I have posted this information in response to a question elsewhere. I have copied it here as I thought you might find it useful. Most importantly it outlines the parameters you need to be aware of/controlling for STZ to work efficiently and consistently. Get these right and it is a very reliable model.

STZ has two intracellular effects accounting for it’s cellular toxicity: DNA alkylation and NAD+/NADH depletion by a PARP dependent mechanism, the latter being suggested to be most important in beta cell toxicity. This matters as it demonstrates that STZ will be toxic to any cell within which it achieves adequate intracellular concentrations.

STZ is taken up into cells by GLUT2, differential expression of GLUT2 by different tissues explaining much (but not all) of the differential tissue toxicity and the well described strain differences. It is a very toxic compound so effective use requires a concentration to be found that results in a toxic concentration in beta cells but a non-toxic concentration in the other tissues that it will enter. In our early use of STZ (20 years ago!) we performed autopsies on animals that died and found liver and kidney to be particularly affected, which is not surprising now that we know about GLUT2 mediated uptake. If the dose is too high widespread toxicity will be seen. Unfortunately the safe and effective window is quite narrow. For mice, we titrate new batches in 20mg/kg steps. Rats require lower doses so 10mg/kg steps may be needed. So the window is small.

This is all fairly basic stuff. What is crucially important is that, just like glucose, STZ exists in α and β anomeric forms. This is not the same as the D/L enantiomers where the whole molecule is a mirror image, but defines the position of a single hydroxyl group: often referred to as up or down! So this is all D-glucose/STZ with two anomers. This is important for a couple of reasons. GLUT2 only transports α glucose and therefore similarly only transports α STZ. It is therefore the concentration of the α anomer that is important in determining the toxicity of a particular preparation. The commercially available preparations have a defined composition, typically approximately 75% α anomer, in the solid compound.

However, as with all such compounds, the α anomeric and β anomeric forms will equilibrate in solution, so as soon as STZ is dissolved equilibration begins. Much of the degradation of STZ is in fact this equilibration with loss of the α anomer with resulting loss of efficacy. True degradation is slow. Early work describes a half life of about 19 minutes. This started to define the rate of equilibration in solution with the functional loss of efficacy relating to loss of α anomer as a result of equilibration. The de la Garza-Rodea paper is of course crucial in this regard and describes this chemistry definitively. What this publication does not take into account is the wider aspects of the biology. From memory I think they used the same does of STZ for immediate administration and delayed administration. Given the above these preparations will inevitably and predictably have different efficacy/toxicity. At equilibrium there is a slight excess of β anomer. The rate at which equilibrium is achieved is of course affected by temperature and the buffer used. So aqueous and citrate buffer, pH4-7, solution at room temperature or kept on ice and any time point for injection from 10 minutes to 3 hours are all possible. What will differ is the percentage of the initial α anomer that is still present in solution and therefore the toxicity of the particular prep at that particular time. STZ equilibrates particularly quickly in aqueous solutions at room temperature, much less quickly in citrate buffer at pH 4-4.5 on ice. The problem with the 2-3 hour equilibrated protocol is that this preparation will have approximately half the concentration of ‘active’ α anomer compared to fresh STZ. Hence the lower incidence of diabetes in many peoples hands and the suggestion that multiple doses are needed. Diabetes can be consistently and reliably induced with a single dose if all the above parameters are considered when deciding on the protocol for your particular experiments. Given your logistic constraints I can see that the equilibrated protocol might work best but do make sure that you work out the correct dose.

I mention this as it is crucially important to recognise these differences as the dose of STZ used by one person may not be right in your model with your logistic constraints.

I should add that the nutritional status of the animal affects the response. This is obvious from the above as α D-glucose will of course compete with α STZ for uptake and the amount of glucose ‘available’ will therefore impact on beta cell toxicity. Administration of carbohydrate post injection has been advocated to counter the hypoglycaemia that can be seen with insulin release from targeted beta cells. This may be true (although we have not found this to be a big problem with correct doses and may relate to overdosing with hepatic toxicity preventing also counter regulatory mechanisms). This post dose carbohydrate may be more important in preventing ongoing toxicity. This was important in our experiments as we were transplanting islets so needed to avoid toxicity to the transplanted tissue. We found transplanted beta cells to be fine at 3 days post injection of STZ. What matters is that this variable is controlled also in your protocol. I would suggest that the best way is to fast animals overnight as discussed elsewhere on Research Gate at length. This may be a problem for the multiple dose regimens.

