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Steven R. Smith, M.D. Professor Molecular Endocrinology Lab Director, Human Phenotyping Core, CNRU Baton Rouge, LA, USA [email protected] labs.pbrc.edu/endocrinology Pennington Biomedical Research Center LSU System tBHQ differentiates adipocytes in vitro Unpublished data to stimulate discussion
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Steven R. Smith, M.D.
ProfessorMolecular Endocrinology LabDirector, Human Phenotyping Core, CNRU
Baton Rouge, LA, [email protected]/endocrinologyPennington Biomedical Research CenterLSU System
tBHQ differentiates adipocytes in vitro
Unpublished data to stimulate discussion
An in-vitro model for early adipocyte differentiationShortfalls 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.
Signaling in human adipocytesin-vitro model systems
(1) human mesenchymalstem cells (hMSC) &
(2) SGBS cells [Wabitsch, 2001]
(3) primary human adipocytes and S.V. cultures
Advantages:– reagents cross-over to
clinical studies– opportunities for
signaling systems that are not present in rodent cell lines; e.g. agouti
Disadvantages:– hMSC more expensive– reagents don’t cross over
into rodent models
adipocyte
Pleuripotentprecursor
(MSC)
Pre-adipocyte
osteoblast
osteocytes
MAPK (ERK1,2 activation), osteoblastic transcription factors
MesenchymalStem Cell
Preadipocyte
“Adipiphage”
Mature Insulin-Sensitive
Adipocyte
C/EBPβ
C/EBPδ
C/EBPα
PPARγC/EBPα
Adipocyte
Osteoblast, chondrocyte, tenocyte...
Other regulatory systems include: Wnt 5a/10, IGF-1, TNF- α, MCRs, et cetera
Human mesenchymal stem cells• Modified the methods of Pittenger, et al.• hMSC isolated from bone marrow• Purified by inverse selection• Differentiate into:
– Neurons– Skeletal muscle– Adipocytes
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)
– Tenocytes– Chondrocytes– Osteoblasts
Human mesenchymal stem cells
hMSC
3T3-L1
FBS, insulin, DEX, IBMX, indomethacinPreconfluent cells
Postconfluent cells FBS, insulin
-2 0 2 8
1 2 3 4 5 6 8 9 10 12 1314 15 16 17 18 19 200 7 2111
-2 0 2 8
1 2 3 4 5 6 8 9 10 12 1314 15 16 17 18 19 200 7 2111
3+1
3+3
one induction
Human mesenchymal stem cells
00.050.1
0.150.2
0.250.3
0.350.4
0.45
10% 5% 2.5% 0.5%Concentration of serum during postconfluence period
OD
3+13+3one induction
0
50
100
150
200
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22days
arbi
trary
uni
ts
leptin mRNA
AIM AIM AIM
GAPDH
C/EBPß
36kDa
48kDa
0 0.5 1 2 3 6 21 Days
53kDa
0 0.5 1 2 3 6 7 8 12 13 14 15 219 Days
PPARγ
0
0.2
0.40.6
0.8
1
control 1.25 2.5 5Concentration [µM]
OD
TroglitazoneBRL 49653
00.050.1
0.150.2
0.250.3
0.35
OD
control(5%FBS)15% rabbitserum
control(5%FBS)50 µMPD98059
0.050.1
0.150.2
0.250.3
0.35
OD
Regulation of adipocyte differentiation
POSITIVE+ PPAR-γ agonists+ ADD/SREBP+ IGF-1+ glucocorticoids+ T3
+ insulin ?+ Wnt pathways+ rabbit serum+ Others !
NEGATIVE- inflammatory
cytokines: TNFα/IFNγ
- BMP’s- MAPK- TGF-β- ADSF/resistin- oxidative stress
hMSC – inhibitors of differentiation
0
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50OD485nm
0 1.25 2.5 5 10 20
6.25
3.12
1.5
0
6.25
3.12
1.5
0
± 1 Standard Error(s)
TNF-α(ng/ml)
IFN-γ(U/ml)
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.
control tBHQ
oltipraz TZD
Oil Red O staining
0.0
0.2
0.4
0.6
0.8
1.0
1.2
control TZD 20µM tBHQ 50µM olti
OD
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 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.
Oltipraz
• Dithiolthione compounds for the treatment of neurological disorders and for memory enhancement – WO109118A2 with a patented synthesis of Oltipraz. (EdenlandHoldings, 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.
Differentiation promoters are sometimescell type specific
• Compound A = tBHQ• Compound B = oltipraz• Both are inducers of ‘endogenous
antioxidant enzymes’ such as NQO, HO1, etc. via nrf2
