This document summarizes previous research on the effects of 1,25-dihydroxyvitamin D3 and retinoic acid on PPARγ expression and insulin resistance in diabetes mellitus type 2. It was found that both metabolites inhibit PPARγ expression and adipocyte differentiation. However, past studies only tested the metabolites individually and on differentiated cells, not mixtures or on pre-adipocytes. The proposed research aims to test mixtures of the metabolites on pre-adipocytes and differentiated cells, as well as conduct transactivation studies to further understand how the metabolites modulate PPARγ and insulin resistance.

1. 1,25-­‐Dihydroxyvitamin
D3
and
Re7noic
Acid
Modula7on
of
PPARγ
Expression
and
Insulin
Resistance
in
Diabetes
Mellitus
II
Brian
R.
Covello

2. Diabetes
Mellitus
II
• 25.8
million
people
• By
2050,
1
in
3
US
adults
will
have
diabetes
(Powers,
2005)
• Leading
cause
of
kidney
failure,
blindness,
non-­‐
accidental
amputa7ons
of
legs
(Powers,
2005)
• Insulin
– Beta
cells,
islet
of
Langerhans
in
Pancreas
– Regulates
glucose
metabolism
• Uptake
of
glucose
• Decrease
in
gluconeogenesis
• Insulin
Resistance
à
DM
II
(Powers,
2005)
• Links
to
obesity
(Wajchemberg,
2000)
• TZDs
are
efficient
an7-­‐diabe7c
drugs

3. PPARγ
• Nuclear
Receptor
• Transcrip7on
factors
for
DNA
• Heterodimerize
with
RXR
• Increase
insulin
sensi7vity
(Liang,
2006)
• Increase
adipocyte
differen7a7on
(Liang,
2006)
• Increase
fat
uptake
• PPARγ1
and
PPARγ2
(isoforms)
• Promoter
usage
and
splicing
(Liang,
2006)
• Co-­‐ac7vators,
co-­‐
repressors,
ligands,
phosphoryla7on
(Liang,
2006)

4. Vitamin
D
• Vitamin
D
is
a
fat
soluble
vitamin
• Vitamin
D
insufficiency
correlated
to
obesity
and
DM
II
• UV
à
epithelial
cells
à
Vitamin
D
à
1,25-­‐
dihydroxyvitamin
D3
à
VDR
(Yoshifumi,
1998)

5. Vitamin
A
• Re7nol
A
is
fat
soluble
vitamin
A
• Fat
soluble?
• 3T3-­‐L1
• Once
inside
cells
metabolized
to
Re7noic
Acid
• Inhibitory
Effect
on
PPAR
gamma
(Yoshifumi,
1998)

6. No7ce
Anything?
ß
PPAR
gamma
Re7noic
Acid
1,25-­‐dihydroxyvitamin
D3
• Limited
supply
of
RXR
receptor
• Compe77on
amongst
nuclear
receptors
for
a
common
heterodimeric
partner
(Yoshifumi,
1998)

7. Previous
Research
• Muta7ons
in
PPAR
gamma
cause
insulin
resistance
(Barroso,
1999)
• TZDs
bind
PPAR
gamma
(Gregoire,
1998)
• 1,25(OH)2D3
has
inhibitory
effect
on
differen7a7on
and
prolifera7on
in
3T3-­‐L1
cells
(Yoshifumi,
1998)
– Inhibi7on
of
PPAR
gamma
expression
• Re7noic
acid
also
slightly
inhibits
PPAR
(Yoshifumi,
1998)
• Tests
were
conducted
on
cells
proceeding
through
differen7a7on
process
• Tests
were
under
the
influence
of
PPAR
ligand
TZD

8. Previous
Research
• In
1988
Ishida
et
al
reported
inhibitory
effect
on
prolifera7on
and
differen7a7on
of
3T3-­‐L1
cells
– 67%
decrease
at
10-­‐8M
1,25(OH)2D3
– Significant
decrease
even
at
10-­‐10M
– Exact
mechanism
was
s7ll
unknown
• 1998
Yoshfumi
et
al
showed
1,25(OH)2D3
inhibited
PPAR
gamma
expression
when
bound
to
TZD
– Vitamin
D
insufficiency
à
More
PPAR
à
More
Adipocyte
Differen7a7on
à
Obesity
à
DM
II

9. Gaps
&
Goals
• No
test
of
mixtures
of
metabolites
has
been
conducted
– So
what?
– Mixtures
are
found
in
vivo
• No
tests
were
conducted
on
3T3-­‐L1
cells
during
pre-­‐adipocyte
stage
• No
tests
on
non-­‐ligand
bound
PPAR
have
been
conducted
• No
transac7va7on
studies
have
been
conducted

