Thyroid Hormone Regulation of Hepatic Genes in Vivo Detected by Complementary DNA Microarray
Xu Feng,
Yuan Jiang,
Paul Meltzer and
Paul M. Yen
Molecular Regulation and Neuroendocrinology Section Clinical
Endocrinology Branch (X.F., P.M.Y.) National Institute of Diabetes
and Digestive and Kidney Diseases
Cancer Genetics Branch
(Y.J., P.M.) National Human Genome Research Institute National
Institutes of Health Bethesda, Maryland 20892
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ABSTRACT
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The liver is an important target organ of thyroid
hormone. However, only a limited number of hepatic target genes have
been identified, and little is known about the pattern of their
regulation by thyroid hormone. We used a quantitative fluorescent cDNA
microarray to identify novel hepatic genes regulated by thyroid
hormone. Fluorescent-labeled cDNA prepared from hepatic RNA of
T3-treated and hypothyroid mice was hybridized
to a cDNA microarray, representing 2225 different mouse genes, followed
by computer analysis to compare relative changes in gene expression.
Fifty five genes, 45 not previously known to be thyroid
hormone-responsive genes, were found to be regulated by thyroid
hormone. Among them, 14 were positively regulated by thyroid hormone,
and unexpectedly, 41 were negatively regulated. The expression of 8 of
these genes was confirmed by Northern blot analyses. Thyroid hormone
affected gene expression for a diverse range of cellular pathways and
functions, including gluconeogenesis, lipogenesis, insulin signaling,
adenylate cyclase signaling, cell proliferation, and apoptosis. This is
the first application of the microarray technique to study hormonal
regulation of gene expression in vivo and should prove to
be a powerful tool for future studies of hormone and drug action.
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INTRODUCTION
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Thyroid hormones (T3 and
T4) have profound effects on metabolism, growth,
and development (1 ). The effects of thyroid hormone are mediated by
thyroid hormone receptors (TRs) that belong to the nuclear hormone
receptor superfamily; these include the steroid hormone
receptors, the retinoic acid receptors, the retinoid X receptors , the
vitamin D receptor, the peroxisome proliferator-activated
receptors, and other orphan receptors (2 3 ). TRs are encoded by
two genes (
and ß) and are expressed as several isoforms (TR
,
TRß1, and TRß2) (2 3 ). TRs bind to thyroid hormone-response
elements in the promoters of target genes. Interestingly,
T3 can positively and negatively regulate
transcription of target genes (2 3 4 ); however, only a few of the
approximately 30 known target genes are negatively regulated. Moreover,
most of these negatively regulated target genes are expressed in the
pituitary or hypothalamus (2 3 4 ) rather than peripheral tissues.
The liver is a major target organ of thyroid hormone. It has been
estimated that approximately 8% of the hepatic genes are regulated by
thyroid hormone in vivo (1 ), and thus the liver is an ideal
tissue to study gene regulation by thyroid hormone. Although a limited
number of individual target genes in the liver have been studied, a
large-scale profile of the target genes regulated by thyroid hormone
has not been undertaken. cDNA microarray hybridization is a powerful
tool to study hormone effects on cellular metabolism and gene
regulation on a genomic scale as it enables simultaneous measurement
and comparison of the expression levels of thousands of genes (5 6 ).
Recently, cDNA microarrays have been used to study the gene expression
due to fibroblast differentiation; oncogenesis; aging and caloric
restriction of mouse muscle; cell cycle in yeast; and differentiation
in Drosophila (7 8 9 10 11 12 13 ). They also have been used in drug
development programs to monitor changes in gene expression due to drug
treatment (14 ). Additionally, two groups recently have used microarrays
to examine broad patterns of gene regulation in leukemias and lymphomas
and correlate the pattern with clinical outcome (15 16 ). Thus far,
microarrays have not been used to assess hormonal regulation of target
genes and their patterns of expression.
