Thiazolidinediones and Bone
Metabolism
3rd Annual Friedman Fellows Symposium
November 13th 2010
Pauline Suwandhi, M.D., Amit Seth, M.D., Ashutosh Pareek, D.O.,
Vanessa Sy, M.D., Leonid Poretsky, M.D., and Donna Seto-Young Ph. D.
Beth Israel Medical Center
Division of Endocrinology and Metabolism
Albert Einstein College of Medicine
New York, U.S.A.
Thiazolidinediones (TZDs)
 TZDs are insulin-sensitizing
agents that are widely prescribed
in the management of type 2
diabetes mellitus.
 TZDs activate the nuclear
receptor super family 
peroxisome-proliferator activator
receptor-g (PPAR-g) by binding to
the peroxisome proliferator
response element (PPRE) and
turning on gene transcription.
The activated genes include
those involved in glucose and
lipid metabolism.
TZD effect on Ovarian Steroidogenesis
 TZDs reduce androgen levels and restore ovulation in
patients with polycystic ovary syndrome (PCOS)
 TZDs also directly reduce estrogen and enhance
progesterone production in human ovarian cell culture
Seto-Young et al. 2005, J Clin Endocrinol Metab 90:6099-6105.
TZDs’ Interaction with Insulin Signaling
Pathways and Effect on Steroidogenic
Regulation
Seto-Young et al., 2007, J Clin Endocrinol Metab 92: 2232-2239
4
TZDs’ Interaction with Insulin Signaling
Pathways and Effect on Steroidogenic
Regulation
 Insulin binds to insulin receptor, activates the tyrosine kinase and
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stimulates insulin receptor substrate-1 (IRS-1) expression
Insulin also activates steroidogenic acute regulatory (StAR) protein
expression which leads to increased progesterone, testosterone and
estrogen synthesis
TZDs interact with PPAR-g which in turn affect components of insulin
signaling pathways.
TZDs indirectly activate insulin receptor, IRS-1 and StAR expression
TZDs increase progesterone production and inhibit testosterone and
estrogen synthesis.
TZDs inhibit aromatase activity.
Seto-Young et al., 2007, J Clin Endocrinol Metab 92: 2232-2239
5
TZDs’ effect on Aromatase Activity
 TZDs inhibit estrogen synthesis
 TZDs have no effect on aromatase mRNA or protein
expression, suggesting no effect on gene
transcription or protein translation.
 In the enzyme kinetics study, TZDs inhibit Vmax and
Km of aromatase, acting as un-competitive inhibitors.
Seto-Young et al. 2010 Manuscript submitted to Hormone and Metabolic
Research.
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TZDs, Estrogen and Bone Fragility
 Menopause, an estrogen-deficient state, is known to be
the cause of osteoporosis; estrogen and its receptor play
a major role in bone metabolism
 Studies of aromatase inhibitors for the treatment of
breast cancer show that letrozole, exemestane and
anastrazole induce a decline of bone mineral density (BMD)
and increase risk of fracture.
Khosla S, 2010, J Clin Endocrinol Metab 95:356-3577
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Literature on the effects of TZDs on
Bone Metabolism
 Clinical trials
• Treatment with troglitazone
•
•
•
•
decreases bone turnover in patients
with DM (Okazaki, et al, 1999)
TZDs induce bone loss in older DM women (Schwartz et al.,
2006)
ADOPT trial reported a higher risk of fractures in DM women
treated with rosiglitazone (Kahn et al., 2006)
Treatment with rosiglitazone decreases alkaline phosphatase (AP)
and osteocalcin but has no effect on bone resorption markers in
DM women (Berberoglu et. al, 2007)
ADOPT trial reported that treatment with rosiglitazone increases
AP and C-terminal telopeptide (CTX) and reduces procollagen
type 1 amino terminal-propeptide (P1NP) (Zinman et al, 2010)
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Literature on the effect of TZDs on Bone
Metabolism - continued
In vitro studies
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•
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•
TZDs mediate gene transcription and differentiation in
mesenchymal progenitor cells to adipocytes and increase
fat accumulation (Johnson et al., 1999)
TZD inhibits the formation of osteoclast-like cell (Okazaki et
al., 1999)
Rosiglitazone increases apoptosis of osteoblasts without
any change in biomarkers of osteocalcin and alkaline
phosphatase (AP) (Soroceanu et al., 2004)
Rosiglitazone decreases osteoblast formation markers procollagen type-1 N-terminal pro-peptide (P1NP) &
osteocalcin, but has no effect on resorption marker type 1
collagen N-telopeptide (NTX) (Grey et al., 2007)
TZDs inhibit TNF-α-mediated osteoclast-like cells
differentiation (Yang et al, 2010)
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Bone Turnover Metabolism
Bone has to undergo modeling and remodeling to maintain its
structure and function.
Modeling and Remodeling
 Modeling/construction : bone formation carried out by
osteoblasts.
 Remodeling/reconstruction : bone resorption carried out
by osteoclasts.
 Both processes influenced by systemic factors : endocrine
(including estrogen level), metabolic and nutritional.
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Hypothesis
TZDs inhibit bone metabolism through:
 aromatase enzyme inhibition
 direct effect on osteoblast/osteoclast
Objective


