Increased cell Proliferation and Reduced Mass Before Diabetes Onset in the Nonobese Diabetic Mouse

1
Increased ß-cell Proliferation and Reduced Mass
Before Diabetes Onset in the Nonobese
DiabeticMouse

• Sreenan, S., A. J. Pick, M. Levisetti, A. C.
  Baldwin, W. Pugh, and K. S. Polonsky. 1999.
  Increased ß-cell proliferation and reduced mass
  before diabetes onset in the nonobese diabetic
  mouse. Diabetes. May 1999 989-996
• Presented by Merlande M. Dieujuste

2
Diabetes mellitus and Type 1 diabetes mellitus

• Diabetes affects the bodys ability to regulate
  blood glucose levels
• There are 4 main types of diabetes
• Insulin dependent diabetes Type 1 is often
  considered an autoimmune disorder
• The inflammation of the islets in the pancreas is
  known as insulitis
• There is no cure for Type 1 diabetes

3
Decline in ß-cells occurs after insulitis or
before diabetes?

• Preventing the malfunction and reduction of
  ß-cells in the pancreas may be crucial in
  preventing Type 1 diabetes
• ß-cell mass as well as their function becomes
  affected as a result of one of the following
• - ß-cell mass and insulin secretion
• progressively declines after insulitis
  occurs
• - A drastic destruction of ß-cells occurs just
• before diabetes

4
Nonobese diabetic-LT/J Mice vs Nonobese
diabetic/Severe immunodeficiency mice

• Nonobese diabetic mice are the best models of
  human Type 1 diabetes
• Female NOD-LT/J mice and NOD/Scid mice chosen and
  matched by age as well as genetic background
• They were studied at 8-9 weeks (non-diabetic), 13
  weeks (non-diabetic), and 18 weeks (diabetic)

5
Preparing pancreas for analysis of ß-cell mass

• Mice were injected with 5-bromo-2 deoxyuridine
  (Brd-U) six hours before euthanization
• 5-7 µm of sections of the pancreas were removed
• ß-cell mass had been analyzed through the
  point-counting morphometry after the following
• ?Insulin primary antibody followed by a
  secondary
• antibody
• ?Sections was developed with 3, 3
  diaminobenzidine (DAB) and counterstained
  with hemotoxylin

6
Analyzing the rate of ß-cell replication

• Brd-U cells as well as the non-ß-cell islets were
  stained to analyze the rate of ß-cell replication
• To detect Brd-U stained cells
• ?Mouse anti-Brd-U antibody followed by anti-
• mouse immunoglobulin G and DAB
• ?To detect the non-ß-cells islets
• ?Cocktail of primary antibodies to glucagon,
• somatostatin, and pancreatic polypeptide

7
ß-cell mass gradually decreases while ß-cell
replication rates increases significantly

• ß-cell mass gradually decreases over time in NOD
  mice
• By 18 weeks, the ß-cell mass of NOD mice was
  significantly lower than NOD/Scid mice
• The rate at which ß-cells replicate was
  significantly greater in NOD mice than NOD/Scid
  mice

8
?-cell mass and ß-cell replication rates of
age-matched NOD mice and NOD/Scid Mice
9
Gradual decrease in ß-cells (A-C) compared to
other islet cells present in the pancreas (D-F)
10
Double staining for insulin and Brd-U as opposed
to double staining for non-ß islet cells and Brd-U

• It is theoretically possible that lymphocytes
  stained by Brd-U could affect result of ß-cell
  replication rate
• A subset of 13 week old pancreas sections of NOD
  mice were double stained for insulin and Brd-U

11
No significant difference between immunostaining
of both non ß-islets and Brd-U cells (A) and
insulin and Brd-U cells (B)
12
Is there a relationship between the changes in
the ß-cell mass and the amount of insulin
secreted from the perfused pancreas?

• Pancreas sections of 13 week old mice were
  perfused with a buffer containing bovine serum
  albumin and variable amounts of glucose
• 20 mmol/l of arginine was administered in the
  presence of 5mmol/l glucose, then 20 mmol/l
  glucose
• Insulin concentrations was measured by a double
  antibody radioimmunoassay using a rat standard
• The slope of glucose potentiation had been
  measured using a one-way analysis of variance

13
Insulin secretion in response to glucose and
arginine progressively declines
14
Does the amount of insulin secreted changes with
an increase in glucose concentrations?

• Glucose concentration was increased in both NOD
  mice and NOD/Scid mice of the same age
• Glucose concentrations was increased from 2
  mmol/l to 26 mmol/l during a 100 minute time-span
• Measurements was made after the first minute and
  after every tenth minute
• 20 mmol/l arginine was added for another 25
  minutes

15
Insulin gradually decreases with an increase in
glucose concentrations
16
ß-cell mass progressively declines at a slow rate

• ß-cells slowly decreases as replication rates
  increase to maintain the appropriate levels
• Secretion of insulin increases as well to
  maintain glucose levels in the blood
• Glucose levels gradually increase, creating toxic
  effects on ß-cell function
• Diabetes occurs when ß-cells can no longer
  replicate in order to maintain their levels, and
  the concentration of insulin secreted declines
• These results may provide a lead to finding a
  method in which Type 1 diabetes can be delayed or
  prevented