We have used a cDNA microarray to study the hepatic gene expression in
hypothyroid mice treated with T3. We have
identified 55 target genes, with 14 up-regulated and 41 down-regulated
by T3. Most of these genes have not previously
been described to be regulated by thyroid hormone. Analyses of their
expression profile revealed thyroid hormone effects on multiple
cellular pathways. This study also is the first application of cDNA
microarray technology to study hormonal regulation of target genes
in vivo.
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RESULTS AND DISCUSSION
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Microarray Analyses
To investigate the effect of thyroid hormone on hepatic gene
expression, RNA from hypothyroid and T3-treated
mice was prepared, labeled with fluorescent dye, and hybridized with
the cDNA microarray as described in Materials and Methods.
The color images of the hybridization results (Fig. 1
) were made by representing the Cy3
fluorescent image as green and Cy5 fluorescent image as
red, and merging the two color images. Similar findings were
observed in a total of six independent microarray studies of livers
from individual mice treated with T3 or
T4.

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Figure 1. A Representative Portion of the Microarray Used to
Detect Hepatic Genes Regulated by T3
A microarray was hybridized to total RNA from hypothyroid mouse liver
(green fluorochrome) and hyperthyroid mouse liver after
6 h treatment with thyroid hormone (red
fluorochrome). RNA from the hypothyroid mouse liver was used to prepare
cDNA labeled with Cy3-deoxyuridine triphosphate (dUTP), and mRNA
hyperthyroid mouse liver was used to prepare cDNA labeled with
Cy5-dUTP. The two cDNA probes were mixed and then simultaneously
hybridized to the microarray. In this image of the subsequent scan,
mRNAs that were more abundant in hypothyroid mouse liver (suppressed by
thyroid hormone) were detected as green spots, whereas
mRNAs that were more abundant in hyperthyroid mouse liver (stimulated
by thyroid hormone) were detected as red spots.
Yellow spots represented genes whose expression did not
vary substantially between the two samples. The arrows
indicate spots representing the following genes: 1. Bcl-3; 2. carbonyl
reductase; 3. B61; 4. membrane-type matrix metalloproteinase; 5. Spot
14.
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We sampled 2225 genes on the cDNA microarray, which represents
approximately 10% of the expressed genes in liver, assuming that the
liver transcriptosome contains 10,00020,000 genes (20 ). We found that
14 genes (0.6%) displayed a greater than 2-fold increase in expression
levels after thyroid hormone treatment whereas 41 genes (2.4%)
displayed more than a 60% decrease in gene expression (Table 1
). This latter finding was surprising
since there are only a few known negatively regulated target genes in
peripheral tissues. Seven of 14 of the positively regulated genes were
not identified previously as thyroid hormone-responsive, whereas 38/41
of the negatively regulated genes were not identified previously as
such. Three genes (malic enzyme,
myosin heavy chain, and myelin
basic protein) have been described previously to be regulated by
T3 but did not show significant induction in our
studies even though they were contained on the microarray. The latter
two genes are predominantly expressed in heart and brain, respectively,
and not significantly expressed in liver (19 ). Weiss et al.
(17 ) recently treated a different strain of mice for 4 days with
T3 and observed induction of malic enzyme mRNA
(17 ). The observed lack of T3-induction of malic
enzyme mRNA may be due to delayed time course of induction (6 h
vs. 4 days) or differences in mouse strains.
The changes in mRNA levels of several known and novel target genes on
the microarray were confirmed by Northern blot analysis (Fig. 2
). Our data are consistent with previous
studies that have shown that cDNA microarrays can predict induction and
repression of gene expression on Northern blots with high reliability
(7 10 ). The major factors affecting the reliability of microarray
analyses are the quality of RNA and the efficiency of labeling.
Therefore, hybridization and microarray analyses were only performed in
samples in which high specific labeling of RNA was achieved. Only genes
that were regulated in three separate experiments comparing mice
treated with propylthiouracil (PTU) ± T3
are included in Table 1
. These results also were confirmed in three
additional experiments in which mice were treated with PTU ±
T4.

