The Expression of GAD in Beta Cells of NOD Mice is Required for the Development of Diabetes

1
The Expression of GAD in Beta Cells of NOD Mice
is Required for the Development of Diabetes

• Susannah A. Hill

2
Background

• Type one diabetes in an early onset and insulin
  dependant disease. Diabetes is caused by an
  attack of pancreatic beta cells by specific T
  cells. The disease is characterized by low levels
  of insulin accompanied by high levels of
  glucagon. This hormone imbalance prevents the
  transport of glucose into cells. Without insulin,
  blood glucose remains abnormally high and cells
  are deprived of their main source of energy.
  Without insulin activating its receptor and
  causing a dimerization, autophosphorylation is
  not achieved and GLUT-4 receptors are not
  recruited and presented at the cell membrane.
  Ultimately, serum glucose at very high levels has
  a toxic effect on the body, damaging the renal,
  nervous, and cardiovascular systems. A study was
  conducted by Yoon et al. 1999 to evaluate the
  effect of GAD protein expression on the incidence
  of diabetes.

3
GAD(Glutamic Acid Decarboxylase)

• GAD is a protein produced by beta islet cells and
  also produced in the brain where it is involved
  in the production of GABA. GAD antibodies are
  among the first recognized antibodies in
  pre-diabetic cells.

4
Control of Autoimmune Diabetes in NOD Mice by GAD
Expression or Suppression in Beta Cells

• Yoon et al. 1999

5
Glutamic Acid DecarboxylaseGAD is a protein
produced by beta islet cells and also produced in
the brain where it is involved in the production
of GABA. GAD antibodies are among the first
recognized antibodies in pre-diabetic cells.
6
A Six Part Study

• The expression of GAD is required to develop
  diabetes
• The suppression of GAD is specific
• GADs action is specific to the beta cell
• GAD acts via diabetogenic T cells
• Other B cell autoantigen-specific T cells are
  dependant upon GAD
• GAD suppressed/expressing B cell grafts

7
The expression of antisense GAD was quantitated
with a Southern blot.This gel shows the
concentrations of antisense GAD in high, medium,
low, and transgene negative mouse lines. (Each
lane corresponds to an ear tag )
8
Histological examination of islets in high,
medium, low, and transgene-negative islets
(blacknormal islets, greyT cell infiltration)
9
Conclusion

• These data indicate that a high amount of
  antisense GAD expression is capable of almost
  eliminating the autoimmune response.

10
The expression of GAD is required to develop
diabetes

• Highly anti-GAD transgenic NOD mice did not
  develop diabetes
• Moderate and low amounts of transgene prevented
  diabetes by 33 and 25 respectively
• Conclusion Beta cell GAD expression is required
  for the development of diabetes

11
The incidence of diabetes at various ages is
shown. The highly transgenic group displays a 0
incidence.
12
The suppression of GAD is specific

• Transgenic mice infected with a viral DNA
  developed diabetes
• Transgene-negative mice infected with viral DNA
  also developed diabetes
• Conclusion The prevention of diabetes in
  transgenic mice is not due to the nonspecific
  effect of an antisense transgene

13
GADs action is specific to the beta cell

• Diabetogenic T cells were able to infiltrate into
  the salivary gland of highly transgenic mice
• Conclusion Autoimmunity is specific to beta cells

14
The difference between islet cells of transgenic
(GAD) and transgene-negative lines (note the
severe destruction in F)
15
The salivary gland cells in both transgenic and
transgene-negative mouse lines demonstrate severe
T cell infiltration, indicating the specificity
of GADs action islets
16
GAD acts via diabetogenic T cells

• 0 of mice receiving splenocytes from highly
  transgenic mice developed diabetes
• 90 of mice receiving splenocytes from transgene-
  negative mice developed diabetes
• Conclusion GAD expression is required for the
  generation of diabetogenic T cells

17
Other beta cell autoantigen-specific T cells are
dependant upon GAD

• Immunization of NOD mice with GAD suppresses T
  cell responses to GAD, heat shock protein 60,
  carboxypeptidase H, and peripherin
• Conclusion The suppression of GAD prevents
  immune responses of other auto- antigens as well
  as GAD

18
The splenic T cell proliferative response to
other islet auto-antigens _at_ 8, 12, and 15 wks of
age (B-D) in the presense and absense of GAD
suppression
19
The resilience of GAD-suppressed beta cells to
attack by grafted diabetogenic T cells

• 0 of mice receiving GAD-suppressed islets
  developed diabetes
• 100 of recipients of GAD-expressing islets
  developed diabetes
• Transplanting env-(an antisense proviral DNA),
  caused islet destruction

20
The effect of GAD suppressed and expressing islet
grafts on blood glucose levels in NOD recipients.
21
Antisense-GAD islets are intact in top
photomicrograph while the bottom displays severe
infiltration by mononuclear cells.
22
Conclusions

• The expression of GAD is required to develop type
  1 diabetes in the NOD mouse
• The resistance of GAD suppressed islets is a
  specific effect
• GAD expression is neccessary for the induction of
  diabetogenic T cells
• These CD4 and CD8 T cells cannot act without GAD