3rd Annual Friedman Fellows SymposiumNovember 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.

Thiazolidinediones and Bone Metabolism

Beth Israel Medical CenterBeth Israel Medical CenterDivision of Endocrinology and Division of Endocrinology and MetabolismMetabolismAlbert Einstein College of Albert Einstein College of MedicineMedicineNew York, U.S.A.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- (PPAR- 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.

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.

TZD effect on Ovarian Steroidogenesis

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TZDs’ Interaction with Insulin Signaling Pathways and Effect on Steroidogenic

Regulation

Seto-Young et al., 2007, J Clin Endocrinol Metab 92: 2232-2239

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TZDs’ Interaction with Insulin Signaling Pathways and Effect on Steroidogenic

Regulation Insulin binds to insulin receptor, activates the tyrosine kinase

and 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- 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

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TZDs’ effect on Aromatase Activity

TZDs inhibit estrogen synthesisTZDs 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 trialsClinical trials• Treatment with troglitazone decreases bone turnover in Treatment with troglitazone decreases bone turnover in

patients with DM (Okazaki, et al, 1999)patients with DM (Okazaki, et al, 1999)• TZDs induce bone loss in older DM women (Schwartz et TZDs induce bone loss in older DM women (Schwartz et

al., 2006)al., 2006)• ADOPT trial reported a higher risk of fractures in DM ADOPT trial reported a higher risk of fractures in DM

women treated with rosiglitazone (Kahn et al., 2006)women treated with rosiglitazone (Kahn et al., 2006)• Treatment with rosiglitazone decreases alkaline Treatment with rosiglitazone decreases alkaline

phosphatase (AP) and osteocalcin but has no effect on phosphatase (AP) and osteocalcin but has no effect on bone resorption markers in DM women (Berberoglu et. al, bone resorption markers in DM women (Berberoglu et. al, 2007) 2007)

• ADOPT trial reported that treatment with rosiglitazone ADOPT trial reported that treatment with rosiglitazone increases AP and C-terminal telopeptide (CTX) and increases AP and C-terminal telopeptide (CTX) and reduces procollagen type 1 amino terminal-propeptide reduces procollagen type 1 amino terminal-propeptide (P1NP) (Zinman et al, 2010)(P1NP) (Zinman et al, 2010)

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In vitro studiesIn vitro studies• TZDs mediate gene transcription and differentiation TZDs mediate gene transcription and differentiation

in mesenchymal progenitor cells to adipocytes and in mesenchymal progenitor cells to adipocytes and increase fat accumulation (Johnson et al., 1999)increase fat accumulation (Johnson et al., 1999)

• TZD inhibits the formation of osteoclast-like cell TZD inhibits the formation of osteoclast-like cell (Okazaki et al., 1999)(Okazaki et al., 1999)

• Rosiglitazone increases apoptosis of osteoblasts Rosiglitazone increases apoptosis of osteoblasts without any change in biomarkers of osteocalcin without any change in biomarkers of osteocalcin and alkaline phosphatase (AP) (Soroceanu et al., and alkaline phosphatase (AP) (Soroceanu et al., 2004)2004)

• Rosiglitazone decreases osteoblast formation Rosiglitazone decreases osteoblast formation markers pro-collagen type-1 N-terminal pro-peptide markers pro-collagen type-1 N-terminal pro-peptide (P1NP) & osteocalcin, but has no effect on (P1NP) & osteocalcin, but has no effect on resorption marker type 1 collagen N-telopeptide resorption marker type 1 collagen N-telopeptide (NTX) (Grey et al., 2007)(NTX) (Grey et al., 2007)

• TZDs inhibit TNF-α-mediated osteoclast-like cells TZDs inhibit TNF-α-mediated osteoclast-like cells differentiation (Yang et al, 2010)differentiation (Yang et al, 2010)

Literature on the effect of TZDs on Bone Metabolism - continued

Bone Turnover Metabolism

Bone has to undergo modeling and remodeling to maintain its structure and function.

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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.

