Aged PROP1 Deficient Dwarf Mice Maintain ACTH
Igor O. Nasonkin1.¤a, Robert D. Ward2.¤b, David L. Bavers3, Felix Beuschlein3,4¤c, Amanda H.
Mortensen1, Catherine E. Keegan2,5, Gary D. Hammer2,3, Sally A. Camper1,2,3*
1Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, United States of America, 2Graduate Program in Cellular and Molecular Biology,
University of Michigan, Ann Arbor, Michigan, United States of America, 3Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan,
United States of America, 4Endocrine Research Unit, University Clinic Munich, Munich, Germany, 5Department of Pediatrics and Communicable Diseases, University of
Michigan Medical School, Ann Arbor, Michigan, United States of America
Humans with PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth
insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in
other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortiso-
lism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the Prop1null(Prop1-/-)
and the Ames dwarf (Prop1df/df) mouse models, but corticotroph and pituitary adrenal axis function have not been
thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that
causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live
with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and
corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis.
Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH
and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated
corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive
hormone loss and hypocortisolism.
Citation: Nasonkin IO, Ward RD, Bavers DL, Beuschlein F, Mortensen AH, et al. (2011) Aged PROP1 Deficient Dwarf Mice Maintain ACTH Production. PLoS
ONE 6(12): e28355. doi:10.1371/journal.pone.0028355
Editor: Giovambattista Pani, Catholic University Medical School, Italy
Received August 31, 2011; Accepted November 7, 2011; Published December 1, 2011
Copyright: ? 2011 Nasonkin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by National Institutes of Health (NIH) grants and fellowships: NIH R37HD30428, R01HD34283 (SAC), T32 GM 07544 and T32 HD
07048 (ION), T32 GM07863 and T32 GM07315 (RDW). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
¤a Current address: National Institutes of Health/NEI, Bethesda, Maryland, United States of America
¤b Current address: University of Houston Law School, Houston, Texas, United States of America
¤c Current address: Division of Endocrine Research, Department of Medicine Innenstadt, University Hospital Munich, Munich, Germany
Congenital pituitary hormone deficiency in humans occurs with
a frequency of approximately 1 in 4000 live births and is caused
primarily by mutations in genes important for pituitary develop-
ment [1,2]. Multiple pituitary hormone deficiency (MPHD) results
from a variety of transcription factor mutations, including
mutations in PROP1, POU1F1 (PIT1), HESX1, LHX3, LHX4,
OTX2, SOX2, SOX3, and GLI2 (reviewed in ). Mutations in
POU1F1 almost always cause deficiencies in GH, prolactin (PRL),
and thyroid stimulating hormone (TSH) in addition to overall
pituitary hypoplasia [4,5,6,7]. Mutations in Prophet of PIT1
(PROP1) are the most common known causes of MPHD in
humans. The hormone deficiencies are similar to those caused by
POU1F1 mutations, except that the deficiencies include reduced
gonadotropin production requiring sex hormone substitution and
there is a strong tendency toward progressive hormone loss leading
to lower circulating adrenocorticotropic hormone (ACTH) later in
11,12,13]. Another interesting difference between PROP1 and
POU1F1 patients is the tendency of patients with PROP1 mutations
to undergo apparent degeneration of the pituitary gland during
childhood [14,15]. Initially, magnetic resonance imaging analysis
may reveal a hyperplastic, or enlarged, pituitary gland, which
usually evolves to a hypoplastic appearance a year or so later. The
progressive hormone loss and transient pituitary hyperplasia
associated with PROP1 mutations are not well understood.
