"This is a problem we are having lately in my lab."

Implication being that this is a change and you normally saw a robust insulin peak at 2 min post-glucose injection. Is this correct?

In peripheral blood samples, it is not easy to "catch" the first phase insulin release. As for the modest insulin rise at 15-20min, that is a function of blood glucose levels. If your glucose loading is sufficiently high to raise the blood glucose to about 400-500mg/dl, you will see an insulin peak. I suspect that at 3gm/kg your peak blood glucose remains at or below 300mg/dl.

If you are interested in recording the first phase insulin release (0-4min), a clean way to do the experiment is to use a catheter in situ or alternatively, perform an isolated pancreatic perfusion.

We usually checked insulin levels at the different time-points of the GTT (0,15,30,60 min). So we didn't perform the GSIS routinely. Now we are starting to use this method since we understand is a more accurate protocol for assessing the insulin response. Some of my lab mates do get a nice insulin peak, but many of us do not. We cannot identify which is the factor affecting our results.

In case of intraperitoneal injection of glucose, there is a small possibility that one may not be delivering the glucose in the desired compartment,which may lead to variability due to the time it make take for all of the glucose to diffuse and reach the blood. 

A possibility is to switch the glucose loading from ip to intravenous- tail vein should work quite nicely.

Joan, I got the same problem with c57bl6J mice. I tried lateral tail vein and submandibular bleedings, I got really high levels of glucose in circulation but did not see an increase of insulin at all. I found a paper from Ronald Kahn´s lab in which  they described acute insulin secretion in vivo. They also mentioned in the text that not all the mice respond to glucose injections.

As Tausif suggests, iv injection could hit harder the beta-cell but also glucose will be cleared faster by the kidney so your time frame would be shorter.

If you are interested in analyzing first phase insulin secretion, the isolated islets perifusion would be the approach that I would use.

Jose, I have performed isolated islet perfusion as well as isolated pancreatic perfusion in the past and the latter yields much cleaner results (if done carefully - as with all surgical techniques, there is a learning curve).

Detecting a rise in insulin during an IPGTT in mice is VERY difficult. We usually take multiple samples in the first 15 minutes of the IPGTT in an effort to catch the first phase, which itself is stressful for the mouse and yields very little sample. A second phase insulin release is not observed in mice during an IPGTT, so there is not much point trying to measure insulin after 30 mins. Even if one can detect a rise in insulin, in general it is very small and can be noisy. If insulin secretion is the most important phenotype to you, switch to oral glucose tolerance tests using the gavage method. 

A major problem in our hands is the ELISA kits used to measure insulin in mice. We have never gotten reliable data from the Alpco kits or any other ELISA based method. More recently we have switched to using Luminex assays, and this seems to be much better. Note that a number of MMPCs use in-house radioactive assays, and not commercial ELISA kits. We have always believed that hemolysis of the plasma sample disrupts the measurement of insulin in ELISA-based methods, but it is tricky to obtain frequent tail blood samples without hemolysis. 

In short, what you are trying to do is very tricky and subject to lots of noise. Good luck!!!

I think because injected glucose do not cross the liver, as it happens when glucose provided by feeding, the insulin response has delay.

My colleagues and I have seen this many times with the i.p gtt in mice, no matter what dose glucose was used, 1-3g per kg lean mass. This is an issue of stress due to handling conscious mice and the fact that by not following the natural oral route of glucose administration you are avoiding the incretin effect. Stress increases catecholamines and they will reduce insulin secretion. We have moved to the oral glucose tolerance test and see 2-3 fold increases in insulin 15min after gavage. Insulin is already elevated even 5min after the gavage, but we always see it back to basal by 45min. We use a fixed 50mg dose of glucose in 200ul water, equivalent to ~2g per kg lean mass. We have seen these consistent results in hundreds of mice using the millipore insulin ELISA. Another tip is to practice gavage the mice with an empty gavage needle for 4-5 consecutive days a week or so before the OGTT, this will familiarize them and reduce the stress of handling. Thore comf

Greg, you are quite correct about better response through oral gavage versus ip or iv route of glucose challenge. Practice for familiarization of animals as well as improving proficiency of the researcher in doing the procedure smoothly, both contribute significantly towards minimizing stress to animals, which causes considerable variations in glucose levels within a group of animals. We have seen these differences between different researchers in my lab - some people were always, for the lack of better terms, more calm themselves and gentle with animals and they consistently produced numbers with less variations. 

The other practical issue is volume of blood one can safely and quickly draw from small animals. To obtain a better curve, initial temptation tends to be to have quite a few time points on the curve. But this is not realistically possible. The usual result ends up showing that it is taking too long to finish drawing blood by the 3rd time point, then one rushes to the next animal in the same group and if the process and times are not carefully planned, the whole experimental scheme starts falling apart. My suggestion will be choose fewer time points, have one additional person to help and do as clean a job as possible to minimize inter-animal variations.  

Exactly Tausif, its all about being calm, confident and efficient. We always perform ogtt's with 2-3 experienced people, take only 3 time pointsand have reduced the sample size, no more than 40ul blood per time point. This gives us just enough plasma for 5ul insulin assay and 5ul NEFA assay. 

Regards

Greg