To examine the effects of TZDs on mouse
osteoblast cells :
 cell growth,
 bone turnover markers,
 pro-collagen expression,
 cell differentiation
To examine whether aromatase inhibition plays a
role in any of the TZD effects on mouse osteoblast
cells
Methods – Culture System
 A commercially available mouse osteoblast cell (MOC)
line, 7F2, from American Type Culture Collection (ATCC),
was co-cultured with or without human granulosa cells
(HGC)
 The cells were then incubated with:
 pioglitazone 25mM
 rosiglitazone 25mM
 Testosterone 1mM
 testosterone 1mM+ pioglitazone 25mM
 testosterone 1mM+ rosiglitazone 25mM
TZDs Inhibit Estradiol Synthesis
p <0.001
p <0.001
Pioglitazone inhibited
estradiol synthesis in the
MOC and HGC co-culture
O p tic a l D e n s ity
(C o m p a re d to D a y 7 C o n tro l)
TZD Effect on MOC-HGC Cell Growth
(Optical Density)
C o n tro l
C o n tro l
T e s to s te ro n e
T e s to s te ro n e
P io g lita zo n e
R o s ig lita zo n e
T e s to s te ro n e + P io g lita zo n e
T e s to s te ro n e + R o s ig lita zo n e
100
100
p<0.02
10
10
0
2
4
6
In c u b a tio n tim e (d a y)
8
0
2
4
6
8
In c u b a tio n tim e (d a y)
TZDs inhibit cell growth. Testosterone can ameliorate the cell growth
inhibition caused by TZDs.
TZD effect on MOC Cell Growth (Optical Density)
O p tic a l D e n s ity
(C o m p a re d to D a y 7 C o n tro l)
100
C o n tro l
T e sto ste ro n e
P io g lita zo n e
T e sto ste ro n e
+ P io g lita zo n e
R o sig lita zo n e
R o sig lita zo n e
+ T e sto ste ro n e
10
2
3
4
5
6
7
In c u b a tio n T im e (D a ys )
Pioglitazone & Rosiglitazone are associated with
decreased MOC growth as measured by optical density.
R
R
R
o
o
o
si
si
g
g
g
g
io
g
io
io
si
P
P
P
C
tr
25
10
5m
m
M
M
m
m
M
M
M
25
m
l
M
o
5m
n
10
g
o
O p tic a l D e n s ity (% C o n tro l
TZDs Affect Cell Growth in a Dose-Dependent
Manner
120
100
80
60
40
20
0
TZD Effect on Osteoblast
Growth/Differentiation
Thiazolinediones
inhibit cell growth and
increase fat
accumulation.
MOC cultures were
stained with Oil Red O
to highlight the
presence of
adipocytes.
TZD Effect on Alkaline Phosphatase
(AP) Activity
M .O .C . + H .G .C .
M .O .C .
A lk a lin e P h o s p h a ta s e S p e c ific A c tivity
A lk a lin e P h o s p h a ta s e S p e c ific A c tivity
140
100
80
p < 0 .0 0 1
60
p < 0 .0 0 1
40
20
S p e c ific A c tivity
S p e c ific A c tivity
120
100
p < 0 .0 2 5
p < 0 .0 2 9
80
60
p < 0 .0 0 1 p < 0 .0 0 1
40
20
0
0
C
P
T
T + P
C e ll S a m p le L y s a te
C o n tro l
P io g lita zo n e
T e sto ste ro n e
T e sto ste ro n e + P io g lita zo n e
C
P
R
T
T + P T + R
C e ll S a m p le L y s a te
C o n tro l
P io g lita zo n e
R o sig lita zo n e
T e sto ste ro n e
T e sto ste ro n e + P io g lita zo n e
T e sto ste ro n e + R o sig lita zo n e
Pioglitazone and Rosiglitazone are associated with decreased AP
activity levels. Addition of testosterone to MOC+HGC co-culture
“protects” AP activity levels from effects of Thiazolidinedione.
TZD Effect on Osteocalcin Synthesis
M .O .C . + H .G .C .
M .O .C .
O s te o c a lc in S p e c ific A c tiv ity
O s te o c a lc in S p e c ific A c tiv ity
180
160
140
100
p < 0 .0 4
S p e c ific A c tiv ity
S p e c ific A c tiv ity
120
120
p < 0 .0 3
80
100
60
40
p < 0 .0 3 7
80
p < 0 .0 0 2
p < 0 .0 0 5
60
p < 0 .0 0 9
40
20
20
0
0
C
P
T
T + P
C e ll S a m p le L y s a te
C o n tro l
P io g lita zo n e
T e sto ste ro n e
T e sto ste ro n e + P io g lita zo n e
C
P
R
T
T + P T + R
C e ll S a m p le L y s a te
C o n tro l
P io g lita zo n e
R o sig lita zo n e
T e sto ste ro n e
T e sto ste ro n e + P io g lita zo n e
T e sto ste ro n e + R o sig lita zo n e
Pioglitazone reduces osteocalcin production in MOC-HGC co-culture
and MOC culture.
Mouse Pro-collagen mRNA expression
Pioglitazone and rosiglitazone inhibit mouse procollagen mRNA expression
Conclusions
 Pioglitazone and rosiglitazone


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
inhibit osteoblast cell growth
decrease bone turnover biomarkers (AP and osteocalcin levels)
decrease mouse pro-collagen mRNA expression
increase differentiation to adipocytes
 Inhibition of aromatase by TZDs does not play a role in the
osteoblast cell growth or in the effects of TZDs on bone
turnover markers, since inhibition of cell growth and the
effects on bone turnover markers were observed in MOC
culture which did not contain granulosa cells.
 Results are consistent with clinical studies showing
increased fracture risk and bone loss in patients with
diabetes treated with TZDs.
Future Research
 To study TZD effect in mouse osteoblasts:
1. bone turnover markers:

RANKL expression
FGF-23 expression
MAPK-Erk1/2 expression
Wnt signaling pathway
2.
bone resorption markers:

Type I collagen cross-linked N- (NTX) or C-telopeptide (CTX)
TZDs effect on RANKL induction of osteoclast differentiation.



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 To study effect of TZDs on mouse osteoclasts
and human osteoblasts.
Thank You!