Several mouse models have been used to dissect the mechanism
of Prop1 action in pituitary development and function. The Ames
dwarf (Prop1df/df) and the Prop1null(Prop1-/-) mouse mutants
recapitulate the human MPHD phenotype in that adult mutants
are profoundly deficient in TSH, GH, PRL, have low circulating
gonadotropins, and pituitary hypoplasia [16,17,18,19]. Studies in
Prop1 mutant mice show that precursor cells fail to colonize the
anterior lobe resulting in reduced cell proliferation and enhanced
apoptosis after birth leading to hypoplasia that becomes evident in
the weeks after birth [20,21]. Prop1 mouse mutants differ from
humans with PROP1 mutations in that the hormone deficits are
PLoS ONE | www.plosone.org1December 2011 | Volume 6 | Issue 12 | e28355
consistently congenital rather than progressive, thyroid hormone
and growth hormone replacement are sufficient for fertility, and
there is no clear evidence for transient pituitary hyperplasia.
The genetic background exerts a considerable influence on the
phenotype of the Prop1 deficient mice, although both alleles,
Prop1df/dfand Prop1-/-, have the same features when normalized for
genetic background . Similarly, humans with the same
mutation in PROP1 can have different clinical presentations
. Enrichment of the 129S1/SvImJ (129) background enhances
the frequency with which newborn Prop1 mouse mutants die of
respiratory distress. The lack of pituitary TSH results in fetal
hypothyroidism, reduced expression of the thyroid hormone
inducible transcription factor TTF1 in the lung, and inadequate
production of surfactants, known target genes of TTF1. The lungs
fail to inflate, causing respiratory distress and lethality .
Increasing the contribution of C57BL/6J (B6) strain background
tended to protect against this survival defect in newborns. Here we
report that the B6 background increases the sensitivity of Prop1
deficient mice to lethality after weaning. The reason for this
juvenile lethality has not been explored.
Corticotroph development does not appear to be affected in the
Prop1 deficient mice, and corticosterone levels are not reduced in
newborn mutants [19,22,23]. Because most PROP1 patients who
have been closely followed appear to have evolving hypocortiso-
lism , and the underlying cause of the juvenile lethality of Prop1
mutant mice is not known, it is necessary to investigate pituitary
adrenal function in young and old Prop1 deficient mice on a
sensitized (B6) genetic background.
We report no evidence for progressive ACTH loss in juvenile and
young adult Prop1 deficient mice. In contrast, our results show
increased serum ACTH and corticosterone levels in young and old
Prop1 mutants. The pituitary-adrenal axis is functional in young
adult Prop1nullmice as demonstrated by elevated activity in response
to restraint stress. Prop1 mutants have significantly reduced blood
glucose levels, as expected for GH deficient animals, which could
trigger the activation of the pituitary-adrenal axis. Untreated
hypoglycemia can cause mortality in both humans and mice .
We conclude that both of the Prop1 mouse alleles we tested on
various genetic backgrounds differ from human patients by
maintaining elevated pituitary adrenal axis activity through 1 year
of age, with no evidence for evolving hypocortisolism.
Materials and Methods
All mice were housed in a 12-h light, 12-h dark cycle with
unlimited access to tap water and Purina 5008 or 5020 chows. All
procedures using mice were approved by the University of
Michigan Committee on Use and Care of Animals, and all
experiments were conducted in accordance with the principles and
procedures outlined in the NIH Guidelines of the Care and Use of
The Prop1Sactm1heterozygous null mice, referred to here as
Prop1+/2, were generated from R1 (129/Sv x 129/Sv-CP) ES cells
by replacing the coding region of exon 1, intron 1, and a portion of
exon 2 with cassettes encoding b-galactosidase and neomycin
resistance (19, 37). The chimeras were mated to C57BL/6J mice
(B6) (The Jackson Laboratories, Bar Harbor, ME) to generate F1
heterozygous animals and were first analyzed on a mixed F2
C57BL/6J-129S1/SvImJ background (B6/129). The F2 Prop1+/2
heterozygous mice were backcrossed to B6 mice for four
generations to establish the Prop1+/2N4 B6 breeding colony,
which is theoretically 93.75% pure B6. Mice used in the study of
pituitary-adrenal function were from the N4 B6 genetic back-
ground unless expressly stated otherwise. Prop1-/-mice were
determined by PCR as previously described [19,20].