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Figure 2. Autoradiograph of Northern Blots Containing Total
RNA (20 µg) from Hypothyroid Mice (C) and Hyperthyroid Mice (T) That
Were Probed with cDNA from Genes That Were Regulated by T3
on Microarray
Molecular weights of the hybridizing bands were consistent with
published transcript sizes. The control signals of 36B4 showed that
similar amounts of RNA samples were loaded.
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Consistent with some of the known metabolic effects of thyroid hormone,
T3 regulated a diverse range of genes that affect
many different aspects of cellular metabolism and function such as
gluconeogenesis, lipogenesis, insulin signaling, adenylate cyclase
signaling, cell proliferation, and apoptosis (Table 1
and Fig. 3
).

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Figure 3. Cellular Pathways and Functions Detected by cDNA
Microarray That Are Regulated by Thyroid Hormone
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Gene Profiles and Novel Target Genes Regulated by
T3
T3 stimulates gluconeogenesis and glucose
production in the liver and thus opposes the action of insulin with
respect to hepatic glucose production (21 22 ). It also is known that
hyperthyroidism worsens glycemic control in diabetic patients (23 24 ).
In accord with these findings, we found an increase in
glucose-6-phosphatase mRNA expression, the key enzyme for conversion of
glucose-6-phosphate to glucose. Additionally, we observed a decrease in
mRNA expression of a critical downstream component of the
insulin-signaling pathway, Akt2 (protein kinase B). Akt2 is a
serine/threonine kinase that is activated by insulin and other growth
factors via phosphatidylinositol 3 kinase (25 26 ). Akt2 has been shown
to stimulate glucose uptake in peripheral tissues via GLUT 4 glucose
transporter and to promote glycogen synthesis in liver by inactivating
glycogen synthase kinase 3 (27 28 29 ). Thus, a decrease in Akt2 activity
would decrease glycogen synthesis. It also is interesting that
T3 decreased capping protein
subunit and
adaptin mRNA which encode proteins that are involved in the
redistribution and internalization of the liganded insulin receptor
(30 31 ). In this connection, it recently was reported that there is
decreased endocytosis of insulin receptors in hepatocytes of diabetic
rats (32 ). T3 also increased insulin-like growth
factor (IGF) binding protein-1 mRNA, which inhibits IGF-I signaling as
reported previously (33 ).
It previously has been shown that thyroid hormone can mimic or enhance
the glycogenolytic and gluconeogenic effects of epinephrine and
glucagon in hepatocytes, and increase intracellular cAMP (21 34 35 36 ).
Consistent with these findings, we observed induction of
ß2-adrenergic receptor mRNA and repression of inhibitory G protein
(Gi) mRNA of the adenylate cyclase cascade (Table 1
). Thus,
T3 has complementary effects on two opposing
pathways that regulate intracellular cAMP. Additionally,
T3 may have other effects on this signaling
cascade as T3 has been reported to redistribute
Gs
from cytosol to the plasma membrane, up-regulate Gs
, and
down-regulate Gi
expression in the plasma membrane, as well as
modulate the activity of phosphodiesterase (37 38 ).
Hepatic lipogenesis is regulated by dietary substrates and hormones
such as insulin and thyroid hormone, which control fuel metabolism and
biosynthesis (1 ). We observed increased mRNA expression of spot 14
(S14), a key protein that regulates lipogenesis that previously was
shown to be induced by T3 (39 ). We also saw
induction of fatty acid transport protein mRNA, which encodes a plasma
membrane protein that is highly expressed in liver that mediates the
transport of long chain fatty acids into cells (40 ). We did not observe
induction of malic enzyme mRNA, which is known to be stimulated by
T3 (17 39 41 ). It is possible that malic enzyme
may have a delayed response to T3 or may be
regulated by Spot 14 (39 42 ). Recently, it has been reported that
T3 also can down-regulate cytochrome p450 4A3
(Cyp4A3) mRNA (43 ) and that Cyp4A10 mRNA is increased in diabetic rats
(44 ). We observed that T3 decreases mitochondrial
acyltransferase Cyp4A10 mRNAs, which, in turn, could reduce fatty acid
oxidation acutely and increase lipogenesis.
