Induction of Type 1 Diabetes Using Streptozotocin on C57/Bl6 Mice

Evie Rogkoti Biological Sciences, St. Cloud State University , MN

Abstract: Results:
In this experiment we used 12 C57/Bl6 mice, half of which we treated with a drug called Streptozotocin,
which was supposed to make the mice’ autoimmune system fail. The failure had to do with the metabolism
Body Weights of C57/Bl6
of glucose which they produce from the food they consumed. After treating the 6 mice with the drug and
the other 6 with vehicle (the fluid that the drug dissolves in) for 5 continuous days we tested their blood BODY
glucose levels by bleeding them from the tail vain and we measured their body weights but also fed them BLOOD GLUCOSE WEIGHT
40,0
. T-test Control Day 0 Control Day 7 Control Day 14 Control Day 21
normally, keeping them under control. We measured blood glucose and weighed the mice every 7 days for 4
* * * Treat Day 0 0.48254558
t-testst Control 0
0.802214
Control 7 Control 14 Control 21

weeks and saved their test results which we used in the end to find out if Streptozotocin made them 35,0 Treat 0
Treat Day 7 0.01553156* 0.03611315*
develop Type 1 Diabetes Mellitus. In the experiment, we studied the mice, sacrificed them and collected 30,0
Treat 7

their spleens to do single-cell suspension so that we could examine their blood cells after washing them Treat Day 14 0.049583865* Treat 14 0.0289446*
through the centrifuging machine. The higher their blood glucose was (hyperglycemic) and the lower their 25,0
Treat Day 21 0.051245176 Treat 21 0.00730412*

Body Weight (g)

body weight decreased, the more they became diabetic making our hypothesis come true, that 20,0

Streptozotocin makes the mice’ autoimmune system fail and kills its β-cells which makes it impossible for control treatment
Above are the tables which show the T-tests of the Blood glucose and the body weights that were
their organs to metabolize glucose. In the end the treated mice that became diabetic were sacrificed and 15,0

used for tests along with some controlled, so that they could be compared and lead us to the correct measured once a week for 4 weeks.
10,0

conclusion.
5,0
*the numbers that are statistically significant of the controlled C57/Bl6 mice versus the treated
Introduction: mice on each day. To be statistically significant, the p value has to be bellow 0,05 (p<0.05) at the
0,0
Day 0 Day 7 Day 14 Day 21
same time point.
Type 1 diabetes mellitus (T1D) is a T-cell-mediated autoimmune disorder resulting from the
selective destruction of insulin producing β-cells of the pancreas. Insulin is a hormone that has extensive Discussion:
* p<0.05 compared to controls at the same time point
effects on metabolism and other body functions and is produced by the β-cells of the islets of Langerhans The graph above shows the body weight of the treated C57/Bl6 mice as it began to develop diabetes due to Streptozotocin versus the control mice who were
in the pancreatic follicles. The pancreas is an organ of the digestive and endocrine system that serves two not treated. As seen, as treated mice began to develop diabetes, their average body weights decreased; whereas the average control body weight increased.
Our hypothesis is that by treating the C57/Bl6 mice with the drug called
major functions: exocrine (producing pancreatic juice containing digestive enzymes) and endocrine
(producing several important hormones, including insulin) streptozotocin, they will develop Type 1 Diabetes, which also means that the mice will
Diabetes often goes undiagnosed to people because many of its symptoms seem so harmless. The have the same signs a diabetic organism has. As it is already known, Type 1 Diabetes has
symptoms include: frequent urination, excessive thirst, extreme hunger, unusual weight loss, increased a direct impact on the body weight and the glucose level of the organism that develops
fatigue, irritability, and blurry vision. The symptoms can be seen in mice as well as in humans.
Autoimmunity means the failure of an organism to recognize its own constituent parts as self Glucose Level of C57/Bl6 Mice the disease. In our experiment, the results that we produced were the exact ones that
which allows an immune response against its own cells and tissues. Any disease that results from such we needed which in this case are unusual weight loss and extremely high glucose levels.
an aberrant immune response is termed an autoimmune disease, for example type 1 diabetes mellitus. It
is also known as either juvenile diabetes or insulin dependent mellitus and is now widely known to be an
400
The results the experiment gave us show that:
organ-specific autoimmune disease. 350

In order to understand the pathogenic mechanism in humans and to find treatments for T1D, we 300

The body weights of the control mice are rising (as they should be since the mice are
have used a specific mouse model to demonstrate T1D. In our experiment we used C57/Bl6 mice and 250