The DF/B-Prop1+/dfstock is not inbred. It was obtained from
Dr. A. Bartke at Southern Illinois University in 1988 and
maintained at University of Michigan. This stock was backcrossed
to B6 to N4 .
Restraint stress and blood collection
Young adult mice (8–10 weeks old, N4 B6) were housed
individually 12 hours prior the experiment, with special precau-
tions to avoid stress associated with noise and cage handling. The
blood samples were collected in the morning (between 9:00am and
10:30am) by retro orbital bleeding in heparinized collection tubes
(Microvette CB300; Sarstedt, Inc., Newton, NC). The retro orbital
bleeding was done in less than one minute after initial mouse
handling to prevent stress-induced corticosterone release. Animals
were subjected to restraint stress for 30 minutes, after which
another blood sampling was performed by the same method
[25,26,27]. Plasma was prepared according to the manufacturer’s
protocol for the Microvette CB300 (Sarstedt).
For ACTH measurements in non-stressed conditions, animals of
various ages were anesthetized with metaphane, rapidly decapi-
tated within less than 1 min from the time of initial handling, and
blood samples collected.
Corticosterone, ACTH, and glucose measurements
ACTH and corticosterone were measured by radioimmunoas-
say (RIA) in plasma using a125I RIA kit (ICN Diagnostics, Costa
Mesa, CA) according to the manufacturer’s protocol . The
blood-glucose measurements were done using a FreeStyle glucose
meter (TheraSense, Alameda, CA). Duplicate measurements were
done for each sample collected. According to manufacturer’s
instructions, glucose levels below 60 mg/dL are considered
evidence of hypoglycemia. Glucose measurements were performed
on 3.5 to 5 week, 5 to 6.5 week, and 8 to 10 week pre- and post-
stressed N4 B6 animals. The device’s lowest sensitivity level is
20 mg/dL (http://www.abbottdiabetescare.com). If glucose levels
were below the level of detection, an arbitrary number of 19 mg/
dL was assigned for the purpose of statistical analysis.
Histology and Immunohistochemistry
Adrenals were collected immediately after euthanizing and
rinsed in ice-cold PBS prior to 1 h fixation in 4% paraformalde-
hyde on ice (diluted in PBS, pH 7.2). Samples were washed in
PBS, dehydrated in a graded series of ethanol, and embedded in
paraffin. Seven-micrometer sections were prepared and either
stained with hematoxylin and eosin. The 20a-hydroxysteroid
dehydrogenase antibody was generously provided by Yacob
Weinstein and used at 1:2000-3000 dilution .
Data were processed and plotted using StatView software
(Abacus Concepts, Inc., Edinburgh, United Kingdom), with the
exception of the qRT-PCR data that was processed using
Microsoft Excel Software. ANOVA (analysis of variance) and
Fisher’s exact test were used to evaluate the data. All data are
shown as +/2 1 SEM (standard error of the mean). P-values of less
than 0.05 were considered to be statistically significant.
