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 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

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 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 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 Jun;92(6): 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 Nov;90(11): Epub 2005 Aug 30.

6. Aromatase

7. TZDs Inhibit Estradiol Synthesis
Seto-Young et al. Hormone and Metabolic Research 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 Clin Endocrinol Metab 95:

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):

12. Thiazolidinedione Use and Bone Loss in Older Diabetic Adults
666 diabetic participants, 83 on TZDs. Age range 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):

13. 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):

14. 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 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 Clin Endocrinol Metab 95(1):134-42

16. In-vitro and Animal Studies on TZDs and Bone Metabolism

17. 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 Endocrinology 140:

18. 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 Oct;183(1):
2. Rzonca SO, et al Endocrinology Jan;145(1):401-6.
3. Ali AA, et al. Endocrinology Mar;146(3):

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.

38. Thank You!