T3 also decreased another downstream insulin
signaling component, PHAS (phosphorylated heat-and acid-stable
protein), which is the 4E(eIF-4E)-binding protein that complexes with
the eukaryotic initiation factor, eIF4E, and inhibits eIF-4E-dependent
translation. Phosphorylation of PHAS by insulin signaling causes
dissociation of PHAS from the PHAS-eIF-4E complex, and leads to
increased translation (45 46 ). Since T3
down-regulated PHAS, it is possible that T3, a
known hepatic mitogen, may have an insulin-mimetic effect with
respect to protein synthesis. On the other hand,
T3 decreases Akt2 mRNA expression and increases
intracellular cAMP, which may decrease phosphorylation of PHAS (46 47 )
and thus inhibit translation. Despite studies of thyroid hormone
regulation of protein synthesis (48 49 ), neither the detailed
mechanism nor the net effects of T3 on
translation are known.
Previous reports have shown that T3 has complex
effects on hepatocyte proliferation and cell survival (50 51 52 ).
Consistent with these effects, we observed changes in genes involved in
apoptosis and cell cycle progression. In particular, genes involved in
cell proliferation such as Bcl3, B61, and kinesin-like protein (Kip1p)
were induced by T3. Bcl3 is an I
B-related
protein that behaves differently than I
B as it can act as a
coactivator for nuclear factor (NF)
B homodimers (53 54 55 ). It
also functions as a coactivator for AP-1 complexes and retinoid X
receptors (56 ) and thus potentially may serve as a coactivator for TR
itself. B61 is a glycosylphosphatidylinositol-linked protein, which
serves as a ligand for Eck receptor protein-tyrosine kinase (57 ) and
may act as a hepatic mitogen. Kip1p participates in the segregation of
chromosomes during mitosis by modulating the movement of spindle
assembly and chromosome distribution (58 ). Recent studies suggest that
T3 stimulation of proliferation may be of
clinical importance in gene therapy as it may promote the transfection
of viral vectors into liver (59, 60).
Although T3 stimulates hepatocyte proliferation,
it also can stimulate apoptosis in hepatocytes in which proliferation
is pharmacologically blocked, and in amphibian tails during
metamorphoses (52 61 ). In this connection, we found that several genes
involved in apoptosis were regulated by T3, as
T3 decreased expression of Akt2 and protein
kinase C (PKC)
mRNA, and increased expression of the PKC inhibitor,
(PKCi) mRNA. Akt2 can induce phosphorylation of caspase-9 to inhibit
its protease activity, as well as activate NF
B (62 ). PKC
, an
atypical form of PKC, has been shown to be critically involved in
important cell functions such as proliferation and cell survival.
PKC
activates I
B kinase B, which then can activate NF
B (63 ).
Additionally, PKCi opposes the action of PKC, so induction of PKCi
would promote apoptosis (64 ).
In our microarray studies, T3 negatively
regulated several genes involved in glycoprotein synthesis such as
ß-galactoside
2,6-sialyltransferase, and
-2,3-sialyltransferase. Of note, hypothyroidism has been shown to
induce
-2,3-sialyltransferase expression in pituitary thyrotrophs
(65 ). The sialyltransferases catalyze the transfer of sialic acid from
cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc)
to terminal positions on sugar chains of glycoproteins and glycolipids
(66 ) and are markers for many tumors (67 68 ). Finally,
T3 negatively regulated genes involved in
cellular immunity, cell matrix, cell structure, endocytosis, and
mitochondrial function which, in turn, may represent novel areas of
T3 action (Table 1
).
To confirm and extend our findings on newly discovered target genes, we
performed a separate time course experiment on T3
regulation of two newly identified target genes, Bcl-3 and
2,3
sialyltransferase. mRNA was measured by Northern blot analyses from
livers harvested from mice at various times after
T3 treatment. Bcl-3 mRNA levels were induced
approximately 10-fold 1 h after T3 treatment
and declined to 5-fold by 3 h (Fig. 4A
).
