quences, referred to as the upstream insulin regula- 
tory element A (IRE-A) and downstream (IRE-B) el- 
ements, respectively. Both elements independently 
conferred a twofold inductive effect of insulin on 
gene expression and together resulted in a three- 
fold effect. 
The gel shift assay was used to detect transacting 
factors that interact with these elements. Both IRE- 
A and IRE-B interacted with insulin-sensitive DNA- 
iMnding proteins. Within 60 min of exposure of 3T3 
adipocytes or H35 hepatoma cells to insulin, the 
activity of these sequence-specific DNA-binding 
proteins is increased at least fourfold. This effect is 
not inhibited by protein synthesis inhibitors. The 
IRE-A DNA-binding protein is induced 10-fold in 
liver and fat during the process of fasting and re- 
feeding rats a high-carbohydrate, low-fat diet, again 
supporting the importance of GAPDH gene regula- 
tion in vivo. Finally, diabetic animals show a de- 
crease in IRE-A DNA-binding activity and a marked 
stimulation of binding activity in extracts derived 
from insulin-treated diabetic animals. 
To examine whether the acquisition of hormone- 
responsive GAPDH gene expression during differ- 
entiation of 3T3 preadipocytes to adipocytes was 
correlated with a change in activity of a DNA-bind- 
ing protein, the laboratory has used the gel mobil- 
ity shift assay to detect the interaction of nuclear 
proteins and IRE-A or IRE-B DNA. DNA binding cor- 
related with the pattern of hormone-regulated 
GAPDH gene expression in vivo and in cultured 
cell lines. The IRE-A DNA-binding protein was 
barely detectable in 3T3 preadipocytes and was in- 
duced markedly by differentiation. The IRE-B DNA- 
binding protein was not detectable in 3T3 pre- 
adipocytes under any conditions but was highly 
responsive to insulin in 3T3 adipocytes. At the same 
time, the SRE-binding domain, which appears to 
mediate the effect of insulin on c-fos and EGR gene 
transcription, was used to show that equivalent 
amounts of SRE-binding activity had been extracted 
from 3T3 preadipocytes and adipocytes. These re- 
sults indicated that the factors that mediate the 
growth-related effects of insulin on gene expres- 
PUBLICATIONS 
sion in 3T3 cells were constitutively present, while 
the factors that mediate the effect of insulin on 
GAPDH gene expression are expressed in differenti- 
ated tissues with lipogenic capacity. Thus the mech- 
anism by which insulin achieves tissue-specific reg- 
ulation of the GAPDH gene involves the induction 
of a novel set of transcription factors in terminally 
differentiated cells. 
The activity of the IRE-A and IRE-B DNA-binding 
proteins is increased acutely and chronically by insu- 
lin. Efforts to understand the mechanisms that un- 
derlie this effect will be greatly facilitated by clon- 
ing these factors. Dr. Alexander-Bridges and her 
colleagues have isolated a clone that expresses a fu- 
sion protein capable of specific interaction with the 
IRE-A. Mutation of bases critical to the DNA-protein 
interaction inhibits binding of the fusion protein 
with IRE-A DNA. Further characterization of this 
clone is under way. Should further studies support 
the identity of this fusion protein as a transcription 
factor, antibodies to this protein will be raised and 
used to study the acute regulation of its DNA-bind- 
ing activity in insulin-sensitive cells. Together these 
antibodies and cDNA clones will be used to exam- 
ine the tissue distribution of this factor and to dis- 
sect the mechanism by which its expression and/or 
regulation are limited to specific tissues. Ultimately 
these studies will lead to an understanding of the 
mechanism by which insulin modulates the expres- 
sion of specific genes involved in the maintenance 
of normal glucose and lipid metabolism. For exam- 
ple, GAPDH gene expression is markedly increased 
in the fat pads of obese Zucker rats but not in other 
insulin-sensitive tissues. With these clones, the reg- 
ulation of IRE-A gene expression in the tissues of 
these animals can be examined. Understanding the 
hormonal control of lipid metabolism at a molecu- 
lar level will provide invaluable insights into the 
mechanisms of obesity, a disease of fundamental 
importance in promoting diabetes. 
Dr. Alexander-Bridges is also Clinical Assistant at 
Massachusetts General Hospital and Assistant Pro- 
fessor of Medicine at Harvard Medical School. 
Books and Chapters of Books 
Alexander, M., Denaro, M., Galli, R., Kahn, B., and Nasrin, N. 1989. Tissue specific regulation of the glyceral- 
dehyde-3-phosphate dehydrogenase gene by insulin correlates with the induction of an insulin-sensitive 
transcription factor during differentiation of 3T3 adipocytes. In Obesity. Towards a Molecular Approach. 
UCLA Symposia on Molecular and Cellular Biology (Bray, G., Ricquier, D., and Spiegelman, B.M., Eds.). 
New York: Wiley-Liss. 
Continued 
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