The Role of The Role of Thiazolidinediones in Bone Thiazolidinediones in Bone

MetabolismMetabolism

David C. Gajzer M.D.David C. Gajzer M.D.Pauline Suwandhi M.D.Pauline Suwandhi M.D.

Yun Feng M.D.Yun Feng M.D.Amit K. Seth M.D.Amit K. Seth M.D.

Leonid Poretsky M.D.Leonid Poretsky M.D.Donna Seto-Young Ph.D.Donna Seto-Young Ph.D.

BackgroundBackground

ThiazolidinedionesThiazolidinediones• Used for treatment of type 2 diabetes mellitusUsed for treatment of type 2 diabetes mellitus

• Increase insulin sensitivity of peripheral tissues Increase insulin sensitivity of peripheral tissues by binding and activating peroxisome by binding and activating peroxisome proliferator activated receptor-proliferator activated receptor-γγ (PPAR- (PPAR-γγ))

Reported Effects of TZDs on Reported Effects of TZDs on Bone MetabolismBone Metabolism

• Inhibited bone formation and mineralization Inhibited bone formation and mineralization (Johnson (Johnson et al., 1999)et al., 1999)

• Decreased osteoblastogenesis, increased bone Decreased osteoblastogenesis, increased bone marrow adiposity in men and women marrow adiposity in men and women (Okazaki et al., (Okazaki et al., 1999; Watanabe et al., 2003)1999; Watanabe et al., 2003)

• Induced bone loss in older diabetic women Induced bone loss in older diabetic women (Schwartz (Schwartz et al., 2006)et al., 2006)

• Decrease in bone osteoblast markers pro-collagen Decrease in bone osteoblast markers pro-collagen type-1 N-terminal pro-peptide (P1NP) and type-1 N-terminal pro-peptide (P1NP) and osteocalcin, but no change in type 1 collagen N-osteocalcin, but no change in type 1 collagen N-telopeptide (NTX) telopeptide (NTX) (Grey et al.,(Grey et al., 2007)2007)

• ADOPT trial reported a higher risk of fractures in ADOPT trial reported a higher risk of fractures in diabetic women randomized to rosiglitazone diabetic women randomized to rosiglitazone compared to metformin and glyburide compared to metformin and glyburide (Kahn et al., (Kahn et al., 2006)2006)

Effects of Thiazolidinediones on Effects of Thiazolidinediones on Aromatase ActivityAromatase Activity

It was recently demonstrated that It was recently demonstrated that thiazolidinediones:thiazolidinediones:

• Exhibited inhibitory effects on aromatase Exhibited inhibitory effects on aromatase enzyme activity by interfering with the enzyme activity by interfering with the androgen substrate binding to aromatase androgen substrate binding to aromatase (Seto-Young et al., 2007; Avtanski et al., 2007)(Seto-Young et al., 2007; Avtanski et al., 2007)

• Reduced estrogen biosynthesis in human Reduced estrogen biosynthesis in human granulosa cellsgranulosa cells(Seto-Young et al., 2007; Avtanski et al., 2007)(Seto-Young et al., 2007; Avtanski et al., 2007)

HypothesisHypothesis

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

ObjectiveObjective

• In human granulosa cells and osteoblast cell co-In human granulosa cells and osteoblast cell co-cultures stimulated with TZDs, compare:cultures stimulated with TZDs, compare:– 1. Bone Turnover Markers:1. Bone Turnover Markers:

Alkaline phosphataseAlkaline phosphatase OsteocalcinOsteocalcin Procollagen type-1 propeptide (P1NP)Procollagen type-1 propeptide (P1NP) Type-1 collagen N-telopeptide (NTX)Type-1 collagen N-telopeptide (NTX)

– 2. Estradiol and Estrone levels2. Estradiol and Estrone levels– 3. Cell Growth 3. Cell Growth – 4. Cell Differentiation4. Cell Differentiation

MethodsMethods• Osteoblast Cells and purified Human Granulosa Cells Osteoblast Cells and purified Human Granulosa Cells

were co-cultured for 2-3 dayswere co-cultured for 2-3 days• Mixed cultures were stimulated with testosterone alone, Mixed cultures were stimulated with testosterone alone,

pioglitazone alone, and testosterone and pioglitazone pioglitazone alone, and testosterone and pioglitazone together.together.

• Estradiol levels were measured using a Estradiol levels were measured using a radioimmunoassay.radioimmunoassay.

• Alkaline Phosphatase levels were measured using an Alkaline Phosphatase levels were measured using an ELISA assay.ELISA assay.

