I performed an Insulin Sensitivity Test on diabetic and control mice. I noticed that after 30 minutes blood glucose level come back to the levels before the insulin administration and this is happening faster in our diabetic mice. This can't only be explained by the turnover of insulin. Does somebody have an idea? Can it be an uncontrolled activity of glycogenolysis and gluconeogenesis?
In an insulin resistant, diet-induced obese C57Bl6 mouse (>10 weeks high fat diet), insulin (IP bolus; 0.75U/kg) lowers glucose slightly, then it increases above baseline, just as you are describing. You can try a slightly higher dose of insulin, however this makes it impossible to DIO or DM2 mice with lean, normal controls because the normal mice will go hypo with an insulin much higher than 0.75. With this dose, basal of 125-150mg/dL drops to ~80mg/dl in a lean adult C57 male mouse (in my hands anyway).
Anyway, what you are seeing is due to the combined effects of insulin resistance (at liver, muscle particularly), elevated glucagon, and defects in the counterregulatory response. Probably high corts as well. And yes, the increase is largely due to hepatic glucose production (mostly due to glycogenolysis)...but the only way to truly assess this is with a hyperinsulinemic, euglycemic clamp.
My best suggestion, if you cant increase the insulin dose during your ITT, is to calculate the AUC from both the ITT and GTT. Also measure basal (fed), postprandial and fasting glucose levels and insulin if possible (there are elisas that take
In an insulin resistant, diet-induced obese C57Bl6 mouse (>10 weeks high fat diet), insulin (IP bolus; 0.75U/kg) lowers glucose slightly, then it increases above baseline, just as you are describing. You can try a slightly higher dose of insulin, however this makes it impossible to DIO or DM2 mice with lean, normal controls because the normal mice will go hypo with an insulin much higher than 0.75. With this dose, basal of 125-150mg/dL drops to ~80mg/dl in a lean adult C57 male mouse (in my hands anyway).
Anyway, what you are seeing is due to the combined effects of insulin resistance (at liver, muscle particularly), elevated glucagon, and defects in the counterregulatory response. Probably high corts as well. And yes, the increase is largely due to hepatic glucose production (mostly due to glycogenolysis)...but the only way to truly assess this is with a hyperinsulinemic, euglycemic clamp.
My best suggestion, if you cant increase the insulin dose during your ITT, is to calculate the AUC from both the ITT and GTT. Also measure basal (fed), postprandial and fasting glucose levels and insulin if possible (there are elisas that take
https://www.researchgate.net/post/Best_time_points_for_blood_glucose_samples_and_concentrations_to_use_during_an_IP_intra-peritoneal_glucose_tolerance_or_insulin_resistance_test_in_C57B6_mice?exp_tc=tprc
It is also important to note that the half-life of insulin is ~10 minutes in mice (Cresto et al., 1977). The half-life of insulin is ranges 226-314 sec in rats (Canas et al., 1995). Therefore, differences in the glucose concentration after the initial fall (i.e. beyond 30 minutes after the insulin bolus) might not reflect an effect on insulin action. Thus, if the only difference in glucose levels among groups occurs at the 120-minute time point, this is not indicative of an effect on insulin action. The rise in glucose from 30 to 120 min reflects the counterregulatory response to hypoglycemia.
The rise in glucose is due to stress that you induce when you handle the mice. You need to increase the insulin dose to overcome that stress induced rise in glucose and lower glucose. Only if glucose falls will you test insulin sensitivity. Its a common problem, but higher insulin will solve this. You may have to rescue low glucose levels in the wt mice by giving ip glucose, or you can show that a lower dose of insulin reduces glucose to a comparable degree.
When do you remove foods before ITT? You can increase the dose up to 1 U/kg at least. It doesn't cause severe hypoglycemia even to chow-fed mice (BW>30g). Just in case, please prepare 10-20% glucose solution to rescue.
Christoph made a good point. Obese rodents are more sensitive to stress and exhibit a hypersecretion of corticosterone 30 min after even mild handling stress (if not acclimated). To overcome this, I usually prefer to work with extremely well handled mice/rats that have been acclimated to both handling and also to IP injections. Both rats and mice acclimate with biweekly handling and periodic injection over several weeks-a few months. I realize this is not always practical, particularly for shorter term studies. Both rats and mice will acclimate with 5-7 days of handling and saline injections. Rats should handled (a lot like you would handle a pet) for several minutes 2x/day. Mice should be acclimated to gentle scuffing and can be kindly handled 1x/d for 1 week. :)
What was the type of insulin and type of insulin administration (ip, iv, ...) ?
The liver plays a important role in blood glucose homeostasis by maintaining the balance between glucose input and output . Insulin inhibits hepatic glucose
output, suppressing gluconeogenesis and glycogenolysis, by inhibiting expression and activity of the key enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase. The insulin resistance is associated with insulinopenic and hyperinsulinemic conditions.
Glucose regulation is driven dissipative chaotic oscillator. With two feedback loops it must be so. Any unusual impulse will cause instability. Insulin without C-protein and GABA is an unusual impulse. Expect the system to bounce. This is normal behaviour for such a system.
If you want a visual image of such a system attach a pendulum to a springy support and watch what happens.
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