I am following the method as described by Hughey et al (www.jove.com/details.php?id=2432) for conducting the hyperinsulinemic-euglycemic clamp in Wistar rats (400g) but the overnight fasted blood (arterial) glucose level varies considerably between rats, from 6-10 mM. Once I commence insulin infusion (2 uL/min, 4mU/kg/min) is it most appropriate to clamp the glucose level at the rat's baseline glucose level or at a fixed amount e.g. 5-5.5 mM?
n.b. the only major modification that we have made to the Hughey method is that we are conducting this procedure under pentobarbital anaesthesia (60 mg/kg), which according to Saha et al (2005; Exp Biol Med 230, 777-784) does not affect glycemia.
Overnight fasted blood glucose levels in excess of 6mM seem high, especially approaching 10mM. This increase may be partly due to the use of anesthesia, which is known to cause some increase in blood glucose level. It would be advisable to aim for levels that are more consistent with normal levels (5-7mM).
Since you would probably like to finish the experiment in ~ 1hr during which you need to establish stable levels, chances are that there will be some variations in blood glucose levels but rather than extending time period in trying to get to a very precise glucose level, say 5.5mM, going with a stable number within an accepted range will serve you better - prolonging the experiment has its own caveats.
Best,
Dear Dr Alam,
thankyou for your thoughts and advise it has been most helpful.
Kind regards,
Damien.
Dear Dr Perry,
I appreciate your advice. Yes for this study we will need to conduct the procedure on anaesthatised animals which seems to be done more often than what I first thought..
Kind regards,
Damien.
I agree with Dr. Perry. Chose a single reasonable value fasting and use it for all your animals
Damien,
It goes without saying that to compare numbers between animals within a group and various groups, one needs to have a glucose level that is as constant as possible. My point was to not waste excessive time to try and get a specific value within the normal range. For example, if you find that a certain rate of infusions of glucose and insulin quickly gets to stable glucose level at 5.7mM, stay with it and match the rest of animals as close to it as you can. There will always be minor differences between various animals within a group - that is the nature of in vivo experiments an dthis is why one has groups of animals to obtain statistically significant difference.
Best,
While anesthesia can cause insulin resistance, my own results suggest that it has a greater effect in the liver, which may be causing the fasting hyperglycemia. I've also seen in a few subgroups of animals that hyperglycemia (even quiet modest elevations in glucose) can induce insulin resistance, and alter insulin dispersion through muscle. For that reason, I agree with the above answers that you should clamp at a similar level for all your animals.
However, I wonder why some animals show a higher fasting glucose level under anesthesia as it is not a consistent effect through all animals - this is something I have not been able to answer yet in my own studies. If possible, it might be worth measuring glucose levels prior to inducing anesthesia, as well as once under anesthesia, in order to determine whether anesthesia itself is elevating glucose levels - and may provide another good rationale for clamping at a lower level.
Cathryn,
At least in my experience, I have not (I'm tempted to say never, but shall refrain from doing so) encountered variations of such magnitude in blood glucose levels of animals that are truly fasted overnight that some reach the limit of kidney to retain glucose and start spilling it in urine.
Here are some things to consider:
1. Change the cage of rat(s) rather than simply removing the food pellets. Some partially eaten food pallets fall through into the cage and some rats are very good in finding them.
2. I have noted more inter-personal differences between various people than a well-trained person with experience in animal handling will observe in a group. I have had people in my lab who consistently measure ~10 mg/dl virtually for every rat compared another person on the same group of animals. Some people are simply better in keeping their animals more calm!
Dear Dr Kolka,
Could you provide details on which anesthetics that you use?
I agree that to gain insight into the variability between animals, it would be important to collect blood glucose samples prior to anesthesia. Unfortunately, I am unable to do this as blood collection in unanesthetized animals isnt something we have approval to do.
I have recently purchased the blood glucose telemetry system from DSI - this technology will enable us to easily answer questions like this. I will try to incorporate this into future study designs.
Kind regards,
Damien.
Sure, I have used both sevoflurane and isoflurane, with propofol for induction.
Also, my work is in dogs, which makes it easier to regulate food intake. The blood glucoses are very consistent in conscious animals under fasting conditions, and we almost never detect hyperglycemia. We do typically take glucose levels prior to anesthesia, and it does seem to be an anesthesia specific effect as they are not hyperglycemic prior to induction of anesthesia.
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