• Optical density was measured.Optical density was measured.– Stimulated plates were trypsinizedStimulated plates were trypsinized– Cells were isolated and then resuspended in 1ml PBS Cells were isolated and then resuspended in 1ml PBS

BufferBuffer– Using spectrophotometry, the optical density of cell Using spectrophotometry, the optical density of cell

solutions was measured at 600 nmsolutions was measured at 600 nm• Cell plates were stained with Hematoxylin, Eosin, and Cell plates were stained with Hematoxylin, Eosin, and

Oil Red O stains.Oil Red O stains.

Estradiol Radioimmuno-AssayEstradiol Radioimmuno-Assay

Time (Hours) Cell Plate Average Estradiol Activity Percent Activity

0 Mouse Osteoblast Cells 0.00 0.0

0Mouse Osteoblast Cells+ Testosterone

0.00 0.0

0Mouse Osteoblast Cells+ Pioglitazone

0.00 0.0

0Mouse Osteoblast Cells+Testosterone+Pioglitazone

0.00 0.0

48 Mouse Osteoblast Cells 0.00 0.0

48Mouse Osteoblast Cells+ Testosterone

4402.07 100%

48Mouse Osteoblast Cells+ Pioglitazone

0.00 0.0

48Mouse Osteoblast Cells+Testosterone+Pioglitazone

785.86 17.9%

96 Mouse Osteoblast Cells 0.00 0.0

96Mouse Osteoblast Cells+ Testosterone

7354.37 100%

96Mouse Osteoblast Cells+ Pioglitazone

0.00 0.0

96Mouse Osteoblast Cells+Testosterone+Pioglitazone

1184.22 16.1%

Pioglitazone is associated with decreased estradiol levels.

ELISA Alkaline Phosphatase ELISA Alkaline Phosphatase AssayAssay

Cell PlatesCell PlatesAverage Alkaline Average Alkaline Phosphatase LevelPhosphatase Level

Alkaline PhosphataseAlkaline Phosphatase Specific ActivitySpecific Activity

Specific ActivitySpecific Activity%%

Mouse Osteoblast &Mouse Osteoblast &Human Granulosa CellsHuman Granulosa Cells 1.6581.658 0.1130.113 100100

Mouse Osteoblast & Mouse Osteoblast & Human Granulosa CellsHuman Granulosa Cells+ Testosterone+ Testosterone

1.5401.540 0.1110.111 100100

Mouse Osteoblast & Mouse Osteoblast & Human Granulosa CellsHuman Granulosa Cells+ Pioglitazone+ Pioglitazone

0.7390.739 0.0600.060 52.752.7

Mouse Osteoblast & Mouse Osteoblast & Human Granulosa CellsHuman Granulosa Cells+ Testosterone+ Testosterone+ Pioglitazone+ Pioglitazone

0.7840.784 0.0660.066 59.859.8

Pioglitazone is associated with decreased alkaline phosphatase levels.

Cell GrowthCell GrowthCell PlatesCell Plates Average Optical Density Average Optical Density

(at 600nm)(at 600nm)

Mouse Osteoblast CellsMouse Osteoblast Cells 1.304 (100%)1.304 (100%)

Mouse Osteoblast Cells Mouse Osteoblast Cells + Testosterone+ Testosterone

1.308 (100%)1.308 (100%)

Mouse Osteoblast Cells Mouse Osteoblast Cells + Pioglitazone+ Pioglitazone

1.162 (89%)1.162 (89%)

Mouse Osteoblast CellsMouse Osteoblast Cells+ Testosterone+ Testosterone+ Pioglitazone+ Pioglitazone

1.227 (94%)1.227 (94%)

Pioglitazone is associated with decreased cell growth as measured by optical density.

Cell DifferentiationCell Differentiation

1st row: Mouse Osteoblast Cells and Human Granulosa Cells cultured with 2% FBS MEM Alpha Medium

2nd row: Cells stimulated with 2% FBS MEM Alpha Medium and Pioglitazone display increased lipogenesis (Oil Red O stain).

3rd row: Cells stimulated with 2% FBS MEM Alpha Medium and Testosterone display increased growth and eosinophily (due to increased cytoplasmatic protein synthesis).

4th 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

Conclusion (Preliminary Conclusion (Preliminary Results)Results)

• TZDs, specifically pioglitazone, may lead to TZDs, specifically pioglitazone, may lead to decreased bone turnover as measured by decreased bone turnover as measured by alkaline phosphatase levels.alkaline phosphatase levels.

• TZDs may lead to decreased estradiol levels.TZDs may lead to decreased estradiol levels.• TZDs may inhibit cell growth of mouse TZDs may inhibit cell growth of mouse

osteoblast cells.osteoblast cells.• TZDs may lead to increased differentation of TZDs may lead to increased differentation of

mouse osteoblast cells into adipocytes.mouse osteoblast cells into adipocytes.• Further research is needed to determine the Further research is needed to determine the

effects of pioglitazone on bone metabolism.effects of pioglitazone on bone metabolism.