Hypothesis

TZDs inhibit bone metabolism through:aromatase enzyme inhibitiondirect 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 25Mrosiglitazone 25MTestosterone 1Mtestosterone 1M+ pioglitazone 25Mtestosterone 1M+ rosiglitazone 25M

TZDs Inhibit Estradiol Synthesis

Pioglitazone inhibited estradiol synthesis in the MOC and HGC co-culture

p <0.001

p <0.001

TZD Effect on MOC-HGC Cell Growth(Optical Density)

TZDs inhibit cell growth. Testosterone can ameliorate the cell growth inhibition caused by TZDs.

Incubation time (day)

0 2 4 6 8

10

100

ControlTestosteroneRosiglitazoneTestosterone + Rosiglitazone

Incubation time (day)

0 2 4 6 8

Op

tic

al

De

ns

ity

(Co

mp

are

d t

o D

ay

7 C

on

tro

l)

10

100

ControlTestosteronePioglitazoneTestosterone + Pioglitazone

p<0.02

TZD effect on MOC Cell Growth (Optical Density)

Pioglitazone & Rosiglitazone are associated with decreased MOC growth as measured by optical density.

Incubation Time (Days)

2 3 4 5 6 7

Op

tica

l Den

sity

(C

om

par

ed t

o D

ay 7

Co

ntr

ol)

10

100

ControlTestosteronePioglitazoneTestosterone + PioglitazoneRosiglitazoneRosiglitazone + Testosterone

TZDs Affect Cell Growth in a Dose-Dependent Manner

Op

tica

l Den

sity

(%

Co

ntr

ol

0

20

40

60

80

100

120

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

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.

M.O.C. + H.G.C.

Alkaline Phosphatase Specific Activity

Sp

ec

ific

Ac

tivi

ty

Cell Sample Lysate

C P T T + P0

20

40

60

80

100

ControlPioglitazoneTestosteroneTestosterone + Pioglitazone

C P R T T + P T + R0

20

40

60

80

100

120

140

ControlPioglitazoneRosiglitazoneTestosteroneTestosterone+PioglitazoneTestosterone+Rosiglitazone

M.O.C.

Alkaline Phosphatase Specific ActivityS

pe

cif

ic A

cti

vity

Cell Sample Lysate

p<0.001 p<0.001

p<0.001p<0.001

p<0.025

p<0.029

TZD Effect on Osteocalcin Synthesis

Pioglitazone reduces osteocalcin production in MOC-HGC co-culture and MOC culture.

M.O.C. + H.G.C. Osteocalcin Specific Activity

Sp

ecif

ic A

ctiv

ity

Cell Sample Lysate

C P T T + P0

20

40

60

80

100

120

ControlPioglitazoneTestosteroneTestosterone + Pioglitazone

C P R T T + P T + R0

20

40

60

80

100

120

140

160

180

ControlPioglitazoneRosiglitazoneTestosteroneTestosterone+PioglitazoneTestosterone+Rosiglitazone

M.O.C. Osteocalcin Specific Activity

Sp

ecif

ic A

ctiv

ity

Cell Sample Lysate

p<0.002 p<0.005

p<0.037

p<0.009

p<0.03

p<0.04

Mouse Pro-collagen mRNA expression

Pioglitazone and rosiglitazone inhibit mouse pro-collagen mRNA expression

Conclusions Pioglitazone and rosiglitazonePioglitazone and rosiglitazone

inhibit osteoblast cell growthinhibit osteoblast cell growth decrease bone turnover biomarkers (AP and osteocalcin decrease bone turnover biomarkers (AP and osteocalcin

levels)levels) decrease mouse pro-collagen mRNA expressiondecrease mouse pro-collagen mRNA expression increase differentiation to adipocytesincrease 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 Results are consistent with clinical studies showing increased fracture risk and bone loss in patients with increased fracture risk and bone loss in patients with diabetes treated with TZDs.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.

To study effect of TZDs on mouse osteoclasts and human osteoblasts.

Thank You!