1. The Role of Thiazolidinediones in Bone Metabolism David C. Gajzer M.D. Pauline Suwandhi M.D. Yun Feng M.D. Amit K. Seth M.D. Leonid Poretsky M.D. Donna Seto-Young Ph.D.

2. Background Thiazolidinediones Used for treatment of type 2 diabetes mellitus Used for treatment of type 2 diabetes mellitus Increase insulin sensitivity of peripheral tissues by binding and activating peroxisome proliferator activated receptor-γ (PPAR-γ) Increase insulin sensitivity of peripheral tissues by binding and activating peroxisome proliferator activated receptor-γ (PPAR-γ)

3. Reported Effects of TZDs on Bone Metabolism Inhibited bone formation and mineralization (Johnson et al., 1999) Inhibited bone formation and mineralization (Johnson et al., 1999) Decreased osteoblastogenesis, increased bone marrow adiposity in men and women (Okazaki et al., 1999; Watanabe et al., 2003) Decreased osteoblastogenesis, increased bone marrow adiposity in men and women (Okazaki et al., 1999; Watanabe et al., 2003) Induced bone loss in older diabetic women (Schwartz et al., 2006) Induced bone loss in older diabetic women (Schwartz et al., 2006) Decrease in bone osteoblast markers pro-collagen type-1 N-terminal pro-peptide (P1NP) and osteocalcin, but no change in type 1 collagen N-telopeptide (NTX) (Grey et al., 2007) Decrease in bone osteoblast markers pro-collagen type-1 N-terminal pro-peptide (P1NP) and osteocalcin, but no change in type 1 collagen N-telopeptide (NTX) (Grey et al., 2007) ADOPT trial reported a higher risk of fractures in diabetic women randomized to rosiglitazone compared to metformin and glyburide (Kahn et al., 2006) ADOPT trial reported a higher risk of fractures in diabetic women randomized to rosiglitazone compared to metformin and glyburide (Kahn et al., 2006)

4. Effects of Thiazolidinediones on Aromatase Activity It was recently demonstrated that thiazolidinediones: It was recently demonstrated that thiazolidinediones: Exhibited inhibitory effects on aromatase enzyme activity by interfering with the androgen substrate binding to aromatase (Seto-Young et al., 2007; Avtanski et al., 2007) Exhibited inhibitory effects on aromatase enzyme activity by interfering with the androgen substrate binding to aromatase (Seto-Young et al., 2007; Avtanski et al., 2007) Reduced estrogen biosynthesis in human granulosa cells (Seto-Young et al., 2007; Avtanski et al., 2007) Reduced estrogen biosynthesis in human granulosa cells (Seto-Young et al., 2007; Avtanski et al., 2007)

5. Hypothesis TZDs may inhibit aromatase enzyme activity in human granulosa cells, possibly leading to reduced estrogen synthesis and consequently increased risk of osteoporosis. TZDs may inhibit aromatase enzyme activity in human granulosa cells, possibly leading to reduced estrogen synthesis and consequently increased risk of osteoporosis.

6. Objective In human granulosa cells and osteoblast cell co-cultures stimulated with TZDs, compare: In human granulosa cells and osteoblast cell co-cultures stimulated with TZDs, compare: –1. Bone Turnover Markers:  Alkaline phosphatase  Osteocalcin  Procollagen type-1 propeptide (P1NP)  Type-1 collagen N-telopeptide (NTX) –2. Estradiol and Estrone levels –3. Cell Growth –4. Cell Differentiation

7. Methods Osteoblast Cells and purified Human Granulosa Cells were co- cultured for 2-3 days Osteoblast Cells and purified Human Granulosa Cells were co- cultured for 2-3 days Mixed cultures were stimulated with testosterone alone, pioglitazone alone, and testosterone and pioglitazone together. Mixed cultures were stimulated with testosterone alone, pioglitazone alone, and testosterone and pioglitazone together. Estradiol levels were measured using a radioimmunoassay. Estradiol levels were measured using a radioimmunoassay. Alkaline Phosphatase levels were measured using an ELISA assay. Alkaline Phosphatase levels were measured using an ELISA assay. Optical density was measured. Optical density was measured. –Stimulated plates were trypsinized –Cells were isolated and then resuspended in 1ml PBS Buffer –Using spectrophotometry, the optical density of cell solutions was measured at 600 nm Cell plates were stained with Hematoxylin, Eosin, and Oil Red O stains. Cell plates were stained with Hematoxylin, Eosin, and Oil Red O stains.

