2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity is particularly striking in adipose tissue, where it causes severe wasting. This phenomenon suggests that TCDD could have effects on adipocyte differentiation, now demonstrated using 3T3-L1 cells as a model system. When cells were treated with 10 nM TCDD before differentiation or during the first two days of induction in the presence of dexamethasone (dex) and isobutylmethylxanthine (IBMX), a reduction occurred in the number of fat cell colonies measured 7–10 days later by Oil Red O staining. Northern blotting showed an accompanying reduction in amounts of mRNA encoding several adipocyte markers. In contrast, when TCDD was added after differentiation, it had no effect on the maintenance of the adipose phenotype. Dose-response and structure-activity relationships were consistent with a process mediated by interaction of TCDD with the Ah receptor. The possibility that TCDD acts by inhibiting the signaling pathways activated by dex and IBMX was investigated. TCDD did not interfere with glucocorticoid-inducible transcription as judged by the unimpaired responsiveness of a transfected reporter construct. Treatment of cells with TCDD augmented the increase in protein kinase A (PKA) activity elicited by either IBMX or forskolin; therefore, if TCDD disrupts the cAMP signaling pathway, it must do so at a step after activation of PKA.

REFERENCES

Ailhaud
G.
,
Grimaldi
P.
,
Negrel
R.
(
1992
).
Cellular and molecular aspects of adipose tissue development.
Annu. Rev. Nutr
12
,
207
233
Bradford
M. M.
(
1976
).
A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding.
Anal. Biochem
72
,
248
254
Brewster
D. W.
,
Matsumura
F.
(
1984
).
TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) reduces lipoprotein lipase activity in the adipose tissue of the guinea pig.
Biochem. Biophys. Res. Commun
122
,
810
817
Burbach
K. M.
,
Poland
A.
,
Bradfield
C. A.
(
1992
).
Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor.
Proc. Nat. Acad. Sci. USA
89
,
8185
8189
Canga
L.
,
Levi
R.
,
Rifkind
A. B.
(
1988
).
Heart as a target organ in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity: decreased-adrenergic responsiveness and evidence of increased intracellular calcium.
Proc. Nat. Acad. Sci. USA
85
,
905
909
Cao
Z.
,
Umek
R. M.
,
McKnight
S. L.
(
1991
).
Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells.
Genes Dev
5
,
1538
1552
Chirgwin
J. M.
,
Przybyla
A. E.
,
MacDonald
R. J.
,
Rutter
W. J.
(
1979
).
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18
,
5294
5299
Cook
J. S.
,
Lucas
J. J.
,
Sibley
E.
,
Bolanowski
M. A.
,
Christy
R. J.
,
Kelly
T. J.
,
Lane
M. D.
(
1988
).
Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and cAMP.
Proc. Nat. Acad. Sci. USA
85
,
2949
2953
Cook
K. S.
,
Hunt
C. R.
,
Spiegelman
B. M.
(
1985
).
Developmentally regulated mRNAs in 3T3-adipocytes: analysis of transcriptional control.
J. Cell Biol
100
,
514
520
Dani
C.
,
Amri
E.-Z.
,
Bertrand
B.
,
Enerback
S.
,
Bjursell
G.
,
Grimaldi
P.
,
Ailhaud
G.
(
1990
).
Expression and regulation of pOb24 and lipoprotein lipase genes during adipose conversion.
J. Cell. Biochem
43
,
103
110
Denison
M. S.
,
Fisher
J. M.
,
Whitlock
J. P.
(
1988
).
Inducible, receptor-dependent protein-DNA interactions at a dioxin-responsive transcriptional enhancer.
Proc. Nat. Acad. Sci. USA
85
,
2528
2532
Diamond
L.
,
O'Brien
T. G.
,
Rovera
G.
(
1977
).
Inhibition of adipose conversion of 3T3 fibroblasts by tumour promoters.
Nature
269
,
247
249
Elks
M. L.
,
Manganiello
V. C.
(
1985
).
A role for soluble cAMP phosphodiesterases in differentiation of 3T3-L1 adipocytes.
J. Cell. Physiol
124
,
191
198
Enan
E.
,
Liu
P. C. C.
,
Matsumura
F.
(
1992
).
2,3,7,8-tetrachlorodibenzo-p-dioxin causes reduction of glucose transporting activities in the plasma membranes of adipose tissue and pancreas from the guinea pig.
J. Biol. Chem
267
,
19785
19791
Enan
E.
,
Matsumura
F.
(
1993
).
2,3,7,8-tetrachlorodibenzo-p-dioxin induced alterations in protein phosphorylation in guinea pig adipose tissue.
J. Biochem. Toxicol
8
,
89
99
Enan
E.
,
Matsumura
F.
(
1994
).
Significance of TCDD-induced changes in protein phosphorylation in the adipocyte of male guinea pigs.
