AMIT SETH, ASHUTOSH PAREEK, VANESSA SY, PAULINE SUWANDHI, ZEV ROSENWAKS, DONNA SETO-YOUNG,

LEONID PORETSKY

Thiazolidinediones and Bone Metabolism: in-vitro

studies

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-γ (PPAR-γ)

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

Rosiglitazone12 weeksResults

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 inType 2 Diabetes

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 – 57.04 yearsResults

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

An Analysis from A Diabetes Outcome Progression Trial (ADOPT)

Effect of Rosiglitazone, Metformin, and Glyburide on Bone Biomarkers in Patients

with Type 2 Diabetes

1605 participants from ADOPT study1 yearResults

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

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.

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 M Rosiglitazone 5, 10 and 25 M Testosterone 1M Testosterone 1M and pioglitazone 5, 10 and 25M Testosterone 1M and rosiglitazone 5, 10 and 25M

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)

Incubation time (day)

0 2 4 6 8

10

100

ControlTestosteroneRosiglitazoneTestosterone + Rosiglitazone

Incubation time (day)

0 2 4 6 8

Op

tical

Den

sit

y

(Co

mp

are

d t

o D

ay 7

Co

ntr

ol)

10

100

ControlTestosteronePioglitazoneTestosterone + Pioglitazone

Pioglitazone Rosiglitazone

p < 0.001

p < 0.001

TZD effect on mouse osteoblast cell (MOB) growth

Op

tic

al

De

nsit

y

(% c

on

tro

l, m

ea

n ±

SE

M)

0

20

40

60

80

100

120

MOB culture

p<0.002)

γp<0.001)

p<0.001)p<0.001)

p<0.001)

TZD effect on cell growth/differentiation

osteoblast

adipocyte

TZD effect on Fatty Acid Uptake

Control Rosiglitazone Pioglitazone

14[C

]-o

leic

aci

d u

pta

ke/o

pti

cal

de

nsi

ty(%

co

ntr

ol,

mea

n ±

SE

M)

0

50

100

150

200

(p<0.03)

(p<0.05)

B

TZD effect on Alkaline Phosphatase Activity

Rosiglitazone (M)

0 5 10 15 20 25 30

Pioglitazone (M)

0 5 10 15 20 25 300

100

200

300

To

tal a

lka

lin

e p

ho

sp

ha

tas

e a

cti

vit

y

(% c

on

tro

l, m

ea

n ±

SE

M)

0

50

100

150

200

250

MOB culture

MOB + HGC co- culture

A B

C D

p<0.001)

(p<0.046)

(p<0.001)

(p<0.042)

(p<0.001)

(p<0.038)

(p<0.001)

(p<0.001)

pp

γ(p<0.001) (p<0.001)

*

*

*

*

Control (no pioglitazone or testosterone) * Pioglitazone

Pioglitazone + 1M Testosterone

Rosiglitazone

Rosiglitazone + 1M Testosterone

Control (no rosiglitazone or testosterone)*

TZD effect on Osteocalcin activity

MOC+HGC Osteocalcin activity

0

20

40

60

80

100

120

140

p<0.001

p<0.046p<0.011

p<0.022

p<0.012

p<0.006

p<0.018

p<0.022

p=0.839

pio : pioglitazonerosi : rosiglitazoneall concentrations are in M

% o

f to

tal o

ste

oca

lcin

activity

MOB+HGC Osteocalcin Activity

TZD effect on Osteocalcin activity

MOC Osteocalcin Activity

0

20

40

60

80

100

120

p<0.001

p<0.001

p<0.001

p<0.001

NS

p<0.05

p<0.001

p<0.001

p<0.05

% o

f to

tal o

ste

oca

lcin

activity

pio : pioglitazonerosi : rosiglitazoneconcentrations are in M

MOB Osteocalcin Activity

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

Pioglitazone (M)

0 5 10 15 20 25 30

50

100

150

200

250

300

Rosiglitazone (M)

0 5 10 15 20 25 30

C DMOB culture

(p<0.001)

(p<0.001)

(p<0.001)

To

tal O

PG

pro

du

cti

on

in t

he

tis

su

e c

ult

ue

me

diu

m(%

co

ntr

ol, m

ea

n ±

SE

M)

0

50

100

150

200A B

MOB + HGC co-culture

(p<0.001)

(p<0.001) (p<0.001)

(p<0.001)

γp

p

p

p

(p<0.001)*

*

*

*

Rosiglitazone

Rosiglitazone + 1M Testosterone

Control (no rosiglitazone or testosterone)*Control (no pioglitazone or testosterone) * Pioglitazone

Pioglitazone + 1M Testosterone

TZD effect on RANKL production

Pioglitazone (M)

0 5 10 15 20 25 30

100

200

300

400

500

600

Rosiglitazone (M)

0 5 10 15 20 25 30

C DMOB culture

(p<0.003)

(p<0.019)

(p<0.041)

To

tal R

AN

KL

pro

du

cti

on

(%

co

ntr

ol, m

ea

n ±

SE

M)

100

200

300

400

500 B

(p<0.047)

(p<0.048)

(p<0.009)

*

**

Control (no pioglitazone or testosterone) * Pioglitazone

Pioglitazone + 1M Testosterone

Rosiglitazone

Rosiglitazone + 1M Testosterone

Control (no rosiglitazone or testosterone)*

A

(p<0.018)

(p<0.003)

MOB + HGC co-culture

Fibroblast Growth Factor – 23 (FGF-23) Regulation

Image sourced from Nature magazine

TZD effect on FGF-23 production

Pioglitazone (M)

0 5 10 15 20 25 30

500

1000

1500

2000

2500

Rosiglitazone (M)

0 5 10 15 20 25 30

To

tal F

GF

-23

pro

du

ctio

n (

% o

f co

ntr

ol,

mea

n ±

SE

M)

200

400

600

800

1000

1200

1400

1600

MOB culture

A

C D

MOB +HGC co-culture B

(p<0.029)

(p<0.023)γ(p<0.021)

(p<0.024)

(p<0.023)

(p<0.041)

(p<0.006)

(p<0.036)

* *

* *

Control (no pioglitazone or testosterone) * Pioglitazone

Pioglitazone + 1M Testosterone

Rosiglitazone

Rosiglitazone + 1M Testosterone

Control (no rosiglitazone or testosterone)*

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!