Prop1 deficiency can cause postnatal lethality
We analyzed the viability of two different mutant alleles of Prop1
on several genetic backgrounds. The Ames dwarf mutant, Prop1df/df,
Prop1 Mutants Maintain ACTH Production
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arose spontaneously on a poorly defined genetic background (DF/
B), and it carries a missense mutation in the homeodomain,
Ser83Pro [18,30]. We generated a null allele, Prop1+/2, on a mixed
background comprised of C57BL/6J (B6) and 129S1/SvImJ (129)
. We frequently observed a crisis in mutant viability after
weaning. On the 129/B6 mixed background 37% (13/35) of the
Prop1-/-animals exhibited lethargy, wasting, and death between 3
and 7 weeks of age. Death usually occurred within 3-5 days of initial
signs of distress.More males wereaffected than females(p=0.03).A
similar wasting and lethality phenotype was also observed in 27%
(6/22) of compound heterozygotes, Prop1df/-, on a mixed back-
We back-crossed both strains, DF/B-Prop1df/+and 129/B6-
Prop1+/2, four times to B6 to be able to compare the phenotypes of
the two alleles on a consistent genetic background. We observed
identical viability of the homozygous mutants for each allele at two
weeks of age: 17.5% Prop1df/dfand 19.5% Prop1-/-for each on N4
B6, p=0.69 . The N4 B6 background, however, increased the
risk of lethality after weaning in homozygotes for both of the Prop1
Prop1 deficient mice exhibit elevated levels of circulating
ACTH and corticosterone
To determine whether the observed post-weaning lethality on
the N4 B6 background could arise from evolving hypocortisolism,
we examined ACTH and corticosterone production. We analyzed
the serum of 3.5 to 5 week old Prop1-/-and normal mice on the N4
B6 background by RIA to address the ability of Prop1 mutant
corticotrophs to secrete ACTH (Fig. 1). There was no evidence
for reduced ACTH production. Although these N4 B6-Prop1-/-
mice showed a trend towards increased serum ACTH compared
to wild type and heterozygote littermates, the difference was not
significant. Western blots revealed similar ACTH protein content
in the pituitary glands of normal and Prop1 mutant mice (data not
To determine whether Prop1 mutants exhibit evolving hypo-
cortisolism at older ages we aged Prop1 mutant animals with three
different genotypes and genetic backgrounds (Prop1-/-, Prop1df/-,
and Prop1df/df) to 7–12 months old and measured both ACTH and
corticosterone. All genotype combinations of Prop1 mutants had
significantly elevated ACTH and corticosterone (Fig. 1). ACTH
levels were 2 to 2.5x elevated in mutants relative to normal
littermates, and the corticosterone levels were even more
dramatically heightened in mutants. Our evidence for up
regulation of the pituitary-adrenal axis in Prop1 deficient mice is
consistent with previous reports of elevated corticosterone in Ames
dwarf mice [31,32], and the increased corticosterone levels we
reported in Prop1-/-newborns . Thus, there is no evidence that
Prop1 mutant mice develop the age related ACTH deficiency and
hypocortisolism that has been observed in some human patients
with PROP1 mutations.
Prop1 deficient mice respond to restraint stress
Stress increases pituitary ACTH release and subsequent
corticosterone secretion by the adrenal gland [27,33,34]. We
exposed Prop1 mutant and normal animals to restraint stress to test
the ability their pituitary-adrenal axis to respond to this challenge
(Fig. 2). Serum corticosterone levels were measured in N4 B6
Prop1-/-, Prop1+/2and Prop1+/+male and female mice at 8–10
weeks of age prior to and following 30 min of restraint stress.
Prop1-/-animals had dramatically elevated basal, serum levels of
corticosterone compared to wild type and Prop1+/2mice (Fig. 2A
and 2B, white bars). Basal corticosterone was 4 fold higher in
nonstressed male mutants than normal littermates, and 3 fold
higher for female mutants vs. normals. Following restraint stress,
males and females exhibited elevated serum
corticosterone compared to Prop1+/-and wild type mice (Fig. 2A
and 2B, black bars). The fold increase in corticosterone from
Figure 1. No evidence for evolving hypocortisolism in Prop1
deficient animals. Blood plasma was collected from 3.5 to 5 wk N4B6
(Panel A) and 34 to 52 wk mixed genetic background (Panel B) animals
from and the circulating ACTH levels were determined by RIA. Males
and females were included together because the individual analysis
showed no difference in the ACTH levels of aged-matched animals of
the same genotype. At 3.5 to 5 weeks Prop1-/-(n=6) animals tended to
have higher circulating levels of ACTH than Prop1+/2(n=10) or Prop1+/+
(n=10) animals, but the difference was not statistically significant (top).
At 34 to 52 weeks three different genotypes of Prop1 mutant animals,
Prop1-/-(n=8), Prop1df/-(n=20), and Prop1df/df(n=12), exhibited an
increase in circulating ACTH levels compared to Prop1+/+(n=9)
(bottom). Values represent the mean ACTH production (pg/mL) 6 SE.