So in summary it is all about understanding the biology and chemistry and as a result getting the dose of the α anomer right for your model.

Hope this helps

what kind of rat model are you using?

from what I know 150mg/kg B.W is way too high. I induced mine at 45-50mg/kg B.W. And we had starved the rats for nearly 6-8 hours.....or overnight.

I too had problems initially. I felt that the important criteria to induce diabetes is its nutritional status. If you understand the logic behind the STZ mode of action you'll understand why.

Additionally it largely depends on the strain the rats you're using. Check the literature on how to induce diabetes with the strain you're using.

All the best.

For type 1 diabetes the dose 40 mg/kg for 5 consecutive days is sufficient but you have to do study within 4 weeks after STZ injection. ( Note: STZ is a light sensitive it will not work if slight exposure to light just check ). if you want to develop Type 2 than you select neonatal induced diabetes model, these neonatal animals slowly develop diabetes after 6 weeks you check glucose level with doing OGTT. If you want to see CNS related clinical signs than you have to wait for long time more than 10 weeks, in this period than animals slowly converted into Type 1 diabetes. My suggestion is Diabetes with obesity is a good model for CNS related studies.

for this you just inject STZ in SD rat pups after weaning the animals fed with high fat diet for 8 weeks or 12 weeks than you see the clinical signs.

We use NOD/SCID or NSG male mice (6-8weeks) for Type 1 induction. STZ was purchased from Sigma. Dose used 120mg/kg after preliminary titration from 100-180mg/ml. The mice turned diabetic (above 20mM) after 4-7 days. STZ was dissolved in 0.1M Citrate buffer (pH4.5) freshly and passed through 0.22uM filter prior to injection (within 15 minutes). We have consistent (90%) take rate.

thanks a lot to all of you who have written in. Indumati and mallikarjun I think it is a bit more tricky when using mice. I believe rats are a lot easier to work with. I do try and keep it well protected from light. Undoubtedly the strain and environmental conditions play a role in induction of all kinds of conditions using drugs.

Hooi, 2 weeks daily injection seems a bit much but it works for you! I have seen people inject for 2 weeks but every alternate day. There is a lot of variation in the doses and induction regimen when you look at the literature. I think I will have to increase the number of injections from 5 at the 50mg/kg dose.

Shu, we have tried a 120mg/kg single injection but not even a single animal got a blood sugar above 7mMol/L. Possibly your strain is NOD/SCID more amenable to this dose? It appears 150 is too high and 120 is too low in our case.We have C57Bl6+CBA background i think a lot more trial and error will ne needed before we can get something going. I think I am going to throw in the towel and get a trangenic line possibly a db/db or ob/ob mouse.

thanks a bunch again...

Some mice require 10 ip injections to become chronically diabetic. We found this to be true in almost all of our female mice regardless of strain. I would suggest you try again with the low dose protocol and increase the number of doses. Most of our animal strains are on the C57BL6 background.

Thanks patricia, i think i may have to increase the injections for sure.

Richa we did study by injecting STZ 50 mg/kg 5 consecutive days to SAM mice three times we get good induction after 10 days.

Single doses work perfectly well if the right dose is used. 150mg/kg is very high for rats as above. However, I note you had a low incidence with 120mg/kg so I wonder about the details of your protocol. The dose needed does depend how long after you make up the STZ it is injected and what buffer you use. If you look at my Research Gate page you will see multiple answers on STZ use including discussion of mechanisms of action, stability, buffers etc. Did you starve the animals overnight? This can make a big difference due to competitive inhibition of STZ uptake into beta cells.

I have posted this information in response to a question elsewhere. I have copied it here as I thought you might find it useful. Most importantly it outlines the parameters you need to be aware of/controlling for STZ to work efficiently and consistently. Get these right and it is a very reliable model.