-2,3-Sialyltransferase mRNA
levels decreased 1 h after T3 treatment and
reached their nadir at 3 h. (Fig. 4B
). It is possible that
-2,3
sialyltransferase and some of the other negatively regulated target
genes identified by our microarray may serve as new markers of thyroid
hormone action outside the pituitary, as well as tools for
understanding the mechanism of negative regulation by nuclear hormone
receptors.

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Figure 4. Northern Blot Analysis of Bcl-3 and
-2,3-Sialyltransferase Expression in Hypothyroid and Hyperthyroid
Mouse Liver
Liver biopsies were taken from PTU-treated mice at the indicated times
(h) after thyroid hormone injection, and RNA was prepared as described
in Materials and Methods. The y axis represents
fold-induction or -repression of mRNA in T3-treated mice
relative to basal levels mRNA levels in hypothyroid mice. The x axis
shows time of sampling after T3 injection. Data are
mean ± SD (n > 3) and were normalized against
the 36B4 mRNA signals. A, Time course of T3 induction of
Bcl-3 in mouse livers. B, Time course of T3 repression of
-2,3-sialyltransferase in mouse livers.
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Although the majority of the cells within the liver are hepatocytes, it
is possible that regulated genes from other cell types (e.g.
endothelial cells, Kupfer cells, red blood cells) may also have been
detected by the microarray. Furthermore, although we studied gene
expression relatively soon after T3
administration (6 h), it is not known whether all the target genes
identified are regulated directly by T3. It is
possible that intracellular factors may be rapidly induced which, in
turn, may regulate some of the target genes we have described. Also,
since this study was performed in vivo, it is possible that
hormones that are regulated by T3 may regulate
some of the target genes. In particular, GH is induced by
T3 in rodents and was found to coregulate a small subset of
T3-induced target genes that were studied by Oppenheimer
and associates (69 ) using two-dimensional protein gel electrophoresis
(69 ). Microarray studies using hypophysectomized mice, GH receptor
knockout mice, or GH antagonists may help clarify this issue. Despite
these potential limitations, our study has demonstrated the utility of
cDNA microarrays to identify novel target genes in vivo and
extend the applicability of this powerful technology beyond
conventional cell culture systems.
In conclusion, we have used cDNA microarray technology to analyze gene
expression changes in mouse liver after administration of thyroid
hormone. We identified 55 target genes, most of which have not been
described previously. Surprisingly, many of these target genes were
negatively regulated. Two recent reports have demonstrated the broad
patterns of gene expression that can occur in human disease (15 16 ).
Similarly, we have observed changes in the patterns of gene expression
at the genomic level after hormone stimulation. Thus, our findings have
enhanced our awareness of the large repertoire of genes and the
multiple cell processes and signaling pathways regulated by thyroid
hormone. It is likely that such complex regulation of gene expression
occurs in other target tissues regulated by thyroid hormone and by
other hormones. The use of microarray technology in living animals is a
powerful tool to study gene regulation in a physiological system. It
will be useful in identifying novel pathways for hormone action and
tumorigenesis. It also should prove valuable for drug design as it will
enable characterization of agonist and antagonist properties of drugs
as well as side effects based upon gene expression patterns.
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MATERIALS AND METHODS
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Animals
Six-week-old mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and maintained in the animal
care facility at NIH according to NIH animal care guidelines.
Hypothyroid mice were fed a low iodine (loI) diet supplemented with
0.15% PTU purchased from Harlan Teklad Co. (Madison, WI) for 4 weeks
(17 ). After 4 weeks on this diet, hyperthyroid mice were injected ip
with 100 mg L-T3 or
T4 per 100 g mouse body weight in 200 µl
PBS. Control mice were injected with the same volume of PBS alone. Six
hours after thyroid hormone injection, mice were killed by cervical
dislocation. Livers were isolated for total RNA isolation. TSH
measurements (kindly performed by Dr. Samuel Refetoff, University of
Chicago, Chicago, IL) indicated that mice treated with PTU according to
this protocol were profoundly hypothyroid before
T3 or T4 administration
(TSH 64110 ng/ml, n = 4; nl 0.020.95 ng/ml).
