3 rd Annual Friedman Fellows Symposium November 13 th 2010 Pauline Suwandhi, M.D., Amit Seth, M.D., Ashutosh Pareek, D.O., Vanessa Sy, M.D., Leonid Poretsky, M.D., and Donna Seto-Young Ph. D. Thiazolidinediones and Bone Metabolism Beth Israel Medical Center Division of Endocrinology and Metabolism Albert Einstein College of Medicine New York, U.S.A.

Thiazolidinediones (TZDs) TZDs are insulin-sensitizing agents that are widely prescribed in the management of type 2 diabetes mellitus. TZDs activate the nuclear receptor super family  peroxisome-proliferator activator receptor-  (PPAR-  by binding to the peroxisome proliferator response element (PPRE) and turning on gene transcription. The activated genes include those involved in glucose and lipid metabolism.

 TZDs reduce androgen levels and restore ovulation in patients with polycystic ovary syndrome (PCOS)  TZDs also directly reduce estrogen and enhance progesterone production in human ovarian cell culture Seto-Young et al. 2005, J Clin Endocrinol Metab 90: TZD effect on Ovarian Steroidogenesis

4 TZDs ’ Interaction with Insulin Signaling Pathways and Effect on Steroidogenic Regulation Seto-Young et al., 2007, J Clin Endocrinol Metab 92:

5 TZDs ’ Interaction with Insulin Signaling Pathways and Effect on Steroidogenic Regulation  Insulin binds to insulin receptor, activates the tyrosine kinase and stimulates insulin receptor substrate-1 (IRS-1) expression  Insulin also activates steroidogenic acute regulatory (StAR) protein expression which leads to increased progesterone, testosterone and estrogen synthesis  TZDs interact with PPAR-  which in turn affect components of insulin signaling pathways.  TZDs indirectly activate insulin receptor, IRS-1 and StAR expression  TZDs increase progesterone production and inhibit testosterone and estrogen synthesis.  TZDs inhibit aromatase activity. Seto-Young et al., 2007, J Clin Endocrinol Metab 92:

6 TZDs’ effect on Aromatase Activity TZDs inhibit estrogen synthesis TZDs have no effect on aromatase mRNA or protein expression, suggesting no effect on gene transcription or protein translation. In the enzyme kinetics study, TZDs inhibit V max and K m of aromatase, acting as un-competitive inhibitors. Seto-Young et al Manuscript submitted to Hormone and Metabolic Research.

7 TZDs, Estrogen and Bone Fragility  Menopause, an estrogen-deficient state, is known to be the cause of osteoporosis; estrogen and its receptor play a major role in bone metabolism  Studies of aromatase inhibitors for the treatment of breast cancer show that letrozole, exemestane and anastrazole induce a decline of bone mineral density (BMD) and increase risk of fracture. Khosla S, 2010, J Clin Endocrinol Metab 95:

8 Literature on the effects of TZDs on Bone Metabolism Clinical trials Clinical trials Treatment with troglitazone decreases bone turnover in patients with DM (Okazaki, et al, 1999) Treatment with troglitazone decreases bone turnover in patients with DM (Okazaki, et al, 1999) TZDs induce bone loss in older DM women (Schwartz et al., 2006) TZDs induce bone loss in older DM women (Schwartz et al., 2006) ADOPT trial reported a higher risk of fractures in DM women treated with rosiglitazone (Kahn et al., 2006) ADOPT trial reported a higher risk of fractures in DM women treated with rosiglitazone (Kahn et al., 2006) Treatment with rosiglitazone decreases alkaline phosphatase (AP) and osteocalcin but has no effect on bone resorption markers in DM women (Berberoglu et. al, 2007) Treatment with rosiglitazone decreases alkaline phosphatase (AP) and osteocalcin but has no effect on bone resorption markers in DM women (Berberoglu et. al, 2007) ADOPT trial reported that treatment with rosiglitazone increases AP and C-terminal telopeptide (CTX) and reduces procollagen type 1 amino terminal-propeptide (P1NP) (Zinman et al, 2010) ADOPT trial reported that treatment with rosiglitazone increases AP and C-terminal telopeptide (CTX) and reduces procollagen type 1 amino terminal-propeptide (P1NP) (Zinman et al, 2010)