* getting older and they are fed normally). However the body weights of the treated mice
Blood Glucose (mg/dL)

treated them with multiple low doses of Streptozotocin, which is a naturally occurring chemical that is * Control Treatment

particularly toxic to the β-cells of the pancreas in mammals. 200

were decreased (ex. from 31.5g it fell to 29,6g even though it was the first week) . So in
150
the case of body weight, our hypothesis came true since people and animals who have
Hypothesis: 100
diabetes lose weight because they cannot produce insulin and do not metabolize the
50

glucose which directly becomes fat that is burned immediately. Because of that, the
Our hypothesis is that 0
Day 0 Day 7 Day 14 Day 21
organism not only does not but on weight, but losses weight instead.
treating mice with
Streptozotocin will cause them * p<0.05 compared to controls at the same time point
On the other hand, the glucose levels of the controlled mice stayed the same, but the
Once again the graph above shows the blood glucose of the treated C57/Bl6with diabetes. We see that as treated mice began to develop diabetes, their average
glucose level increased; whereas the average control glucose level decreased. glucose levels of the treated mice rose significantly since they could not metabolize the
to develop Type 1 Diabetes. glucose they consumed (ex the glucose level of the treated mice was 156mg/Dl on day 7
and rose to 265mg/Dl by day 14). The β-cells were destroyed because of the
Materials and Methods: streptozotocin and without them and their product insulin; the glucose that is consumed
Materials and methods cannot be metabolized.
•Mice
We used inbred C57/B16 mice that kept under non-specific pathogenic free conditions at the animal •Sacrificing the mice and single-cell Discussion:
Conclusion:
lab. These mice were aged matched (2-5 months age), males in body weight from 25-35g. (The suspension
studies were approved by the local Institutional Animal Care and Use Committee)
•Induction of diabetes and the treatments
We opened the mice and collected their
After the tests we completed upon the C57/Bl6 mice, we came to the conclusion that
To induce diabetes in the male mice we used Streptozotocin, injected i.p. at a dose of 40mg/kg body spleens. Ste procedure was completed by we expected. Not only the numbers of the blood glucose rising in the treated-with-
weight (bw) daily for 5 consecutive days. So we injected 6 mice with Streptozotocin and another 6 putting the spleens into PBS, (so they can
injected with only vehicle (the fluid that the drug gets dissolved in). Both treatments started on day
Streptozotocin mice compared to the controlled mice, but also the decrease in the
0 and both treated and controlled animals were monitored regularly. Blood glucose levels sampled
be kept safe) and they were placed on ice. treated C57/Bl6 mice’s body weight gave us evidence that they have the right
from tail vain blood were monitored on a weekly basis using handheld glucometer and mice were Next, we smashed the spleen and placed symptoms to be considered diabetic.
defined as diabetic when blood glucose levels in C57/B16 mice exceeded 10mmol/l , them into 500ml of PBS. By putting the We treated the 6 mice with the drug called Streptozotocin for which we made the
respectively.Mice defined as diabetic were used immediately after becoming overly diabetic.
mixture into the Centrifuging machine, got hypothesis that it will destroy the mice’s β-cells which produce insulin, making their
•To explain better…
At first we had 12 mice that we divided in 2 groups of 6 mice. The mice we used were the rid of the bps, the red cells and some organs unable to metabolize the glucose which would eventually make them diabetic.
C57/B16.The first group was treated with Streptozotocin (Treated group)so that they can become other cells that weren’t needed and By testing and observing both the controlled and the treated mice (so that we can
diabetic and the second group (controlled group) was only being injected with vehicle (the fluid
stayed on the surface, and kept only the compare), we see that our hypothesis was correct and that the treated mice were
that the drug gets dissolved in). For 5 consecutive days the mice were treated by the students and diabetic. So we now know that Streptozotocin, a drug that causes autoimmune
every week they were bled and their body weights were measured. Throughout this first weeks white cells. We repeated the procedure
diabetes to the organism, worked on the mice and made them develop Type 1
procedure we lost one of the controlled mice. The next week the mice were bled and measured for 5 times so that the red cells were
again.We didn’t see any big change in the glucose level on day 7 but on day 14 the numbers of Diabetes by killing the cells that are responsible for the normal glucose levels making
glucose in the treated mice were increased (as we see in the chart). On day 14 we sacrificed 6 mice,
completely washed out and so that we them hyperglycemic.
3 from the controlled group and 3 from the treated group, and we did single-cell suspension. could later work on the white cells
Mouse caging system