Molecular and Cellular Biology, January 2001, p. 319-329, Vol. 21, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.319-329.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 022151; Department of Internal Medicine III, University of Leipzig, 04103 Leipzig,2 and Department of Internal Medicine I, Medical University of Lübeck, 23538 Lübeck,3 Germany; and Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain4
Received 31 May 2000/Returned for modification 12 July 2000/Accepted 11 October 2000
The most widely distributed members of the family of insulin
receptor substrate (IRS) proteins are IRS-1 and IRS-2. These proteins participate in insulin and insulin-like growth factor 1 signaling, as well as the actions of some cytokines, growth hormone, and prolactin. To more precisely define the specific role of
IRS-1 in adipocyte biology, we established brown
adipocyte cell lines from wild-type and IRS-1 knockout (KO) animals.
Using differentiation protocols, both with and without insulin,
preadipocyte cell lines derived from IRS-1 KO mice exhibited a marked
decrease in differentiation and lipid accumulation (10 to 40%)
compared to wild-type cells (90 to 100%). Furthermore, IRS-1 KO
cells showed decreased expression of adipogenic marker proteins,
such as peroxisome proliferator-activated receptor gamma (PPAR
),
CCAAT/enhancer-binding protein alpha (C/EBP
), fatty acid
synthase, uncoupling protein-1, and glucose transporter 4. The
differentiation deficit in the KO cells could be reversed almost
completely by retrovirus-mediated reexpression of IRS-1, PPAR
, or
C/EBP
but not the thiazolidinedione troglitazone.
Phosphatidylinositol 3-kinase (PI 3-kinase) assays performed at various
stages of the differentiation process revealed a strong and transient
activation in IRS-1, IRS-2, and phosphotyrosine-associated PI
3-kinase in the wild-type cells, whereas the IRS-1 KO cells showed
impaired phosphotyrosine-associated PI 3-kinase activation, all of
which was associated with IRS-2. Akt phosphorylation was reduced in
parallel with the total PI 3-kinase activity. Inhibition of PI 3-kinase
with LY294002 blocked differentiation of wild-type cells. Thus,
IRS-1 appears to be an important mediator of brown adipocyte
maturation. Furthermore, this signaling molecule appears to exert its
unique role in the differentiation process via activation of PI
3-kinase and its downstream target, Akt, and is upstream of the effects
of PPAR
and C/EBP
.
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