Thiazolidinediones and Bone
Metabolism: in-vitro studies
AMIT SETH, ASHUTOSH PAREEK, VANESSA SY,
PAULINE SUWANDHI, ZEV ROSENWAKS, DONNA
SETO-YOUNG, LEONID PORETSKY
Contents
 Background



Thiazolidinediones
Thiazolidinediones, Aromatase and Estrogen
Bone turnover
 Bone turnover and thiazolidinediones


Clinical studies
In-vitro and Animal studies
 Current project



Hypothesis
Methods
Results
Thiazolidinediones
Thiazolidinediones (TZDs)
 TZDs bind peroxisome-proliferator activator receptor-(PPAR-)
 Once cis-retinoic acid binds RXR, the PPAR-RXR heterodimer
undergoes a conformation change activating peroxisome proliferator
response elements, leading to gene transcription.
 The activated genes include those involved in glucose and lipid
metabolism.
TZD, Aromatase, and Estrogen
 TZDs inhibit estrogen synthesis
 In the enzyme kinetics study, TZDs inhibit Vmax but not Km
of aromatase, acting as non-competitive inhibitors
 TZDs have no effect on aromatase mRNA or protein
expression, suggesting no effect on gene transcription or
protein translation.
Rosiglitazone and Pioglitazone Alter Aromatase Kinetic Properties in Human Granulosa Cells. Araki T, Varadinova M, Goldman M, Rosenwaks Z,
Poretsky L, Seto-Young D. PPAR Research. Accepted September 2011. In Press.
Rosiglitazone and pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase. Seto-Young et
al. Hormone and Metabolic Research. 2011 Apr;43(4):250-6
Interactions among peroxisome proliferator activated receptor-gamma, insulin signaling pathways, and steroidogenic acute regulatory protein in
human ovarian cells. Seto-Young D, Avtanski D, Strizhevsky M, Parikh G, Patel P, Kaplun J, Holcomb K, Rosenwaks Z, Poretsky L. J Clin Endocrinol
Metab. 2007 Jun;92(6):2232-9. Epub 2007 Mar 20.
Direct thiazolidinedione action in the human ovary: insulin-independent and insulin-sensitizing effects on steroidogenesis and insulin-like growth
factor binding protein-1 production. Seto-Young D, Paliou M, Schlosser J, Avtanski D, Park A, Patel P, Holcomb K, Chang P, Poretsky L. J Clin
Endocrinol Metab. 2005 Nov;90(11):6099-105. Epub 2005 Aug 30.
Aromatase
TZDs Inhibit Estradiol Synthesis
Seto-Young et al. Hormone and Metabolic Research. 2011 Apr;43(4):250-6
Estrogen and Bone Fragility
 Menopause is associated with osteoporosis and
decreased estrogen levels
 Studies of aromatase inhibitors for the treatment of
breast cancer show that letrozole, exemestane and
anastrozole induce a decline in bone mineral density
(BMD) and increase risk of fracture
Khosla S, 2010 J Clin Endocrinol Metab 95:356-357.
Bone Metabolism
TZDs and Bone Metabolism –
Clinical Studies
Short Term Treatment with Troglitazone Decreases
Bone Turnover in Patients with Type 2 DM
 33 diabetic patients (17 female, 16 male)
 Troglitazone 400mg/day
 Duration : 4 weeks
 Result : reduction of both serum total and bone-
specific alkaline phosphatase (AP)
Okazaki R et al, 1999 Endocrine Journal 46(6):795-801.
Thiazolidinedione Use and Bone Loss in Older
Diabetic Adults
 666 diabetic participants, 83 on TZDs. Age range 70-79 years.
 Analyzed data from the Health, Aging, and Body Composition
observational study
 22 on troglitazone, 30 on pioglitazone and 31 on rosiglitazone
 Duration : 4 years
 Result :