8. Estradiol Radioimmuno-Assay Time (Hours) Cell PlateAverage Estradiol ActivityPercent Activity 0 Mouse Osteoblast Cells 0.000.0 0 Mouse Osteoblast Cells + Testosterone 0.000.0 0 Mouse Osteoblast Cells + Pioglitazone 0.000.0 0 Mouse Osteoblast Cells +Testosterone +Pioglitazone 0.000.0 48 Mouse Osteoblast Cells 0.000.0 48 Mouse Osteoblast Cells + Testosterone 4402.07100% 48 Mouse Osteoblast Cells + Pioglitazone 0.000.0 48 Mouse Osteoblast Cells +Testosterone +Pioglitazone 785.8617.9% 96 Mouse Osteoblast Cells 0.000.0 96 Mouse Osteoblast Cells + Testosterone 7354.37100% 96 Mouse Osteoblast Cells + Pioglitazone 0.000.0 96 Mouse Osteoblast Cells +Testosterone +Pioglitazone 1184.2216.1% Pioglitazone is associated with decreased estradiol levels.

9. ELISA Alkaline Phosphatase Assay Cell Plates Average Alkaline Phosphatase Level Alkaline Phosphatase Specific Activity Specific Activity Specific Activity % Mouse Osteoblast & Human Granulosa Cells 1.6580.113100 Mouse Osteoblast & Human Granulosa Cells + Testosterone 1.5400.111100 Mouse Osteoblast & Human Granulosa Cells + Pioglitazone 0.7390.06052.7 Mouse Osteoblast & Human Granulosa Cells + Testosterone + Pioglitazone 0.7840.06659.8 Pioglitazone is associated with decreased alkaline phosphatase levels.

10. Cell Growth Cell Plates Average Optical Density (at 600nm) Mouse Osteoblast Cells 1.304 (100%) Mouse Osteoblast Cells + Testosterone 1.308 (100%) Mouse Osteoblast Cells + Pioglitazone 1.162 (89%) Mouse Osteoblast Cells + Testosterone + Pioglitazone 1.227 (94%) Pioglitazone is associated with decreased cell growth as measured by optical density.

11. Cell Differentiation 1 st row: Mouse Osteoblast Cells and Human Granulosa Cells cultured with 2% FBS MEM Alpha Medium 2 nd row: Cells stimulated with 2% FBS MEM Alpha Medium and Pioglitazone display increased lipogenesis (Oil Red O stain). 3 rd row: Cells stimulated with 2% FBS MEM Alpha Medium and Testosterone display increased growth and eosinophily (due to increased cytoplasmatic protein synthesis). 4 th row: Cells treated with 2% FBS MEM Alpha Medium plus both Testosterone and Pioglitazone show less growth and increased lipogenesis than those treated with Testosterone only. Day 5 of Stimulation Increased Lipogenesis Increased Growth and Eosinophily Less Growth, Increased Lipogenesis compared to Testosterone Only Plates

12. Conclusion (Preliminary Results) TZDs, specifically pioglitazone, may lead to decreased bone turnover as measured by alkaline phosphatase levels. TZDs, specifically pioglitazone, may lead to decreased bone turnover as measured by alkaline phosphatase levels. TZDs may lead to decreased estradiol levels. TZDs may lead to decreased estradiol levels. TZDs may inhibit cell growth of mouse osteoblast cells. TZDs may inhibit cell growth of mouse osteoblast cells. TZDs may lead to increased differentation of mouse osteoblast cells into adipocytes. TZDs may lead to increased differentation of mouse osteoblast cells into adipocytes. Further research is needed to determine the effects of pioglitazone on bone metabolism. Further research is needed to determine the effects of pioglitazone on bone metabolism.