3rd Annual Friedman Fellows Symposium

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Thiazolidinediones and Bone Metabolism

• 3rd Annual Friedman Fellows Symposium
• November 13th 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.

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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-g
  (PPAR-g) 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.

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TZD effect on Ovarian Steroidogenesis

• 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
  906099-6105.

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TZDs Interaction with Insulin Signaling Pathways
and Effect on Steroidogenic Regulation
Seto-Young et al., 2007, J Clin Endocrinol Metab
92 2232-2239
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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-g 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 2232-2239

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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 Vmax
  and Km of aromatase, acting as un-competitive
  inhibitors.
• Seto-Young et al. 2010 Manuscript submitted to
  Hormone and Metabolic Research.

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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
  95356-3577

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Literature on the effects of TZDs on Bone
Metabolism

• Clinical trials
• 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)
• 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)
• 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)

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Literature on the effect of TZDs on Bone
Metabolism - continued

• In vitro studies
• 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)
• 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)
• TZDs inhibit TNF-a-mediated osteoclast-like cells
  differentiation (Yang et al, 2010)

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

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Hypothesis

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

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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

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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 25mM
• rosiglitazone 25mM
• Testosterone 1mM
• testosterone 1mM pioglitazone 25mM
• testosterone 1mM rosiglitazone 25mM

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TZDs Inhibit Estradiol Synthesis
Pioglitazone inhibited estradiol synthesis in the
MOC and HGC co-culture
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TZD Effect on MOC-HGC Cell Growth (Optical
Density)
TZDs inhibit cell growth. Testosterone can
ameliorate the cell growth inhibition caused by
TZDs.
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TZD effect on MOC Cell Growth (Optical Density)
Pioglitazone Rosiglitazone are associated with
decreased MOC growth as measured by optical
density.
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TZDs Affect Cell Growth in a Dose-Dependent Manner
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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.
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TZD Effect on Alkaline Phosphatase (AP) Activity
Pioglitazone and Rosiglitazone are associated
with decreased AP activity levels. Addition of
testosterone to MOCHGC co-culture protects AP
activity levels from effects of Thiazolidinedione.
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TZD Effect on Osteocalcin Synthesis
Pioglitazone reduces osteocalcin production in
MOC-HGC co-culture and MOC culture.
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Mouse Pro-collagen mRNA expression
Pioglitazone and rosiglitazone inhibit mouse
pro-collagen mRNA expression
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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.

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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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Thank You!