ROLE OF IRE-ABP, A POTENTIAL REGULATOR OF THE SWITCH 
FROM THE FASTED TO THE REFED STATE 
Maria C. Alexander-Bridges, M.D., Ph.D., Assistant Investigator 
When a fasted animal is fed a high-carbohydrate/ 
low-fat diet, an adaptive response occurs that results 
in a decrease in glucose production and an increase 
in glucose utilization and storage. This process is 
initiated by the hormone insulin. Insulin rapidly in- 
creases the activity of rate-limiting enzymes that reg- 
ulate glucose uptake, glycogen synthesis, and lipo- 
genesis and inhibits the activity of enzymes that 
increase production of glucose. Over a longer pe- 
riod of time, insulin reinforces these changes in en- 
zymatic activity by altering gene transcription. Thus 
insulin resets the phenotype of the cell so that the 
cell is optimally prepared to carry out energy stor- 
age functions. The marked alterations in enzymatic 
activity observed in insulin-deficient diabetes result 
from atrophy of these pathways. 
The work in Dr. Alexander-Bridges' laboratory has 
focused on defining the distal components in the 
signaling pathway of insulin action on expression of 
metabolically active genes. Insulin stimulates tran- 
scription of the glyceraldehyde-3-phosphate dehy- 
drogenase (GAPDH) gene in 3T3 adipocytes and 
H35 hepatoma cells. IRE-A, an insulin response ele- 
ment located between nucleotides —480 to —435 in 
the upstream region of this gene, specifically inter- 
acts with IRP-A, an insulin-induced DNA-binding 
complex. The ability of adipocyte IRP-A to bind 
IRE-A DNA correlates with the ability of this element 
to confer insulin-responsive gene transcription. In 
transgenic animals, GAPDH-growth hormone fu- 
sion genes are regulated by nutritional manipula- 
tion, such as fasting and refeeding in vivo. These 
observations show that regulation of GAPDH gene 
expression is mediated at the level of transcription 
in vivo. The magnitude of the effect on transcrip- 
tion correlates well with the effect of similar manip- 
ulations on IRP-A-binding activity in the fat and 
liver of animals that have been fasted and refed a 
high-carbohydrate diet and in diabetic animals 
treated with insulin. 
Insulin is known to regulate simultaneously the 
transcription of genes that control diverse aspects of 
energy metabolism in lipogenic tissues, e.g., fat and 
liver. The existence of a common transcription fac- 
tor that coordinates this adaptive response has been 
hypothesized for some time. 
A protein that binds the GAPDH IRE-A element, 
IRE-ABP, has been cloned. IRE-ABP binds and acti- 
vates transcription through the GAPDH IRE-A ele- 
ment in cell lines that do not express IRP-A. Two 
forms of the factor have been isolated. One en- 
hances, the other inhibits, insulin-sensitive gene 
transcription through the IRE-A element in insulin- 
sensitive cell lines. 
The high-mobility group (HMG) box do- 
main of the IRE-ABP is 68% identical to the testis- 
determining factor, SRY, a gene isolated from the 
sex-determining region on the Y chromosome, and 
is 98% identical to an autosomal gene that was iso- 
lated during the process of screening a whole mouse 
embryo cDNA library for 5/?F-related sequences. 
IRE-ABP and the SRY protein share DNA-binding 
specificity for IRE-A. IRE-ABP binds IRE-A DNA with 
sequence specificity that overlaps that of the adipo- 
cyte IRP-A nuclear extract complex. The sequence 
in IRE-A DNA that is contacted by IRE-ABP and SRY is 
identical to the sequence 5'-Py-CTTTG(A/T)-3', pre- 
viously defined by Dr. Katherine Jones and her col- 
leagues as a consensus motif contained in several T 
cell-specific genes that are bound with high affinity 
by TCF- 1 a (T cell factor 1 a) . Thus diverse members 
of the HMG family of proteins modulate transcrip- 
tion through a similar spectrum of sequences that 
contain this core motif. 
Insulin simultaneously activates processes that re- 
sult in glucose uptake and utilization and inhibits 
processes that lead to glucose production. Recent 
work has explored the possibility that IRE-ABP plays 
a role in coordinating this adaptive response to a 
glucose load. Identification of a conserved motif for 
IRE-ABP provides a way to search for important phys- 
iological targets of the IRE-ABP and SRY-like family 
of transcriptional regulators. The proposed consen- 
sus sequence for HMG proteins has been identified 
in the upstream region of genes that are regulated 
positively [amylase, the insulin-sensitive glucose 
transporter 4 (GLUT4)] and negatively [phospho- 
enolpymvate carboxykinase (PEPCK)] by insulin. 
This laboratory has shown that IRE-ABP footprints 
these sequences and activates transcription through 
these sites in cells that do not express IRE-ABP. Thus 
Dr. Alexander-Bridges and her colleagues conclude 
that IRE-ABP could bind these sites and regulate 
these genes in vivo. 
Insulin regulates GAPDH gene transcription in a 
tissue-specific manner. Regulation by nutritional 
manipulations occurs in fat and liver but not mus- 
cle. This observation correlates with the fact that 
IRE-ABP mRNA is expressed in fat and liver, but not 
muscle, and is induced in the liver of refed rats. 
CELL BIOLOGY AND REGULATION 
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