*, P,0.01; **, P,0.005; ***, P,0.0005. Corticosterone levels were
measured in serum from aged male Prop1+/+(n=4), Prop1df/df(n=4),
Prop1df/-(n=11), and Prop1-/-(n=7) mice (Panel C). All three genotypes
of Prop1 deficient mice show elevated basal levels of corticosterone
compared to wild type. Prop1df/dfmice have statistically higher basal
levels of corticosterone compared to Prop1df/-or Prop1-/-mice. Values
represent the mean corticosterone (ng/mL of blood) 6 SE. *, P,0.005;
**, P,0.0005; ***, P,0.05.
Prop1 Mutants Maintain ACTH Production
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basal to post-stress measurements is less for Prop1-/-animals
compared to the wild type (2-3 fold compared to 11-16 fold).
While this could be described as a blunted response, the absolute
value of circulating corticosterone following restraint was higher in
mutants than normal littermates. Post stress, the corticosterone
values for male and female mutants were 504 +/2 29 ng/ml and
normal littermates were 373 +/2 18 ng/ml. The post stress values
in the 500 ng/ml range may be the maximal response. Thus, there
is no evidence for impaired pituitary-adrenal axis function.
Prop1 mutant adrenal glands are enlarged relative to
ACTH is important for the development and growth of the
adrenal gland in mice and other mammals [35,36,37]. The
adrenal weights of N4 B6 Prop-/-mice were compared to Prop1+/2
and wild type to determine the consequence of elevated ACTH on
adrenal growth. The absolute size of the adrenal gland is smaller in
the Prop1-/-dwarf males compared to wild type. However, the
ratio of adrenal weight to body weight is actually increased in the
Prop1-/-males compared to wild type (Fig. 3). This is consistent
with the chronically elevated ACTH secretion in Prop1-/-mice.
The mouse adrenal gland is comprised of the adrenal medulla,
which is important for the production of catecholamines such as
norepinepherine and epinephrine, and the adrenal cortex which is
important for corticosterone biosynthesis and contains the zona
glomerulosa and zona fasciculata . We examined adrenal develop-
ment and morphology in N4 B6 normal and Prop1-/-male and
female mice at 3.5, 5, 8, 10 weeks of age. The zona fasciculata and
zona glomerulosa are morphologically indistinguishable in normal
and mutant mice (Fig. 3, and data not shown). The adrenal X-
zone is typically present between the zona fasciculata and medulla
throughout postnatal development and then regresses in male
mice starting at 3 weeks of age and in females during the first
pregnancy . The X-zone is not well understood, but it is
thought to be analogous to the fetal zone in the human adrenal
gland. Growth of the X-zone is regulated by pituitary gonadotro-
pins and activin . The X-zone is marked by 20a-hydroxyste-
roid dehydrogenase immunostaining and is present but smaller in
female Prop1-/-mice at 5 wks and nearly undetectable at 8 wks 
(Fig. 3). Thus, the X-zone is formed but is underdeveloped and
apparently regresses early in Prop1 mutants.
We used Western blotting to evaluate the levels of steroidogenic
enzymes in Prop1-/-adrenals (data not shown). Similar levels of 21-
hydroxylase enzyme, which is important for corticosterone
biosynthesis [42,43], steroidogenic acute regulator protein (StAR),
which mediates the acute steroidogenic response  and the
p450 cholesterol side chain cleavage protein (SSC) [45,46], were
observed in Prop1-/-adrenals compared to Prop1+/2or wild type.
These results are consistent with functioning adrenal glands in
Prop1 deficiency causes low blood glucose
We hypothesized that reduced glucose levels secondary to
growth hormone deficiency could cause the elevated basal levels of
ACTH and corticosterone in the blood of Prop1 deficient mice.