J. Biochem. Toxicol
9
,
159
170
Feinberg
A. P.
,
Vogelstein
B.
(
1983
).
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal. Biochem
132
,
6
13
Fourney
R. M.
,
Miyakoshi
J.
,
Day
R. S.
,
Paterson
M. C.
(
1988
).
Northern blotting: efficient RNA staining and transfer.
Focus
10
,
5
7
Gasiewicz
T. A.
,
Rucci
G.
(
1991
).
-Naphthoflavone acts as an antagonist of 2,3,7,8-tetrachloro-dibenzo-p-dioxin by forming an inactive complex with the Ah receptor.
Mol. Pharmacol
40
,
607
612
Graves
R. A.
,
Tontonoz
P.
,
Ross
S. R.
,
Spiegelman
B. M.
(
1991
).
Identification of a potent adipocyte-specific enhancer: involvement of an NF-1-like factor.
Genes Dev
5
,
428
437
Green
H.
,
Kehinde
O.
(
1974
).
Sublines of mouse 3T3 cells that accumulate lipid.
Cell
1
,
113
116
Green
H.
,
Meuth
M.
(
1974
).
An established pre-adipose cell line and its differentiation in culture.
Cell
3
,
127
133
Green
H.
,
Kehinde
O.
(
1976
).
Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells.
Cell
7
,
105
113
Gregoire
F.
,
de Broux
N.
,
Hauser
N.
,
Heremans
H.
,
van Damme
J.
,
Remacle
C.
(
1992
).
Interferon-and interleukin-1 inhibit adipoconversion in cultured rodent preadipocytes.
J. Cell. Physiol
151
,
300
309
Hayashi
I.
,
Nixon
T.
,
Morikawa
M.
,
Green
H.
(
1981
).
Adipogenic and anti-adipogenic factors in the pituitary and other organs.
Proc. Nat. Acad. Sci. USA
78
,
3969
3972
Hoffman
E. C.
,
Reyes
H.
,
Chu
F.
,
Sander
F.
,
Conley
L. H.
,
Brooks
B. A.
,
Hankinson
O.
(
1991
).
Cloning of a factor required for activity of the Ah (dioxin) receptor.
Science
252
,
954
958
Hotamisligil
G. S.
,
Shargill
N. S.
,
Spiegelman
B. M.
(
1993
).
Adipose expression of tumor necrosis factor-: direct role in obesity-linked insulin resistance.
Science
259
,
87
91
Hudson
L. G.
,
Toscano
W. A.
,
Greenlee
W. F.
(
1985
).
Regulation of epidermal growth factor binding in a human keratinocyte cell line by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Toxicol. Appl. Pharmacol
77
,
251
259
Ignotz
R. A.
,
Massague
J.
(
1985
).
Typetransforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.
Proc. Nat. Acad. Sci. USA
82
,
8530
8534
Kaerenlampi
S. O.
,
Eisen
H.
,
Hankinson
O.
,
Nebert
D. W.
(
1983
).
Effects of cytochrome P1-450 inducers on the cell-surface receptors for epidermal growth factor, phorbol 12,13-dibutyrate, or insulin of cultured mouse hepatoma cells.
J. Biol. Chem
258
,
10378
19383
Kasturi
R.
,
Joshi
V. C.
(
1982
).
Hormonal regulation of stearoyl coenzyme A desaturase activity and lipogenesis during adipose conversion of 3T3-L1 cells.
J. Biol. Chem
257
,
12224
12230
Kirchgessner
T. G.
,
Svenson
K. L.
,
Lusis
A. J.
,
Schotz
M. C.
(
1987
).
The sequence of a cDNA encoding lipoprotein lipase: A member of the lipase gene family.
J. Biol. Chem
262
,
8463
8466
Kuri-Harcuch
W.
,
Wise
L. S.
,
Green
H.
(
1978
).
Interruption of the adipose conversion of 3T3 cells by biotin dificiency: differentiation without triglyceride accumulation.
Cell
14
,
53
58
Kuri-Harcuch
W.
(
1982
).
Differentiation of 3T3-F442A cells into adipocytes is inhibited by retinoic acid.
Differentiation
23
,
164
169
Madhukar
B. V.
,
Brewster
D. W.
,
Matsumura
F.
(
1984
).
Effects of in vivo-administered 2,3,7,8-tetrachlorodibenzo-p-dioxin on receptor binding of epidermal growth factor in the hepatic plasma membrane of rat, guinea pig, mouse, and hamster.
Proc. Nat. Acad. Sci. USA
81
,
7407
7411
Olsen
H.
,
Enan
E.
,
Matsumura
F.
(
1994
).
Regulation of glucose transport in the NIH 3T3 L1 preadipocyte cell line by TCDD.
Environ. Health Perspect
102
,
454
458
Pairault
J.
,
Green
H.
(
1979
).
A study of the adipose conversion of suspended 3T3 cells by using glycerophosphate dehydrogenase as differentiation marker.
