Molecular Mechanisms of Tissue-Specific 
Hormonal Regulation of Gene Expression 
Maria C. Alexander-Bridges, M.D., Ph.D. — Assistant Investigator 
Dr. Alexander- Bridges is also Assistant Professor of Medicine at Harvard Medical School and Clinical As- 
sistant at Massachusetts General Hospital. She received her M.D. and Ph.D. degrees from Harvard Univer- 
sity Medical School, where she was a member of the Harvard-MIT Health Sciences and Technology pro- 
gram, which is geared toward students interested in academic medicine. She developed an abiding interest 
/■ in hormonal regulation of cellular metabolism and, as a graduate student in physiology, investigated the 
I mechanism of insulin- stimulated phosphorylation of cellular proteins. Dr. Alexander- Bridges then served 
as an intern and resident at the Johns Hopkins University. After subspecialty training in endocrinology 
at Massachusetts General Hospital, she was a postdoctoral fellow with Howard Goodman. 
ONGOING studies in our laboratory are 
aimed at elucidating the mechanism of insu- 
lin action on the expression of enzymes that reg- 
ulate cell growth and metabolism. We have 
focused particularly on regulation of the glycer- 
aldehyde- 3 -phosphate dehydrogenase (GAPDH) 
gene in adipose tissue and liver. Insulin induces 
GAPDH mRNA levels 8-fold in cultured 3T3-L1 
adipocytes and 10-fold in fat or liver tissue iso- 
lated from rats fasted and then fed a high-carbo- 
hydrate, low-fat diet. Expression of the GAPDH 
gene is markedly decreased in primary adipocytes 
isolated from diabetic animals and induced above 
basal levels upon replacement of insulin. The ef- 
fect of insulin on this gene is tissue specific; 
GAPDH mRNA is not regulated in muscle. Study 
of insulin regulation of this gene provides a 
marker of the metabolic effects of insulin on gene 
expression. 
Activation of GAPDH gene expression in insu- 
lin-responsive tissues correlates with the pres- 
ence of insulin-responsive DNA-binding proteins 
that bind specifically to elements in the 5' flank- 
ing region of the GAPDH gene and confer insulin- 
responsive gene expression to the chlorampheni- 
col acetyltransferase gene. Within 60 minutes of 
exposure of 3T3 adipocytes or H35 hepatoma 
cells to insulin, the activity of these sequence- 
specific DNA-binding proteins is increased two- 
to fourfold. The insulin-responsive element A 
(IRE-A) DNA-binding protein (IRP-A) is induced 
four- to eightfold in liver and fat during the pro- 
cess of refeeding a fasted rat a high-carbohydrate, 
low-fat diet, a process known to increase circu- 
lating glucose and insulin levels, resulting in the 
induction of glycolytic and lipogenic enzymes. 
IRP-A binding is inhibited in the fat pads of dia- 
betic animals and is induced above normal levels 
when insulin is administered to diabetic animals. 
In muscle, where GAPDH activity is not rate limit- 
ing, IRP-A binding is not detectable. These obser- 
vations support the importance of GAPDH gene 
regulation in vivo. 
The Southwestern screening technique was 
used to clone a gene that encodes a specific IRP-A 
DNA-binding protein. The cloned cDNA has been 
used to examine the mechanism by which insulin 
chronically regulates expression of the GAPDH 
gene. This clone is expressed in liver and fat, but 
not in muscle, which provides an explanation of 
the tissue-specific regulation of GAPDH gene ex- 
pression. Expression of IRP-A mRNA is inhibited 
in diabetes and up-regulated with insulin replace- 
ment; expression is induced during the process 
of fasting and refeeding. In contrast, 1 hour of 
insulin exposure of cells does not appear to alter 
expression of the IRP-A mRNA. Thus it appears 
that the activity of this factor is regulated acutely 
by a post-translational modification and chroni- 
cally by an alteration in gene expression. 
The mechanism by which insulin's initial in- 
teraction with its cell surface receptor tyrosine 
kinase stimulates intracellular processes is un- 
clear. Several enzymes that are regulated by insu- 
lin have been found to undergo phospho-dephos- 
pho intercon versions. Binding of IRP-A protein to 
DNA is dependent on phosphorylation. To define 
the steps in the signal transduction pathway of 
insulin action on this transcription factor, we 
will use antibodies that specifically interact with 
IRP-A protein and examine the effect of insulin 
on the phosphorylation state of the IRP-A protein. 
IRP-A contains a binding domain that is perfectly 
conserved across species from rat to yeast and an 
acidic domain that is capable of activating gene 
transcription. These domains are surrounded by 
putative phosphorylation sites for insulin-sensi- 
tive kinases. Future efforts will be aimed at deter- 
mining whether any of these putative phosphory- 
lation domains is critical for activation of DNA 
binding or transcriptional activity. 
Southern analysis with the conserved IRP-A 
binding domain indicates that this gene belongs 
to a large family of related genes. Over the next 
year, the potential for interaction between family 
members will be explored. In the process, the 
regions of IRP-A that contact DNA and the regions 
that are required for protein-protein interactions 
will be defined. 
9 
