Thiazolidinediones and Bone Metabolism: in-vitro studies Amit Seth, Ashutosh Pareek, Vanessa Sy, Pauline Suwandhi, Zev Rosenwaks, Donna Seto-Young, Leonid Poretsky

Contents Background Bone turnover and 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.

DM participants were divided to 2 groups : Rosiglitazone 12 weeks 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

Effect on Alkaline Phosphatase (AP) and/or osteocalcin In-vitro Studies 1st author TZD Effect Effect on Alkaline Phosphatase (AP) and/or osteocalcin Johnson (1999) In-vitro (Mouse Osteoblast Cells) Rosiglitazone (10, 25 and 50μM)  Gene transcription differentiation to adipocytes  AP 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) 20mg/g/day  osteoblast-specific gene expression Not tested Afshan Ali (2005) 25mg/g/day  of osteoblast differentiation and bone formation 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) Pioglitazone Rosiglitazone p < 0.001 p < 0.001

TZD effect on mouse osteoblast cell (MOB) growth

TZD effect on cell growth/differentiation osteoblast adipocyte

TZD effect on Fatty Acid Uptake

TZD effect on Alkaline Phosphatase Activity

TZD effect on Osteocalcin activity MOB+HGC Osteocalcin Activity

TZD effect on Osteocalcin activity 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

TZD effect on RANKL production

Fibroblast Growth Factor – 23 (FGF-23) Regulation Image sourced from Nature magazine

TZD effect on FGF-23 production

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!