Proc. Nat. Acad. Sci. USA
76
,
5139
5142
Poland
A.
,
Knutson
J. C.
(
1982
).
2,3,7,8-Tetrachloro-dibenzo-p-dioxin and related aromatic hydorcarbons: examination of the mechanism of toxicity.
Annu. Rev. Pharmacol. Toxicol
22
,
517
554
Reyes
H.
,
Reisz-Porszasz
S.
,
Hankinson
O.
(
1992
).
Identification of the Ah receptor nuclear translocator protein (arnt) as a component of the DNA binding form of the Ah receptor.
Science
256
,
1193
1195
Rubin
C. S.
,
Hirsch
A.
,
Fung
C.
,
Rosen
O. M.
(
1978
).
Development of hormone receptors and hormonal responsiveness in vitro.
J. Biol. Chem
253
,
7570
7578
Russell
T. H.
,
Ho
R.-J.
(
1976
).
Conversion of 3T3 fibroblasts into adipose cells: triggering of differentiation by prostaglandin F2and 1-methyl-3-isobutyl xanthine.
Proc. Nat. Acad. Sci. USA
73
,
4516
4520
Santostefano
M.
,
Merchant
M.
,
Arellano
L.
,
Morrison
V.
,
Denison
M. S.
,
Safe
S.
(
1993
).
-Naphtoflavone-induced cyp1a1 gene expression and cytosolic aryl hydrocarbon receptor transformation.
Mol. Pharmacol
43
,
200
206
Schmidt
W.
,
Poll-Jordan
G.
,
Loffler
G.
(
1990
).
Adipose conversion of 3T3-L1 cells in a serum-free culture system depends on epidermal growth factor, insulin-like growth factor-I, corticosterone, and cyclic AMP.
J. Biol. Chem
265
,
15489
15495
Smith
P. J.
,
Wise
L. S.
,
Berkowitz
R.
,
Wan
C.
,
Rubin
C. S.
(
1988
).
Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes.
J. Biol. Chem
263
,
9402
9408
Spiegelman
B. M.
,
Frank
M.
,
Green
H.
(
1983
).
Molecular cloning of mRNA from 3T3 adipocytes.
J. Biol. Chem
258
,
10083
10089
Spiegelman
B. M.
,
Choy
L.
,
Hotamisligil
G. S.
,
Graves
R. A.
,
Tontonoz
P.
(
1993
).
Regulation of adipocyte gene expression indifferentiation and syndromes of obesity/diabetes.
J. Biol. Chem
268
,
6823
6826
Spiegelman
B. M.
,
Hotamisligil
G. S.
(
1993
).
Through thick and thin: wasting, obesity, and TNF.
Cell
73
,
625
627
Stahl
B. U.
,
Beer
D. G.
,
Weber
L. W. D.
,
Rozman
K.
(
1993
).
Reduction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is due to decreased mRNA levels.
Toxicology
79
,
81
95
Taylor
M. J.
,
Lucier
G. W.
,
Mahler
J. F.
,
Thompson
M.
,
Lockhart
A. C.
,
Clark
G. C.
(
1992
).
Inhibition of acute TCDD toxicity by treatment with anti-tumor necrosis factor antibody or dexamethasone.
Toxicol. Appl. Pharmacol
117
,
126
132
Torti
F. M.
,
Dieckman
B.
,
Beutler
B.
,
Cerami
A.
,
Ringold
G. M.
(
1985
).
A macrophage factor inhibits adipocyte gene expression: an in vitro model of cachexia.
Science
229
,
867
869
Torti
F. M.
,
Torti
S. V.
,
Larrick
J. W.
,
Ringold
G. M.
(
1989
).
Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta.
J. Cell Biol
108
,
1105
1113
Whitelaw
M.
,
Pongratz
I.
,
Wilhelmsson
A.
,
Gustafsson
J.-A.
,
Poellinger
L.
(
1993
).
Ligand-dependent recruitment of the arnt coregulator determines DNA recognition by the dioxin receptor.
Mol. Cell. Biol
13
,
2504
2514
Whitlock
J. P.
(
1990
).
Genetic and molecular aspects of 2,3,7,8 tetrachlorodibenzo-p-dioxin action.
Annu. Rev. Pharmacol. Toxicol
30
,
251
277
Williams
I. H.
,
Polakis
S. E.
(
1977
).
Differentiation of 3T3-L1 fibroblasts to adipocytes: the effect of indomethacin, prostaglandin E1 and cyclic AMP on the process of differentiation.
Biochem. Biophys. Res. Commun
77
,
175
186
Wise
L. S.
,
Green
H.
(
1979
).
Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells.
J. Biol. Chem
254
,
273
275
Zezulak
K. M.
,
Green
H.
(
1986
).
The generation of insulin-like growth factor-1-sensitive cells by growth hormone action.
Science
233
,
551
553
This content is only available via PDF.