It seems that you dystroyed the most beta cells and post prandial glucose control. But, some of beta cells are still working normally. However, because of woking islets are lesser than the physiologic state, they need to more time to postprandial glucose control.

Habib, thank you for your suggestions, however it almost 3 weeks and it has been the same for almost 3 weeks

Dear Opeyemi J Olatunji What you have experienced in those diabetic rats is a common phenomenon in drug (alloxan/streptozotocin)-induced diabetes in rodents. It is a major drawback of drug-induced diabetes. This occurred as a result of regeneration of the beta cells of the pancreas. That is why it is very important to always utilize an appropriate dose for induction of diabetes.

I think the problem arose from the dose you administered. If you search the literature, you will observe the optimal dose of streptozotocin for successful induction of diabetes is 55-65 mg/kg. Any dose less than that increases the rate of pancreas regeneration in the rats leading to normalization of blood glucose while a dose greater than that may likely result in severe hyperglycaemia and leading to increased mortality rate.

Dear Omotayo Erejuwa, Thanks so much, however, there are also several literatures that utilizes HFD and a low dose of STZ (25-40 mg/kg) to induce diabetes. As a matter of fact all my previous antidiabetic studies using STZ was a combination of HFD and low dose STZ (35 mg/kg) and the models were successful without this kind of experience

Dear Opeyemi J Olatunji The animals you used previously were not the same as the ones in this your current study. Just like it is in humans so also in animals. Biological variations do exist in humans so also in rodents.

Dear Opeyemi Olatunji, what you observed is a common phenomena with both alloxan and streptozotocin induced models of diabetes (though greater in alloxan model of diabetes).

Key things to consider when administering STZ to experimental animals:

Timeliness of administration: Due to the instability of STZ in solution, it is recommended to administer it within 10 mins of dissolution (and protected from sunlight) and the potency of STZ (in solution) decreases with increase in time. If you did not adhere strictly to this, you are bound to experience variations such as you saw in the diabetogenic effect of STZ in the rats.

Type of buffer and pH of the buffer solution in which you dissolved the STZ: From my experience, I found dissolution of STZ in citrate buffer more effective than in normal saline when administered to experimental rats. In my review paper, I reported that for optimum results, STZ is best to be administered at fasting

state and freshly prepared, dissolved in citrate buffer (pH 4.4-4.5).

Another thing that can also lead to variations in the diabetogenic effect of STZ as you experienced is the age of the animals. If there are variations in the age of the animals administered STZ, then you will also expect variations in its diabetogenic effect.

From your explanations, it is either that the treatment is negatively impacting on their blood glucose (and as a way of validation, I suggest you check the glycemic effect of the treatment on normal rats) or the animals were not in the same diabetic state at the beginning of the experiment. According to you, majority of the animals had blood glucose levels >350 mg/dL three days post STZ induction. But you did not state the upper and lower limits.

Finally, given the low dose of STZ you administered (40 mg/kg), the beta cells of some of the animals in your diabetic model may have regenerated, leading to lower glycemic values than their earlier values.

Lastly, if all of the things I mentioned were taken care of by you, then it is also a possibility that the STZ you are using is getting weak (although you did not state if it was newly purchased or if you have been using it for sometime).

Dear Chinedum Ogbonnaya Eleazu, Thank you so much for the comments, well going by what you said, the STZ was freshly purchased, dissolved in freshly prepared citrate buffer pH 4.5. In addition, 36 rats were induced with diabetes and this phenomenon was observed in only 1 group, which is the group that received no treatment. All other groups had none of these variations. Most of the comments you mentioned were adhered to, the upper limit was 485 mg/dl and lower limits (356 mg/dl) 3 days post STZ administration.

For diabetic groups, you have to check the FBG after 7 days of induced diabetic. if the FBG is still in the normal rate then you can induced diabetic again. but I would like to suggest to reduce the dose of STZ. please, refer the below article

https://www.sciencedirect.com/science/article/abs/pii/S096399692030572X

Dear Opeyemi J Olatunji . As you mentioned, everything was done by the book. Though, do not forget that it is a type 2 and not a type 1 diabetes model. This means that fasting glucose levels will be little above normal levels and postprandially will be high. If, after fasting, glucose seems normal and does not depict grey zone levels (120-140 mg/dL) apply a dexamethasone stimulating test, measure an area under the curve (AUC) and compare with other groups. Another solution is insulin to be measured but it is not costless.If there is still variabillity, the particular group could be exluded from the study and repeat the procedure. Let us not forget that β cells can be regenerated, especially at low streptozotocin doses and streptozotocin models in general may give variabillity in results. Last but not least, a fasting glucose level of 150 mg/dL is not characterized as normal according to the American Diabetes Association.

I have different idea about induce diabetes model using alloxan and STZ, first you have to specify which model you need? Type 1 or 2. Second both alloxan and STZ causing injury of beta cells and this might be reversible or irreversible injury (Recovery), which depend on dose and duration, justifying the normal results. Third neither alloxan nor STZ induce type 2 model, since most causes of type 2 are secondary besides the genetic causes, therefore it is difficult to indice type 2 DM model.