STZ has two intracellular effects accounting for it’s cellular toxicity: DNA alkylation and NAD+/NADH depletion by a PARP dependent mechanism, the latter being suggested to be most important in beta cell toxicity. This matters as it demonstrates that STZ will be toxic to any cell within which it achieves adequate intracellular concentrations.

STZ is taken up into cells by GLUT2, differential expression of GLUT2 by different tissues explaining much (but not all) of the differential tissue toxicity and the well described strain differences. It is a very toxic compound so effective use requires a concentration to be found that results in a toxic concentration in beta cells but a non-toxic concentration in the other tissues that it will enter. In our early use of STZ (20 years ago!) we performed autopsies on animals that died and found liver and kidney to be particularly affected, which is not surprising now that we know about GLUT2 mediated uptake. If the dose is too high widespread toxicity will be seen. Unfortunately the safe and effective window is quite narrow. For mice, we titrate new batches in 20mg/kg steps. Rats require lower doses so 10mg/kg steps may be needed. So the window is small.

This is all fairly basic stuff. What is crucially important is that, just like glucose, STZ exists in α and β anomeric forms. This is not the same as the D/L enantiomers where the whole molecule is a mirror image, but defines the position of a single hydroxyl group: often referred to as up or down! So this is all D-glucose/STZ with two anomers. This is important for a couple of reasons. GLUT2 only transports α glucose and therefore similarly only transports α STZ. It is therefore the concentration of the α anomer that is important in determining the toxicity of a particular preparation. The commercially available preparations have a defined composition, typically approximately 75% α anomer, in the solid compound.

However, as with all such compounds, the α anomeric and β anomeric forms will equilibrate in solution, so as soon as STZ is dissolved equilibration begins. Much of the degradation of STZ is in fact this equilibration with loss of the α anomer with resulting loss of efficacy. True degradation is slow. Early work describes a half life of about 19 minutes. This started to define the rate of equilibration in solution with the functional loss of efficacy relating to loss of α anomer as a result of equilibration. The de la Garza-Rodea paper is of course crucial in this regard and describes this chemistry definitively. What this publication does not take into account is the wider aspects of the biology. From memory I think they used the same does of STZ for immediate administration and delayed administration. Given the above these preparations will inevitably and predictably have different efficacy/toxicity. At equilibrium there is a slight excess of β anomer. The rate at which equilibrium is achieved is of course affected by temperature and the buffer used. So aqueous and citrate buffer, pH4-7, solution at room temperature or kept on ice and any time point for injection from 10 minutes to 3 hours are all possible. What will differ is the percentage of the initial α anomer that is still present in solution and therefore the toxicity of the particular prep at that particular time. STZ equilibrates particularly quickly in aqueous solutions at room temperature, much less quickly in citrate buffer at pH 4-4.5 on ice. The problem with the 2-3 hour equilibrated protocol is that this preparation will have approximately half the concentration of ‘active’ α anomer compared to fresh STZ. Hence the lower incidence of diabetes in many peoples hands and the suggestion that multiple doses are needed. Diabetes can be consistently and reliably induced with a single dose if all the above parameters are considered when deciding on the protocol for your particular experiments. Given your logistic constraints I can see that the equilibrated protocol might work best but do make sure that you work out the correct dose.

I mention this as it is crucially important to recognise these differences as the dose of STZ used by one person may not be right in your model with your logistic constraints.

I should add that the nutritional status of the animal affects the response. This is obvious from the above as α D-glucose will of course compete with α STZ for uptake and the amount of glucose ‘available’ will therefore impact on beta cell toxicity. Administration of carbohydrate post injection has been advocated to counter the hypoglycaemia that can be seen with insulin release from targeted beta cells. This may be true (although we have not found this to be a big problem with correct doses and may relate to overdosing with hepatic toxicity preventing also counter regulatory mechanisms). This post dose carbohydrate may be more important in preventing ongoing toxicity. This was important in our experiments as we were transplanting islets so needed to avoid toxicity to the transplanted tissue. We found transplanted beta cells to be fine at 3 days post injection of STZ. What matters is that this variable is controlled also in your protocol. I would suggest that the best way is to fast animals overnight as discussed elsewhere on Research Gate at length. This may be a problem for the multiple dose regimens.