Reduction of bone mineral density (BMD) 0.67% per year
in women
Schwartz AV et al, 2006 J Clin Endocrinol Metab 91(9):3349-54.
Rosiglitazone Decreases Serum Bone-Specific Alkaline
Phosphatase Activity in Postmenopausal Diabetic Women
 56 obese postmenopausal, newly diagnosed DM
women vs 26 non-DM healthy control
 DM participants were divided to 2 groups :

Treatment with rosiglitazone arm vs diet arm
 Rosiglitazone
 12 weeks
 Results
 Reduction of serum total and bone-specific AP with treatment
with rosiglitazone
 No change in osteocalcin
Berberoglu Z et al, 2007 J Clin Endocrinol Metab 92(9):3623-30
Rosiglitazone-Associated Fractures in
Type 2 Diabetes
An Analysis from A Diabetes Outcome Progression Trial (ADOPT)
 1,840 women and 2,511 men from 488 centers, 17
countries (645 women & 811 men on rosiglitazone)
 Age range 30-75 years old, mean age 56.1 – 57.0
 4 years
 Results


Increased cumulative incidence of fractures in women
treated with rosiglitazone (15.1%) compared with 7.3% with
metformin, 7.7% with glyburide.
No difference in men
Kahn SE et al, 2008 Diabetes Care 31(5):845-51
Effect of Rosiglitazone, Metformin, and Glyburide on
Bone Biomarkers in Patients with Type 2 Diabetes
 1605 participants from ADOPT study
 1 year
 Results
 Women:
↑ osteoclast activity marker – C-terminal telopeptide for type 1
collagen (CTX)
  osteoblast activity markers : Procollagen type 1 N-propeptide
(P1NP) and bone AP


Men:

No change in osteoclast activity marker but  osteoblast activity
markers
Zinman B et al, 2010 J Clin Endocrinol Metab 95(1):134-42
In-vitro and Animal Studies on
TZDs and Bone Metabolism
In-vitro Studies
1st author
Johnson (1999)
In-vitro (Mouse
Osteoblast Cells)
TZD
Effect
Effect on Alkaline
Phosphatase (AP)
and/or osteocalcin
Rosiglitazone
(10, 25 and 50μM)
 Gene transcription
 AP
differentiation to
adipocytes
Johnson TE, et al. 1999 Endocrinology 140:3245-3254
Animal Studies
All the studies demonstrated reduced bone mineral density (BMD) and
increased fat content histologically.
1st author
TZD
Effect
Effect on
biomarkers
Soroceanu (2004)
Animal study
(mice)
Rosiglitazone
3mg/kg
 osteoblast apoptosis
 of osteoblast population
No change in osteoclast activity
No effect in AP in
serum, but  in AP
activity on staining
Rzonca (2004)
Animal study
(mice)
Rosiglitazone
20mg/g/day
 osteoblast-specific gene
expression
Not tested
Afshan Ali (2005)
Animal study
(mice)
Rosiglitazone
25mg/g/day
 of osteoblast differentiation
and bone formation
Not tested
1. Sorocéanu MA, et al J Endocrinol. 2004 Oct;183(1):203-16.
2. Rzonca SO, et al Endocrinology. 2004 Jan;145(1):401-6.
3. Ali AA, et al. Endocrinology. 2005 Mar;146(3):1226-35.
Hypothesis
 TZDs inhibit bone metabolism through:

Aromatase inhibition

Direct effect on osteoblasts/osteoclasts
Objective
 To examine the effects of TZDs on mouse osteoblast cells
(MOB) alone or co-cultured with human granulosa cells
(HGC)

Cell growth

Cell differentiation

Bone turnover markers : AP, Osteocalcin, FGF-23, and
Receptor Activator of Nuclear Factor Kappa-B Ligand
(RANKL)
 To examine whether aromatase inhibition plays a role in
any of the TZD effects on mouse osteoblast cells (MOB)
Methods
 Commercially available mouse osteoblast cell (MOB) line,
7F2 from American Type Culture Collection (ATCC) was
cultured with or without human granulosa cells (HGC)
 Cells were then incubated with