Prop1 deficient mice produce very few somatotrophs and lack
detectable circulating GH [18,47]. GH has pleiotropic functions
that involve many target organs. In the liver GH activates the
production of insulin-like growth factor 1 (Igf1) . Quantitative
RT-PCR measurements revealed a 50-fold decrease in Igf1
expression in the Prop1-/-mouse livers compared to wild type
(data not shown). Growth hormone is important for metabolism
and glucose homeostasis though its role in modulating Igf1
production . GH deficiency can cause hypoglycemia in
rodents and humans . We performed blood glucose measure-
ments on a variety of different Prop1 mutant genotypes at several
ages (Fig. 4). At 3.5 to 5 wks the blood-glucose level of Prop1-/-
mice (N4 B6 background) is similar to that of heterozygous
littermates and wild types, 140 +/2 14 mg/dL vs. 177 +/2
16 mg/dL, p=0.048 (Fig. 4A). By 5 to 6.5 weeks however, the N4
B6 Prop1-/-mice had approximately two-fold lower blood-glucose
levels than either Prop1+/+or Prop1df/+mice, 80 +/2 18 vs. 162 +/
2 22 mg/dL, respectively (Fig. 4B). Thus, mutants this age have
borderline hypoglycemia since a level of less than 60 mg/dL is
considered clinically hypoglycemic. Mice affected by wasting were
clearly hypoglycemic with blood glucose at 36 +/2 9 mg/dL
(Fig. 4B). Moreover, the corticosterone levels in wasting mice 5 to
6.5 wk old mice are strikingly elevated: 2.9 fold relative to wild
type and 1.9 fold relative to healthy mutants (Fig. 4C). The
corticosterone values are 136 +/2 40 in Prop1+/+(N=11), 193 +/
2 55 in Prop1df/-(N=12), and 211 +/2 36 in healthy Prop1-/-
(N=12), and 393 +/2 86 in sick Prop1-/-mice, (N=7). The very
high corticosterone levels support the idea that the wasting
phenotype is not due to failure of the pituitary adrenal axis. The
elevated levels are consistent with a response to metabolic stress,
but it is difficult to determine whether the cachexia is the cause or
the effect of severe hypoglycemia.
Figure 2. Elevated basal corticosterone levels in young adult
Prop1 deficient mice become higher in response to restraint
stress. RIA analysis of circulating corticosterone was carried out on
serum from 8 to 10 week males (A) and females (B) of segregating the
Prop1 null allele at N4 B6 prior to (white bars) and following restraint
stress (black bars). Male Prop1-/-(n=6) had significantly elevated basal
and post-stress levels of corticosterone compared to Prop1+/2(n=7)
and Prop1+/+(n=3). Values represent the mean corticosterone (ng/mL
of blood) 6 SE. *, P,0.0001. Female Prop1-/-(n=3) mice had both
elevated basal and post-stress levels of corticosterone compared to
Prop1+/2(n=5) and Prop1+/+(n=6). Values represent the mean
corticosterone (ng/mL of blood) 6 SE. *, P,0.005.
Prop1 Mutants Maintain ACTH Production
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The low glucose levels persist in older Prop1 deficiency mice.
At 8–10 weeks the N4 B6 Prop1-/-mice had lower glucose levels
(ave. 117 +/2 4 mg/dL) than controls (178 +/2 10 mg/dL)
(Fig. 4D). All genotype combinations of Prop1 mutants had
reduced serum glucose levels at older ages, 34–52 weeks,
although the levels were not low enough to be considered
clinically hypoglycemic: 186 +/2 14 mg/dL for Prop1+/+, 139
+/2 6 Prop1df/df, 128 +/2 5 Prop1df/-, and 128 +/2 4 mg/dL
for Prop1-/-(Fig. 4E). Thus, all genotype combinations of
mutants have significantly lower glucose levels after 5 wks
(p,0.0001), with the lowest levels in wasting mice.
We tested whether N4 B6 Prop1-/-mutants would respond to
30 min restraint stress with elevated glucose levels (Fig. 4D).
The pre- and post-stress values for mutants were 117 +/2 4 and
242 +/2 23 mg/dL, and the pre- and post-stress control values
were 178 +/2 10 and 382 +/2 22. Although the mutants
responded with elevated blood glucose, their post-stress glucose
levels were lower than control littermates. The fold change pre-
and post-stress, however, was similar in mutants and normal
littermates. These results demonstrate that Prop1 deficiency
causes a reduction in circulating glucose levels, but this
deficiency does not block the elevation of blood glucose in
response to stress.