Ioannis Margaritis A rats with a fasting blood glucose of 150 mg/dL (even 200 mg/dL except a mild diabetes model is desired) is not considered diabetic. The diabetes status in rodents is a bit different from that of humans. ADA classification refers to humans.

Amar Mohamed Ismail A low dose of alloxan or streptozotocin combined high fat and/or high carbohydrate/sugar (preferably sucrose or fructose) diet can be used to develop type 2 diabetes model.

Opeyemi J Olatunji As it is you cannot make any valid scientific conclusion on the effect of your treatment on diabetes because your treatment needs to be compared with the control.

If I were you, I would discard the diabetic control rats and take another set of new animals and then induce diabetes afresh. I would randomize them into all the groups including those receiving treatment but the diabetic control group will have more of the new diabetic rats because of obvious reason. All the best.

Opeyemi J Olatunji

I will suggest that you look into your experimental design again and see what you did not get right. Just a tip, you first of all induce diabetes, and then select the diabetic rats to work with. Remember that you will need to allow some time to monitor those that would later become diabetic.

BW,

Egbuna

Dear Omotayo, type 2 patients have higher insulin level but the insulin dependent cells not respond to insulin, due to recycling of the resptors, trafficking of GluT4, and insulin resistance. Since both Alloxan and STZ caused beta cells injury reducing insulin level this is just induced hyperglycemia, therefore not considered as type 2 DM.

Low dose of STZ partially destroys beta cells therefore the cells can regenerate resulting in the normalization of blood glucose levels.

Dear Amar Mohamed Ismail Kindly re-read my response. I wrote that the combination of alloxan/streptozotocin and high fat/sugar diet can be used to induce type 2 diabetes. Alloxan/streptozotocin induces beta cell dysfunction while high fat/sugar diet induces impaired glucose tolerance and insulin resistance. These are the main features of type 2 diabetes mellitus.

Dear Omotayo thank you, high fat and sugar diet causing obesity and insulin resistance which lead to impaired glucose tolerance as you said, this is a not type 2, since any hypolipidemic drug, exercise and control diet lowering blood glucose. Therefore any drugs used for dyslipedemia and obesity, have hypoglycemic effect but not considered as antidiabetic. Please this is my email: [email protected] we need more discussions.

It is not surprising to see odd results in such experiments on animals, simply because of variable response to different stress situations. However, most of induced D M in experimental models are of type I diabetes, which produce unexpected responses as we have noticed in our experience using alloxan as diabetes inducing chemical.Fasting glucose is regulated by insulin produced outside the pancreas ( as recent studies have shown), which takes longer time to reduce blood glucose that after loading glucose by diet. This may the results you have seen with STZ in your study.

Dear Amar Mohamed Ismail It does not seem you responded to my post.

This is what I wrote '

I wrote that the combination of alloxan/streptozotocin and high fat/sugar diet can be used to induce type 2 diabetes. Alloxan/streptozotocin induces beta cell dysfunction while high fat/sugar diet induces impaired glucose tolerance and insulin resistance. These are the main features of type 2 diabetes mellitus.'

Where did I mention obesity?

What is insulin resistance?

What is type 2 diabetes?

What differentiates type 1 diabetes from type 2 diabetes?

Dear Amar Mohamed Ismail Kindly go through these articles

The Characterization of High-Fat Diet and Multiple Low-Dose Streptozotocin Induced Type 2 Diabetes Rat Model https://www.hindawi.com/journals/jdr/2008/704045/

Evaluation of type 2 diabetic mellitus animal models via interactions between insulin and mitogen‑activated protein kinase signaling pathways induced by a high fat and sugar diet and streptozotocin

Dear Omotayo Erejuwa I really like to exchange opinions with everyone in here because we all get better. In the case of glucose values, 150 mg/dL is not considered diabetic per se, unless a glucose tolerance test proves insulin resistance or it could be pre-diabetes. That is the reason why I mentioned that Opeyemi J Olatunji should proceed so. The model he used, is for type 2 diabetes induction, so glucose values close to normal are not rare in this type of protocol. Furthermore Masiello et al., (1998) characterizes rats with glucose values of 155 mg/dL as hyperglycemic. If the researcher wants clearly diabetic animals, then animals with values lower than 180mg/dL could be excluded from the study. I forgot to mention that human glucose meters that are mainly used for the first glucose concentration determination of an experiment, show less than real values because they are designed for human red blood cells and not for rats. So a value of 150mg/dL in the human glucosemeter could be 170mg/dL in reality. To sum up a fasting glucose value of 150 mg/dL is for further investigation and not necessarily normal.

Dear Ioannis Margaritis there is something you did not get. Opeyemi encountered a problem in the diabetic control only. Other groups had higher blood sugar levels.