9 In vitro studies In vitro studies TZDs mediate gene transcription and differentiation in mesenchymal progenitor cells to adipocytes and increase fat accumulation (Johnson et al., 1999) TZDs mediate gene transcription and differentiation in mesenchymal progenitor cells to adipocytes and increase fat accumulation (Johnson et al., 1999) TZD inhibits the formation of osteoclast-like cell (Okazaki et al., 1999) TZD inhibits the formation of osteoclast-like cell (Okazaki et al., 1999) Rosiglitazone increases apoptosis of osteoblasts without any change in biomarkers of osteocalcin and alkaline phosphatase (AP) (Soroceanu et al., 2004) Rosiglitazone increases apoptosis of osteoblasts without any change in biomarkers of osteocalcin and alkaline phosphatase (AP) (Soroceanu et al., 2004) Rosiglitazone decreases osteoblast formation markers pro- collagen type-1 N-terminal pro-peptide (P1NP) & osteocalcin, but has no effect on resorption marker type 1 collagen N-telopeptide (NTX) (Grey et al., 2007) Rosiglitazone decreases osteoblast formation markers pro- collagen type-1 N-terminal pro-peptide (P1NP) & osteocalcin, but has no effect on resorption marker type 1 collagen N-telopeptide (NTX) (Grey et al., 2007) TZDs inhibit TNF-α-mediated osteoclast-like cells differentiation (Yang et al, 2010) TZDs inhibit TNF-α-mediated osteoclast-like cells differentiation (Yang et al, 2010) Literature on the effect of TZDs on Bone Metabolism - continued

Bone Turnover Metabolism Bone has to undergo modeling and remodeling to maintain its structure and function.

11 Modeling and Remodeling Modeling/construction : bone formation carried out by osteoblasts. Remodeling/reconstruction : bone resorption carried out by osteoclasts. Both processes influenced by systemic factors : endocrine (including estrogen level), metabolic and nutritional.

Hypothesis TZDs inhibit bone metabolism through: aromatase enzyme inhibition direct effect on osteoblast/osteoclast

Objective  To examine the effects of TZDs on mouse osteoblast cells : cell growth, bone turnover markers, pro-collagen expression, cell differentiation  To examine whether aromatase inhibition plays a role in any of the TZD effects on mouse osteoblast cells

Methods – Culture System  A commercially available mouse osteoblast cell (MOC) line, 7F2, from American Type Culture Collection (ATCC), was co-cultured with or without human granulosa cells (HGC)  The cells were then incubated with: pioglitazone 25  M rosiglitazone 25  M Testosterone 1  M testosterone 1  M+ pioglitazone 25  M testosterone 1  M+ rosiglitazone 25  M

TZDs Inhibit Estradiol Synthesis Pioglitazone inhibited estradiol synthesis in the MOC and HGC co-culture p <0.001

TZD Effect on MOC-HGC Cell Growth (Optical Density) TZDs inhibit cell growth. Testosterone can ameliorate the cell growth inhibition caused by TZDs. p<0.02

TZD effect on MOC Cell Growth (Optical Density) Pioglitazone & Rosiglitazone are associated with decreased MOC growth as measured by optical density.

TZDs Affect Cell Growth in a Dose-Dependent Manner

TZD Effect on Osteoblast Growth/Differentiation Thiazolinediones inhibit cell growth and increase fat accumulation. MOC cultures were stained with Oil Red O to highlight the presence of adipocytes.

TZD Effect on Alkaline Phosphatase (AP) Activity Pioglitazone and Rosiglitazone are associated with decreased AP activity levels. Addition of testosterone to MOC+HGC co-culture “protects” AP activity levels from effects of Thiazolidinedione.

TZD Effect on Osteocalcin Synthesis Pioglitazone reduces osteocalcin production in MOC-HGC co-culture and MOC culture.

Mouse Pro-collagen mRNA expression Pioglitazone and rosiglitazone inhibit mouse pro- collagen mRNA expression

Conclusions  Pioglitazone and rosiglitazone  inhibit osteoblast cell growth  decrease bone turnover biomarkers (AP and osteocalcin levels)  decrease mouse pro-collagen mRNA expression  increase differentiation to adipocytes  Inhibition of aromatase by TZDs does not play a role in the osteoblast cell growth or in the effects of TZDs on bone turnover markers, since inhibition of cell growth and the effects on bone turnover markers were observed in MOC culture which did not contain granulosa cells.  Results are consistent with clinical studies showing increased fracture risk and bone loss in patients with diabetes treated with TZDs.

Future Research To study TZD effect in mouse osteoblasts:  bone turnover markers: RANKL expression FGF-23 expression MAPK-Erk1/2 expression Wnt signaling pathway  bone resorption markers: Type I collagen cross-linked N- (NTX) or C-telopeptide (CTX) TZDs effect on RANKL induction of osteoclast differentiation. To study effect of TZDs on mouse osteoclasts and human osteoblasts.

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