The main goal of this research was to study the pituitary-
adrenal axis in two different mutant Prop1 alleles on different
genetic backgrounds to detect any evidence of ACTH deficiency
and subsequent hypocortisolism. If ACTH deficiency were
detected, then the mice would correspond to the findings of
acquired hypocortisolism in human MPHD patients with lesions in
the PROP1 gene [11,12,13]. We found no evidence for reduced
pituitary-adrenal axis function in Prop1 mutant mice. Instead, the
pituitary adrenal axis is activated, including both elevated ACTH
and corticosterone in the setting of blood low glucose levels. These
results are consistent with reports for DF/B-Prop1df/dfmice .
The GH deficiency of Prop1 mutant mice is associated with
reduced transcription of Igf1 in the liver, reduced blood glucose
levels, and activation of the pituitary adrenal axis. Despite these
metabolic alterations, affected mice are able to mount a stress
response yielding further elevations of ACTH, glucocorticoids,
and circulating glucose. Thus, we find no evidence of impaired
pituitary-adrenal axis function in Prop1 deficient mice for either the
df or null alleles on the backgrounds and ages tested. While we
cannot rule out the possibility that some combination of
parameters could provoke hypocortisolism in Prop1 mutant mice
Figure 3. Adrenal glands of Prop1 deficient mice are not hypotrophic. Adrenal glands were dissected from 5 and 8 week old female N4 B6
Prop1+/+and Prop1-/-mice, fixed, embedded, sectioned, and stained with hemotoxylin and eosin (Panels A, C, E, G) and immunostained for 20a-
hydroxysteroid dehydrogenase  and developed with diaminobenzidine (brown, Panels B, D, F, H) to visualize the X-zone (brackets). The ratio of
adrenal weight to body weight (Panel I) was increased in Prop1-/-(n=5) compared to Prop1+/2(n=6) or Prop1+/+(n=3) N4 B6 male mice at 8 to 10
wks. Values represent the mean adrenal weight (mg) per body weight (g) 6 SE. *, P,0.0001; **, P,0.0005.
Prop1 Mutants Maintain ACTH Production
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, it appears that evolving ACTH deficiency is a feature that
distinguishes mutant mice from the human patients with PROP1
Both Prop1 null and df mutant mice have the lowest circulating
glucose levels of 25-75 mg/dl between weaning and adulthood,
which is sometimes associated with lethality of unknown cause. We
observed the highest susceptibility to lethality after 5 wks on the B6
strain background, irrespective of the Prop1 mutant allele. Normal
B6 mice have a lower body weight and food intake than many
other strains during the time when Prop1 mutant lethality occurs
(Jax phenome database; http://www.jax.org/phenome). It is
possible that severe hypoglycemia contributes to the increased
susceptibility of Prop1 mutants to lethality on the B6 background,
although other differences in metabolism may be responsible. For
Figure 4. Prop1-defiency results in low blood glucose levels. Blood glucose levels were measured in normal and Prop1 mutant mice at four
ages. (A) Basal glucose levels in 3.5 to 5 week Prop1-/-mice (n=6) were lower than Prop1+/+(n=10) and Prop1+/2(n=10) mice from mixed genetic
backgrounds, but the difference was not statistically significant at this age. (B) On mixed genetic backgrounds the blood-glucose measurements from
5 to 6.5 wk old Prop1+/+(n=6) and Prop1df/df(n=6) were normal, but Prop1df/-(n=3), Prop1-/-healthy (n=12) and Prop1-/-wasting (n=7) mice had
significantly decreased blood-glucose levels. Values represent the mean blood glucose levels (mg glucose/dL blood) 6 SE. *, P,0.01; **, P,0.005. (C)
The low glucose levels in mutants shown in panel B are associated with elevated corticosterone levels (ng corticosterone/ml blood +/2 SE.) (D)
Blood-glucose levels were measured in 8 to 10 week old mice of the N4 B6 background prior to (white bars) and following restraint stress (black bars).
