Thiazolidinediones and Bone Metabolism: in-vitro studies

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

2. 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

3. Thiazolidinediones

4. 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.

5. 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.

6. Aromatase

7. TZDs Inhibit Estradiol Synthesis Seto-Young et al. Hormone and Metabolic Research. 2011 Apr;43(4):250-6

8. 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 ClinEndocrinolMetab95:356-357.

9. Bone Metabolism

10. TZDs and Bone Metabolism – Clinical Studies

11. 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.

12. 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 ClinEndocrinolMetab91(9):3349-54.

13. 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 ClinEndocrinolMetab92(9):3623-30

14. Rosiglitazone-Associated Fractures inType 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

15. 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 ClinEndocrinolMetab95(1):134-42

16. In-vitro and Animal Studies onTZDs and Bone Metabolism

17. In-vitro Studies Johnson TE, et al. 1999 Endocrinology 140:3245-3254

18. 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.

19. Hypothesis • TZDs inhibit bone metabolism through: • Aromatase inhibition • Direct effect on osteoblasts/osteoclasts

20. 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)

21. 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

22. 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

23. TZDs Inhibit Estradiol Synthesis MOB+HGC Culture

24. TZD effect on mouse osteoblast cell (MOB)-HGC cell growth (optical density) Pioglitazone Rosiglitazone p < 0.001 p < 0.001

25. TZD effect on mouse osteoblast cell (MOB) growth

26. TZD effect on cell growth/differentiation osteoblast adipocyte

27. TZD effect on Fatty Acid Uptake

28. TZD effect on Alkaline Phosphatase Activity

29. TZD effect on Osteocalcin activity MOB+HGC Osteocalcin Activity

30. TZD effect on Osteocalcin activity MOB Osteocalcin Activity

31. Osteoblast, Osteoclast, Osteoprotegerin (OPG) and Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) • Osteoblast produces RANK ligand and OPG • OPG blocks RANK ligand

32. TZD effect on OPG production

33. TZD effect on RANKL production

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

35. TZD effect on FGF-23 production

36. 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

37. 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.

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