So in summary it is all about understanding the biology and chemistry and as a result getting the dose of the α anomer right for your model.

Hope this helps

Further to my previous post I have attached some data to support the statement that single doses work. These are data in C57BL6/J mice not rats, but given the comment above with regard to BL6 mice thought this might be of interest. For mice we titrate new batches of STZ in 20mg/kg steps. I have attached data from one such experiment. The formatting has scrambled a bit but the Y-axis is fasting glucose at day 3 post injection. The other important data is that 40% mortality was seen in the 180mg/kg group and no mortality was seen in any other group. All mice in the 160mg/kg group remained diabetic for >100 days. You will see that the dose window is quite small. With such a narrow window control of the parameters mentioned above is clearly critical. However get this right and single doses work well.

Dear Richard thank you for taking the time and re-posting this stuff to my question. I appreciate it tremendously.

I know some painfully accurate titrations will have to be done but after a few attempts one's enthusiasm begins to wane especially when i was looking for a quick and easy methods and this is far from what my lab really does :( I will try and persevere.

I fast the animals 4 hours before single or multiple injections. I do provide them with sucrose water following injections. I use the citrate buffer (pH 4.5) at room temp. Weigh the drug (with foil on tube) take it to animal room add predetermined amount of buffer , dissolve (1-2 mins) and inject from 1st mouse to last within 15 mins. I weigh all the mice and calculate volumes before-hand. I do not foil cover the syringe because i draw up 3 doses in the same syringe so i have to be able to observe the markings, may be i should do it individually and cover the syringes too.

hopefully 2014 will be more successful than 2013 was...

Hang on in there! Protocol sounds pretty good. Do not worry about foil covering the syringe. We never do and if you inject three animals per syringe full and all are done within 15 minutes this will NOT be a reason for failures. This does become a quick and easy method once you have the protocol worked out. I think you may need to starve for more than 4h. We routinely do overnight starve. Just take chow away last thing at night and do injections next morning. As you have seen we do not find we need sucrose post injections as hypoglycaemia has not been a problem. It might reduce efficacy as the glucose released will inhibit ongoing STZ uptake. In theory this would simply affect the dose of STZ you need to use in your protocol so once dose worked out would not be a problem but I worry that the intake might vary and therefore introduce an uncontrolled variable into the protocol. There is no right or wrong for this one, but important to be aware of potential effects. You can see the approach needed to get this right: know the variables affecting your model, control these, avoid the urban myths!

We also do not cover the syringes with foil. As long as the injections are completed within 10-15 minutes of dissolving the STZ in buffer you should be fine. We have never starved our mice prior to injection.

Dear Richard Smith, can you upload again the STZ graph but instead of word document do it with pdf file? I could not open the file you shared. Thanks

Mam, from my point of view first of all it is not good to use direct dose of STZ for inducing dibetic , because just increasing blood glucose cant be called diabetic without checking the insulin level and even lipid level , because this diabetic rat model cant mimic the human diabetic model , because mostly hyperlipedimic human are more prone to diabeties , so its better to make rat first hyperlipedimic by giving high fat diet along with STZ,.

I agree! As much discussed on Research Gate, STZ induced diabetes only reproduces the hyperglycaemia and so is not T1DM and certainly not T2DM. I advocate use of the term STZ induced diabetes. However, if we think carefully about the scientific question we are asking this may be fine. If the only question is whether a given treatment lowers blood sugar then this model may be OK, remembering of course that insulin resistance and partial beta cell function may not be replicated.

Indeed, as both of you state the use of any model to study any human disease has its shortcomings. Also comorbidities often play a role. I have to say i am looking to into some other genetic mouse models to see if i can get into that.

So, what model could reproduce type 1 diabetes?

Our lab routinely use the 5x50mg/kg dose STZ to induce diabetes in male C57BL6 mice. We make up the STZ daily as the compound breaks down rapidly and thus is not as diabetogenic

see the link already discussed: https://www.researchgate.net/post/Can_I_dissolve_Streptozotocin_STZ_in_distilled_water_to_induce_diabetes_in_rats