Prop1-/-(n=8) mice had decreased basal and post-stress blood-glucose levels compared to Prop1+/+(n=9) and Prop1+/2(n=11). Values represent the
mean blood glucose levels (mg glucose/dL blood) 6 SE. *, P,0.0001; **, P,0.0005. (E) Blood-glucose levels in 34 to 52 wk old mice on mixed genetic
background were decreased in all genotypes of Prop1 mutants, Prop1df/df(n=4), Prop1df/-(n=11), Prop1-/-(n=7), compared to normals, Prop1+/+
(n=4). Values represent the mean blood glucose levels (mg glucose/dL blood) 6 SE. *, P,0.005; **, P,0.0005; ***, P,0.0001.
Prop1 Mutants Maintain ACTH Production
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example, the livers of healthy Prop1 deficient mice resemble livers
of normal fasted mice, and sickly mutant livers are more affected
(data not shown) . The Prop1 mutants that survive to
adulthood have significantly longer life spans than their normal
littermates, like other strains with reduced insulin like growth
factor activity .
The lower glucose levels we observed in Prop1 deficient mice are
consistent with clinical data from human patients with GH
deficiency. Approximately 5% of humans with GH deficiency also
had hypoglycemia, and 10% of the hypoglycemic patients died
. Another study showed that approximately 3% (37/1366) of
GH deficient children died and that 24% (9/37) of those who died
suffered from severe hypoglycemia . Pituitary aplasia also
causes severe hypoglycemia, thus representing a serious life
threatening problem in neonates with MPHD if not quickly
treated [55,56]. Differences in the GH signaling pathway involving
AKT2 can cause hypoglycemia, seizures and death [57,58]. The
reason for the individual variation in susceptibility to severe
hypoglycemia and lethality in humans and mice are not known.
We found no evidence for disruption of the pituitary-adrenal
axis in Prop1 deficient mice. In direct contrast to the human
MPHD cases with progressive ACTH loss and hypocortisolism,
Prop1 deficient mice exhibit elevated ACTH and corticosterone
and reduced glucose levels at 6 mo and 1 yr of age. Young adult
Prop1 deficient mice respond to restraint stress with further
elevation of ACTH, corticosterone and glucose levels, and show
no reduction in adrenal content of steroidogenic enzymes,
indicating that the pituitary-adrenal axis can react functionally
to this challenge. In addition, the adrenals of the Prop1-/-mice are
enlarged relative to normal mice when normalized to body weight,
as expected for chronic ACTH secretion in rodents and other
mammals, including primates [36,37]. Finally, sickly, young Prop1
mutants have even higher corticosterone levels than healthy
The basis for the evolving nature of the hormone deficiencies,
including hypocortisolism, in human PROP1 deficient patients
remains elusive. It is tempting to speculate that it arises from
depletion of progenitors, but species differences in function are
also possible. Genetic background affects the viability of young
Prop1 deficient mice, largely due to different responses of target
organs to pituitary hormone deficiency. Multiple Prop1 mutant
alleles and genetic backgrounds support elevated ACTH and
corticosterone levels and lower glucose levels that persist with age.
Although mice with MPHD have been invaluable for understand-
ing the molecular basis for human disorders of hormone-deficiency
and dwarfism, pituitary growth, and pituitary cell specification,
they may be less pertinent for understanding the nature of
progressive hormone deficiency that characterizes humans with
We thank D.B. Hales for the generous gift of the StAR antibody, Walter
Miller for providing us with the p450c21  and p450scc antibodies ,
and Yacob Weinstein for the 20a-hydroxysteroid dehydrogenase antibody
. We also thank Mary Anne Potok for Figure 4.
Conceived and designed the experiments: ION FB CEK SAC. Performed
the experiments: ION RDW DLB FB AHM. Analyzed the data: ION
RDW DLB FB CEK GDH SAC. Wrote the paper: ION RDW SAC.
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