RNA Preparation and Labeling
Total RNA was isolated from mouse livers by RNeasy kit
(QIAGEN, Chatsworth, CA) and further purified by TRIZOL
reagent (Life Technologies, Inc., Gaithersburg, MD). Total
RNA (100 µg) was converted to cDNA by using SuperScript II RNA
reverse transcriptase (Life Technologies, Inc.) as
previously described (10 ). RNA isolated from hypothyroid mice was used
to prepare cDNA probes labeled with Cy3-deoxyuridine triphosphate
(dUTP) dUTP (Amersham Pharmacia Biotech, Piscataway, NJ),
and RNA isolated from hyperthyroid mice was used to prepare cDNA
labeled with Cy5-dUTP (Amersham Pharmacia Biotech).
Labeled cDNA was purified using MicroCon 30 (Amicon, Inc., Beverly,
MA).
cDNA Microarray
The cDNA microarrays contained 2,225 elements derived from
murine EST clones obtained from Research Genetics, Inc.
(Huntsville, AL) as previously described (10 ). PCR products generated
from these clones were printed onto glass slides as previously
described (9 10 ).
Hybridization and Scanning
Labeled cDNA from the hypothyroid mouse and either
T3- or T4- treated mice
were hybridized to a 1.0-cm2 microarray under a
14 x 14 mm glass coverslip overnight at 60 C in a custom-built
hybridization chamber, and fluorescence intensities were analyzed by a
custom-designed laser confocal microscope as previously described (10 18 ). Image analysis was performed using DEARRAY software
(10 ).
Northern Blotting
Total RNA was prepared from mouse liver using TRIZOL reagent
(Life Technologies, Inc.) according to the manufacturers
instructions. Total RNA (20 µg) was separated on 1%
agarose-formaldehyde gel and then transferred to a nylon transfer
membrane (Schleicher & Schuell, Inc., Keene, NH). The
blots were probed with gel purified
-[32P]
dCTP-labeled fragments and exposed to a Biomax film(Eastman Kodak Co., Rochester, NY) at -70 C. mRNA signals were quantified
using ImageQuant software (Molecular Dynamics, Inc.,
Sunnyvale, CA), and normalized with corresponding thyroid
hormone-insensitive 36B4 signals. Fold-inductions were determined from
hyperthyroid mice signal values divided by hypothyroid mice signal
values within the same experiment.
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The European Journal of Endocrinology Prize
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The European Journal of Endocrinology Prize is awarded during
the European Congress of Endocrinology to a candidate who has
significantly contributed to the advancement of knowledge in the field
of endocrinology through publication.
The prize consists of a certificate and Euro 7,250 plus travelling
expenses and will be presented during the Vth European Congress of
Endocrinology to be held in Turin, Italy from June 913, 2001.
Nominations should be submitted to the Chief Editor of the European
Journal of Endocrinology, Professor Paolo Beck-Peccoz, Istituto di
Scienze Endocrine, Piano Terra, Padiglione Granelli, IRCCS, Via
Francesco Sforza 35, 20122 Milan, Italy, by December 31, 2000. For more
detailed information please check our website at: www.eje.org or
contact our office at the following e-mail address: eie@nikotron.com.
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ACKNOWLEDGMENTS
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The authors would like to thank Drs. Leonard Kohn, Joseph Rall,
Marc Reitman, Simeon Taylor (NIDDK), and Drs. Samuel Refetoff and Roy
Weiss (University of Chicago) for helpful discussions.
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FOOTNOTES
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Address requests for reprints to: Paul M. Yen, M.D. Molecular Regulation and Neuroendocrinology Section, Clinical Endocrinology Branch, NIDDK, NIH, Bethesda, Maryland 20892.
Received for publication January 11, 2000.
Revision received February 21, 2000.
Accepted for publication February 24, 2000.
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