − | |||
| − | Remove food from mice for about 6h. | + | ==Protocol== |
| − | Typically starve the mice at 9AM and aim to start injections at | + | * Remove food from mice for about 6h. |
| − | Weigh mice, mark tails if necessary and | + | * Typically starve the mice at 9AM and aim to start injections at 3PM |
| − | Prepare pyruvate syringes with 10 uL per g mouse | + | * Weigh mice, mark tails if necessary, and take a fasting glucose measurement via a tail clip. |
| − | At approximately 1 min intervals, inject appropriate amount of pyruvate into | + | * Prepare pyruvate syringes with 10 uL per g mouse mass (''i.e.'' for a 30g mouse, 300 uL). Ideally, injections are per lean mass, not total mass if the animals differ substantially in adiposity. This is since higher levels of adiposity would result in larger pyruvate injections and potentially inaccurate higher glucose responses. |
| − | Immobilize mouse and restrain tail with one hand | + | * At approximately 1 min intervals, inject appropriate amount of pyruvate into intraperitoneal cavity of the mouse. |
| − | Aim needle between the midline and the hip bone | + | * Immobilize mouse and restrain tail with one hand |
| − | Insert syringe (do not inject) into cavity | + | * Aim needle between the midline and the hip bone |
| − | Eject syringe. | + | * Insert syringe (do not inject) into the cavity |
| − | 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. | + | * Eject syringe. |
| − | Analyse data by both % change from fasting glucose and absolute values. | + | * At desired intervals (normally 15, 30, 45, 60, 75, 90, 105, and 120 min), take blood glucose measurements from the 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. |
| + | * Analyse data by both % change from fasting glucose and absolute values. You may also want to calculate the initial rate of glucose increase (see [[Rate of drop analysis|this protocol]] for details), or the area under the glucose-time curve. | ||
Latest revision as of 17:50, 6 April 2022
Pyruvate tolerance tests are typically done to understand the rates of pyruvate conversion into glucose (as an indirect measure of gluconeogenesis). The assay is relatively straightforward, we fast mice for ~6h to normalize glycemia then perform intraperitoneal injections of pyruvate. That pyruvate is converted into glucose, primarily by the liver. The rate of glucose production from pyruvate is indicated by the peak, or area under the curve of the glucose-time curve. A couple of key caveats. Several substrates could be used for this including pyruvate, glycerol, lactate, alanine, or glutamine, but pyruvate appears to be the most common in the literature. Second, you should always perform a glucose tolerance test to interpret these results. This is because if you have impaired glucose disposal, you will also have elevated disposal of pyruvate-elicited glucose. That would mean that glucose intolerance could be interpreted as excessive pyruvate-glucose conversion. While this assay is relatively easy and inexpensive, the gold standard of glucose production in vivo is to monitor glucose production during a euglycemic clamp either in response to insulin (suppressive to gluconeogenesis) or glucagon (promoting it).
Materials
- 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]
- Scale or echoMRI to determine mouse mass or mouse lean mass
- Beaker for weighing mice
- Syringes
- 2.5g pyruvate in 10 mL PBS, dissolve then adjust pH to 7.3-7.5 - This will correspond to 2.5 g/kg injections.
- Timer
Protocol
- Remove food from mice for about 6h.
- Typically starve the mice at 9AM and aim to start injections at 3PM
- Weigh mice, mark tails if necessary, and take a fasting glucose measurement via a tail clip.
- Prepare pyruvate syringes with 10 uL per g mouse mass (i.e. for a 30g mouse, 300 uL). Ideally, injections are per lean mass, not total mass if the animals differ substantially in adiposity. This is since higher levels of adiposity would result in larger pyruvate injections and potentially inaccurate higher glucose responses.
- At approximately 1 min intervals, inject appropriate amount of pyruvate into intraperitoneal cavity of the mouse.
- Immobilize mouse and restrain tail with one hand
- Aim needle between the midline and the hip bone
- Insert syringe (do not inject) into the cavity
- Eject syringe.
- At desired intervals (normally 15, 30, 45, 60, 75, 90, 105, and 120 min), take blood glucose measurements from the 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.
- Analyse data by both % change from fasting glucose and absolute values. You may also want to calculate the initial rate of glucose increase (see this protocol for details), or the area under the glucose-time curve.