Pioglitazone 5, 10 and 25 mM
Rosiglitazone 5, 10 and 25 mM
Testosterone 1mM
Testosterone 1mM and pioglitazone 5, 10 and 25mM
Testosterone 1mM and rosiglitazone 5, 10 and 25mM
Methods
 Cell growth was measured with optical density and light
microscopy
 Estradiol, Osteoprotegerin (OPG), FGF-23, and RANKL
were measured with ELISA
 Alkaline phosphatase (AP) was measured with
spectrophotometry
 Osteocalcin was measured with RIA
TZDs Inhibit Estradiol Synthesis
MOB+HGC Culture
TZD effect on mouse osteoblast cell (MOB)-HGC cell
growth (optical density)
Optical Density
(Compared to Day 7 Control)
Control
Testosterone
Pioglitazone
Testosterone + Pioglitazone
Control
Testosterone
Rosiglitazone
Testosterone + Rosiglitazone
100
100
Pioglitazone
Rosiglitazone
p < 0.001
p < 0.001
10
10
0
2
4
6
Incubation time (day)
0
8
2
4
6
Incubation time (day)
8
TZD effect on mouse osteoblast cell (MOB) growth
120
100
80
p<0.002)
60
p<0.001)
p<0.001)
p<0.001)
40
p<0.001)
20
on
Pi tro
og l
5
M
Pi
og
10
M
Pi
og
25
M
R
os
ig
5
R
M
os
ig
10
M
R
os
ig
25
M
0
C
Optical Density
(% control, mean ± SEM)
MOB culture
TZD effect on cell growth/differentiation
osteoblast
adipocyte
TZD effect on Fatty Acid Uptake
B
 (p < 0 .0 3 )
150
 (p < 0 .0 5 )
100
50
14
[C ]-o le ic a c id u p ta k e /o p tic a l d e n s ity
(% c o n tro l, m e a n ± S E M )
200
0
C o n tro l
R o s ig lita zo n e
P io g lita zo n e
TZD effect on Alkaline Phosphatase Activity
250
A
Control (no pioglitazone or testosterone)
*
*
Pioglitazone
Pioglitazone + 1 M Testosterone
B
Control (no rosiglitazone or testosterone)
Rosiglitazone
Rosiglitazone + 1 M Testosterone
200
MOB + HGC co- culture
Total alkaline phosphatase activity
(% control, mean ± SEM)
150
(p<0.001)
(p<0.001)
*
*
100
50
(p<0.046)
(p<0.042)
p<0.001)
(p<0.001)
0
300
D
C
MOB culture
200
p
100
p
*
*
(p<0.001)
(p<0.038)
(p<0.001)
0
0
5
10
15
20
Pioglitazone ( M)
25
30
(p<0.001)
0
5
10
15
20
Rosiglitazone ( M)
25
30
TZD effect on Osteocalcin activity
M O C + H G C O s te o c a lc in a c tiv ity
MOB+HGC
Osteocalcin Activity
140
p = 0 .8 3 9
100
80
p < 0 .0 1 8
p < 0 .0 1 1
60
p < 0 .0 1 2
p < 0 .0 4 6
p < 0 .0 2 2
p < 0 .0 0 1
40
p < 0 .0 2 2
p < 0 .0 0 6
20
+
T
+
T
R
R
o
o
si
si
2
1
5
0
5
io
T
+
P
+
T
P
io
2
0
n
ro
e
st
o
st
e
T
p io : p io g lita z o n e
ro s i : ro s ig lita z o n e
a ll c o n c e n tra tio n s a re in m M
1
e
5
si
R
o
R
o
si
2
0
1
5
2
io
P
io
P
o
n
tr
1
o
0
l
0
C
% of total osteocalcin activity
120
TZD effect on Osteocalcin activity
M O C O s te o c a lc in A c tiv ity
MOB
Osteocalcin Activity
120
NS
% of total osteocalcin activity
100
80
p < 0 .0 5
p < 0 .0 0 1
p < 0 .0 0 1
60
p < 0 .0 5
p < 0 .0 0 1
p < 0 .0 0 1
