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Steven R. Smith, M.D.
                              tBHQ differentiates
Professor
Molecular Endocrinology Lab
            ...
An in-vitro model for early
adipocyte differentiation
Shortfalls with current models:
  – Most are murine
     • F442A
   ...
Signaling in human adipocytes
in-vitro model systems       Advantages:
   (1) human mesenchymal        – reagents cross-ov...
osteocytes

                             osteoblast


               MAPK (ERK1,2 activation), osteoblastic transcription ...
Mesenchymal
 Stem Cell         Osteoblast, chondrocyte, tenocyte...


              Preadipocyte



                      ...
Human mesenchymal stem cells
   •   Modified the methods of Pittenger, et al.
   •   hMSC isolated from bone marrow
   •  ...
Human mesenchymal stem cells
3T3-L1
                -2   0   2             8


                                0 1 2 3 4 5...
Human mesenchymal stem cells
     0.45                                           3+1
                                     ...
leptin mRNA
                  200
                           AIM             AIM                  AIM
                  15...
1                                    Troglitazone
                                           BRL 49653
     0.8
     0.6

...
Regulation of adipocyte
      differentiation
POSITIVE                NEGATIVE
  +   PPAR-γ agonists     - inflammatory
  ...
hMSC – inhibitors of differentiation
                                 ± 1 Standard Error(s)
               2.50
          ...
We have identified two compounds
that work as well as the TZD’s to
promote fat cell differentiation in vitro.
Our model sy...
control    tBHQ




oltipraz   TZD
Oil Red O staining

     1.2


     1.0

     0.8
OD




     0.6


     0.4


     0.2

     0.0
           control      ...
Oltipraz
• a dithiole derivate

• 4-Methyl-5-pyrazinyl-3H-1,2-dithiole-3-
  thione, CAS: 64224-21-1
• anti-helmintic
Oltipraz
• Oltipraz is also an anti-carcinogen.
  Safety data from a large clinical trial:
  ’Protective alterations in ph...
Oltipraz
• Dithiolthione compounds for the treatment
  of neurological disorders and for memory
  enhancement – WO109118A2...
Differentiation promoters are sometimes
             cell type specific

