Steven R. Smith, M.D.

ProfessorMolecular Endocrinology LabDirector, Human Phenotyping Core, CNRU

Baton Rouge, LA, USAsmithsr@pbrc.edulabs.pbrc.edu/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Unpublished data to stimulate discussion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