p < 0 .0 0 1
p < 0 .0 0 1
40
20
0
n
co
tr o
l
P io
10
P io
25
Ro
si
10
Ro
si
25
Te
p io : p io g lita z o n e
r o s i : r o s ig lita z o n e
c o n c e n tr a tio n s a r e in m M
s
te
to s
ro n
e
P
T+
io
10
P
T+
io
25
T
o
+R
si
10
T
o
+R
si
25
Osteoblast, Osteoclast, Osteoprotegerin (OPG) and Receptor
Activator of Nuclear Factor Kappa-B Ligand (RANKL)
•
Osteoblast
produces RANK
ligand and OPG
•
OPG blocks
RANK ligand
TZD effect on OPG production
*
*
Control (no pioglitazone or testosterone)
Pioglitazone
Pioglitazone + 1mM Testosterone
Total OPG production in the tissue cultue medium
(% control, mean ± SEM)
200
A
Control (no rosiglitazone or testosterone)
Rosiglitazone
Rosiglitazone + 1mM Testosterone
B
MOB + HGC co-culture
150
p
100
p
*
*
(p<0.001)
(p<0.001)
50
(p<0.001)
(p<0.001)
0
300
C
D
MOB culture
250
200
p
p
150
100
*
(p<0.001)
*
(p<0.001)
50
(p<0.001)
0
5
10
15
20
Pioglitazone ( M)
25
30
(p<0.001)
0
5
10
15
20
Rosiglitazone ( M)
25
30
TZD effect on RANKL production
*
*
Control (no pioglitazone or testosterone)
Pioglitazone
Pioglitazone + 1mM Testosterone
Total RANKL production (% control, mean ± SEM)
500
A
Control (no rosiglitazone or testosterone)
Rosiglitazone
Rosiglitazone + 1mM Testosterone
B
MOB + HGC co-culture
(p<0.003)
400
(p<0.048)
300
200
(p<0.018)
(p<0.047)
100
600
*
C
D
MOB culture
(p<0.009)
500
(p<0.019)
400
300
(p<0.041)
(p<0.003)
200
100
*
0
*
5
10
15
20
Pioglitazone ( M)
25
30
0
5
10
15
20
Rosiglitazone ( M)
25
30
Fibroblast Growth Factor – 23 (FGF-23) Regulation
Image sourced from Nature magazine
TZD effect on FGF-23 production
*
*
Control (no pioglitazone or testosterone)
Pioglitazone
Pioglitazone + 1mM Testosterone
Total FGF-23 production (% of control, mean ± SEM)
1600
A
Control (no rosiglitazone or testosterone)
Rosiglitazone
Rosiglitazone + 1mM Testosterone
B
MOB +HGC co-culture
1400
1200
(p<0.029)
(p<0.024)
1000
800
600
(p<0.023)
(p<0.021)
400
200
*
2500
*
C
D
MOB culture
(p<0.036)
(p<0.041)
2000
1500
1000
(p<0.023)
(p<0.006)
500
*
0
5
10
15
20
Pioglitazone ( M)
25
30
*0
5
10
15
20
Rosiglitazone ( M)
25
30
Conclusion
 Pioglitazone and rosiglitazone affect bone
metabolism by :

Inhibiting osteoblast growth

Increasing differentiation to adipocytes

Increasing fatty acid uptake

Reducing both AP and osteocalcin activity

Reducing OPG production

Increasing RANK ligand production

Increasing FGF-23 production
Conclusion
 The net effect of TZDs on mouse osteoblast cells is
decreased bone formation.
 The effects of pioglitazone and rosiglitazone on
osteoblast are not mediated by aromatase inhibition:
experiments with MOB cultures show similar results
with MOB and HGC co-cultures.
Thank You!