• Compound A = tBHQ
• Compound B = oltipraz
• Bot...
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  1. 1. Steven R. Smith, M.D. tBHQ differentiates Professor Molecular Endocrinology Lab adipocytes in vitro Director, Human Phenotyping Unpublished data to stimulate Core, CNRU discussion Baton Rouge, LA, USA smithsr@pbrc.edu labs.pbrc.edu/endocrinology Pennington Biomedical Research Center LSU System
  2. 2. An in-vitro model for early adipocyte differentiation Shortfalls with current models: – Most are murine • F442A • 3T3L1 – Most examine ‘committed ’ cells; i.e. preadipocytes. – Intuitive to believe that the step from mesenchymal stem cell to preadipocyte is a regulated event.
  3. 3. Signaling in human adipocytes in-vitro model systems Advantages: (1) human mesenchymal – reagents cross-over to stem cells (hMSC) & clinical studies (2) SGBS cells – opportunities for [Wabitsch, 2001] signaling systems that (3) primary human are not present in rodent adipocytes and S.V. cell lines; e.g. agouti cultures Disadvantages: – hMSC more expensive – reagents don’t cross over into rodent models
  4. 4. osteocytes osteoblast MAPK (ERK1,2 activation), osteoblastic transcription factors Pleuripotent precursor (MSC) adipocyte Pre-adipocyte
  5. 5. Mesenchymal Stem Cell Osteoblast, chondrocyte, tenocyte... Preadipocyte “Adipiphage” Mature Insulin-Sensitive Adipocyte Adipocyte C/EBPα C/EBPβ C/EBPδ PPARγ C/EBPα Other regulatory systems include: Wnt 5a/10, IGF-1, TNF- α, MCRs, et cetera
  6. 6. Human mesenchymal stem cells • Modified the methods of Pittenger, et al. • hMSC isolated from bone marrow • Purified by inverse selection • Differentiate into: – Neurons – Tenocytes – Skeletal muscle – Chondrocytes – Adipocytes – Osteoblasts 1. Pittenger MF, et al.: Multilineage potential of adult human mesenchymal stem cells. Science 284:143-147, 1999 2. Human Mesenchymal Stem Cells as an in Vitro Model for Human Adipogenesis Lenka Janderová, Michele McNeil, Angela N. Murrell, Randall L. Mynatt and Steven R. Smith Obesity Research 11:65-74 (2003)
  7. 7. Human mesenchymal stem cells 3T3-L1 -2 0 2 8 0 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19 20 21 hMSC 3+1 3+3 one induction Preconfluent cells FBS, insulin, DEX, IBMX, indomethacin Postconfluent cells FBS, insulin
  8. 8. Human mesenchymal stem cells 0.45 3+1 3+3 0.4 one induction 0.35 0.3 0.25 OD 0.2 0.15 0.1 0.05 0 10% 5% 2.5% 0.5% Concentration of serum during postconfluence period
  9. 9. leptin mRNA 200 AIM AIM AIM 150 arbitrary units 100 50 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 days 0 0.5 1 2 3 6 21 Days C/EBPß 48kDa GAPDH 36kDa 0 0.5 1 2 3 6 7 8 9 12 13 14 15 21 Days PPARγ 53kDa
  10. 10. 1 Troglitazone BRL 49653 0.8 0.6 OD 0.4 0.2 0 control 1.25 2.5 5 Concentration [µM] 0.35 0.35 0.3 0.3 0.25 control 0.25 control 0.2 (5%FBS) 0.2 OD OD (5%FBS) 0.15 15% rabbit 0.15 50 µM serum PD98059 0.1 0.1 0.05 0.05 0
  11. 11. Regulation of adipocyte differentiation POSITIVE NEGATIVE + PPAR-γ agonists - inflammatory + ADD/SREBP cytokines: + IGF-1 TNFα/IFNγ + glucocorticoids - BMP’s + T3 - MAPK + insulin ? - TGF-β + Wnt pathways - ADSF/resistin + rabbit serum - oxidative stress + Others !
  12. 12. hMSC – inhibitors of differentiation ± 1 Standard Error(s) 2.50 0 2.25 2.00 1.5 IFN-γ 1.75 3.12 (U/ml) OD 485nm 1.50 6.25 1.25 1.00 0.75 0.50 0.25 0 0 1.25 2.5 5 10 20 TNF-α (ng/ml)
  13. 13. We have identified two compounds that work as well as the TZD’s to promote fat cell differentiation in vitro. Our model system is human mesenchymal stem cells. Our read-out is oil-red-O staining which measures lipid storage.
  14. 14. control tBHQ oltipraz TZD
  15. 15. Oil Red O staining 1.2 1.0 0.8 OD 0.6 0.4 0.2 0.0 control TZD 20µM tBHQ 50µM olti
  16. 16. Oltipraz • a dithiole derivate • 4-Methyl-5-pyrazinyl-3H-1,2-dithiole-3- thione, CAS: 64224-21-1 • anti-helmintic
  17. 17. Oltipraz • Oltipraz is also an anti-carcinogen. Safety data from a large clinical trial: ’Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China’ J Natl Cancer Inst. 1999 Feb 17;91(4):347-54. • potential drug for the treatment of Alzheimer´s disease and other neurodegenerative disorders.
  18. 18. Oltipraz • Dithiolthione compounds for the treatment of neurological disorders and for memory enhancement – WO109118A2 with a patented synthesis of Oltipraz. (Edenland Holdings, Baybush, Ireland now Hollis-Eden) • The original US patent from Rhône-Poulenc (Aventis): 1,2-Dithiole derivatives – US4110450 which is based on a French patent from 1963.
  19. 19. Differentiation promoters are sometimes cell type specific • Compound A = tBHQ • Compound B = oltipraz • Both are inducers of ‘endogenous antioxidant enzymes’ such as NQO, HO1, etc. via nrf2
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