10. Stage
1
• Test
1,25(OH)2D3
and
Re7noic
Acid
combina7ons
in
pre-­‐adipocytes
Treat
3T3-­‐L1
cells
Control:
No
tx
Dish
1:
1x10-­‐9
M
1,25-­‐dihydroxyvitamin
D3
Dish
2:
1x10-­‐9
M
re7noic
acid
Dish
3:
1x10-­‐6
M
1,25-­‐dihydroxyvitamin
D3
Dish
4:
1x10-­‐6
M
re7noic
acid
Dish
5:
1x10-­‐9
M
each
of
1,25-­‐dihydroxvitamin
D
and
re7noic
acid
Dish
6:
1x10-­‐6
M
each
of
1,25-­‐dihydroxvitamin
D
and
re7noic
acid
Dish
7:
1x10-­‐9
M
of
1,25-­‐dihydroxvitamin
D3
and
1x10-­‐6
M
re7noic
acid
Dish
8:
1x10-­‐6
M
of
1,25-­‐dihydroxyvitamin
D3
and
1x10-­‐9
M
re7noic
acid
Times:
0hours
(control),
8
hours,
20
hours,
40
hours
Hypothesis:
Mixed
concentra7ons
=
Greater
Inhibi7on
&
Less
D3
needed
for
inhibi7on
than
previously
suggested

11. Stage
2
• Induce
differen7a7on
of
3T3-­‐L1
cells
into
adipocytes
• Test
mixture
of
metabolites
on
induced
cells
• Conduct
GDPH
Assay
– Glycerol
3-­‐Phosphate
Dehydrogenase
• Direct
link
to
obesity
and
diabetes

12. Stage
3
• Transac7va7on
studies
• Up-­‐regula7on
– adipocyte
faly
acid-­‐binding
protein
–
acyl-­‐CoA
synthase
– lipoprotein
lipase
– c-­‐Cbl
associa7ng
protein
–
phosphoenolpyruvate
carboxykinase
– faly
acid
transport
protein
– insulin
receptor
substrate
2
(Wajchenberg,
2000)

13. Methods
• Western
Blot
– Protein
Lysate
– Enumera7on
through
spectrophotometry
– SDS-­‐PAGE
à
PVDF
Membrane
– Polyclonal
Ab
and
Secondary
Ab
w/
BCIP
– Counterstain
Ac7n
• Immunofluorescence
– Characterize
structural
changes
(nuclear
receptor)
(Bogazzi,
2007)
(Yoshifumi,
1998)

14. Sources
Barroso,
I.
B.
(1999).
Dominant
nega7ve
muta7ons
in
human
ppar
gamma
associated
with
sever
insulin
resistance,
diabetes
mellitus
and
hypertension.
Le(ers
to
Nature,
402(23),
880-­‐889.
Liang,
G.
L.
(2006).
Peroxisome
proliferator
ac7vated
receptor
gamma
as
a
drug
target
in
the
pathogenesis
of
insulin
resistance.
Pharmacology
&
Therapeu<cs,
111(4),
145-­‐173.
Ishida,
Y.
(1988).
Possible
involvement
of
1,25-­‐dihydroxyvitamine
d3
in
prolifera7on
and
differen7a7on
of
3t3-­‐l1
cells.
Biochemical
and
Biophysical
Research
Communica<ons,
151(3),
1122-­‐1127.
Yoshifumi.
(1998).
Counterac7on
of
re7noic
acid
and
1,25-­‐dihydroxyvitamin
d3
on
up-­‐regula7on
of
adipocyte
differen7a7on
with
ppar
ligand,
an
an7diabe7c
thiazolidinedione,
in
3t3-­‐l1
cells.
Pharmacology
Le(ers,
62(14),
205-­‐211.
Powers,
A.
C.
(2005).
Chapter
323.
Diabetes
mellitus.
In
D.
L.
Kasper,
A.
S.
Fauci,
D.
L.
Longo,
E.
Braunwald,
S.
L.
Hauser,
&
J.
L.
Jameson,
Harrison’s
principles
of
internal
medicine
(16th
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The
McGraw-­‐Hill
Companies,
Inc.
Wajchenberg,
B.
L.
(2000).
Subcutaneous
and
visceral
adipose
7ssue:
their
rela7on
to
the
metabolic
syndrome.
Endocr
Rev
21,
697–738.
Bogazzi,
F.
(2007).
Abnormal
expression
of
ppar
gamma
isoforms
in
the
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7ssue
of
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with
cushing's
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7-­‐12.