The Hospital for Sick Children (J.B., C.F., D.R.), Program in Cell Biology, and Institute of Medical Science and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5G 1X8; and Departments of Pathology (S.A.), University of Toronto and Laboratory Medicine and Pathobiology, University Health Network, Toronto, Ontario, Canada, M5G 2C4
Address all correspondence and requests for reprints to: Dr. Daniela Rotin, Program in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8. E-mail: drotin{at}sickkids.on.ca
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ABSTRACT |
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INTRODUCTION |
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RESULTS |
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We have analyzed neuroendocrine tissues in PTP knockout mice. The
adrenal medulla and pulmonary neuroendocrine bodies (NEBs) appeared
normal in PTP
(-/-), PTP
(+/-), and PTP
(+/+) mice, based on
gross appearance at autopsy, histological evaluation, and in the case
of pulmonary NEBS, on immunohistochemistry with calcitonin gene-related
peptide (data not shown). Subsequent evaluation focused on the
abnormalities noted in the pituitary, endocrine pancreas, and
enteroendocrine system of the gut.
Pituitary Histology and Immunohistochemistry
The anterior pituitary was hypoplastic relative to the
intermediate lobe in all three cohorts of PTP(-/-) animals. As we
previously reported, in many of the PTP
(-/-) neonates the
posterior pituitary was also hypoplastic (10). A prominent
intermediate cleft was still evident in all PTP
(-/-) newborns, as
compared with wild type (WT) controls (Fig. 1
, A and B). The cleft forms during
embryonic development of the adenohypophysis but by birth has become
negligible. Its marked persistence in the PTP
(-/-) neonate
suggests developmental delay of the adenohypophysis.
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To determine whether the significantly decreased immunopositivity for
GH or PRL in the young knockout animals was attributable to apoptosis
of somatotrophs and lactotrophs, we performed TUNEL assays (DNA nick
end labeling of tissue sections). In neither the GH-deficient
PTP(-/-) neonates, nor the PRL- depleted 2- to 3-wk-old
PTP
(-/-) mice, was apoptosis detected in the pituitaries (data not
shown).
Pit-1 is a transcription factor that is necessary for the development
of the somatotroph stem cell and its subsequent differentiation
into thyrotrophs, lactotrophs, and somatotrophs (12, 14).
To determine whether the decreased GH and PRL immunoreactivity was
secondary to a reduction in Pit-1 expression and the subsequent failure
of somatotroph/lactotroph lineage development, we examined Pit-1
immunoreactivity. The relative number of Pit-1-positive cells was not
reduced in those PTP(-/-) mice, which showed markedly decreased
GH, and absent PRL immunoreactivity (Fig. 4
, A and B). Double immunostaining for
Pit-1 and TSH in these PTP
(-/-) cohorts revealed a population of
cells immunoreactive for Pit-1 only, showing that not all
Pit-1-positive cells are thyrotrophs (Fig. 4C
). This suggests that in
the PTP
(-/-) neonate and the 2- to 3-wk-old wasting PTP
(-/-)
mice there are committed somatotrophs and/or lactotrophs incapable of
hormone expression. Alternatively, arrest in pituitary differentiation
may occur just before the emergence of somatotrophs and lactotrophs in
these PTP
(-/-) mice.
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To prove that the GH deficiency was a major contributing factor to the
neonatal mortality, we administered human GH (Humatrope,
Eli Lilly & Co., Indianapolis, IN) to a
series of newborn litters (Fig. 5A).
Survival analysis demonstrated that the PTP
(-/-) neonatal
mortality rate was significantly reduced when animals were treated with
GH compared with saline control, or when left untreated (Fig. 5B
).
Histological analysis of PTP
(-/-) pituitaries at the completion of
therapy (5 d of age) revealed normal GH immunoreactivity (data not
shown). BG levels of Humatrope-treated PTP
(-/-)
neonates that completed the treatment course were also normal (5.6
± 1.0 mmol/liter). Therefore, it appears that PTP
(-/-) neonatal
death is secondary to hypoglycemia, which is contributed to by GH
deficiency.
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To determine whether the reduction in islet mass was due to apoptosis,
we performed a TUNEL assay on representative animals from each of the
PTP(-/-) cohorts. There was no increased rate of apoptosis in the
PTP
(-/-) mice (data not shown).
To determine whether the islet hypoplasia and decreased insulin in the
PTP(-/-) mice were the result of GH deficiency, we assessed islet
histology and insulin immunoreactivity in the GH-treated animals. Both
insulin immunoreactivity and islet morphology normalized in the
PTP
(-/-) newborns and the 2- to 3-wk-old wasting PTP
(-/-)
mice receiving GH (data not shown).
BG And Serum Bicarbonate Levels in 2- to 3-wk-Old Animals
In view of the hypoinsulinemia noted in the wasting 2- to 3-wk-old
PTP(-/-) mice, we speculated that they might be succumbing to
diabetic ketoacidosis or a hyperglycemic hyperosmolar state. We
therefore measured BG levels and serum bicarbonate levels via arterial
blood gas analysis. Serum ketone levels, anion gap, and serum
osmolality could not be determined because of an insufficient volume of
serum available from the wasting PTP
(-/-) mice. BG levels of the
PTP
(-/-) mice were 4.6 ± 0.40 mmol/liter; BG levels of the
PTP
(+/+) controls were 9.0 ± 1.48 mmol/liter. Serum
bicarbonate levels of the PTP
(-/-) mice were 19.2 ± 2.38
meq/liter and were not significantly different from PTP
(+/+)
controls (20.7 ± 2.02 meq/liter). Therefore, despite the
hypoinsulinemia, enough insulin was present to prevent hyperglycemia
and the initiation of ketogenesis.
The BG level of the PTP(+/+) mice represents a random fed
level. The wasting 2- to 3-wk-old PTP
(-/-) mice had no milk in the
gut at autopsy. Thus, the BG level of the PTP
(-/-) mice in this
experiment represents a fasting level and is therefore lower than that
of the fed controls.
Gut Histology And Immunohistochemistry
On hematoxylin-eosin (H & E) section, mild villous
trophy was occasionally evident in the small and large intestine of
the PTP(-/-) mice (data not shown). Immunohistochemistry revealed
significant variability in the expression of gut hormones by the
enteroendocrine system (Table 1
). Most
profoundly affected were the wasting 2- to 3-wk-old PTP
(-/-) mice.
This entire cohort (100%) exhibited a decrease in cells containing
serotonin, some with total lack of immunoreactivity for this gut
hormone (Fig. 8
). Secretin, gastrin,
somatostatin, glucagon, and cholecystokinin (CCK) were decreased in
50% to 75% of mice. Peptide YY (PYY) was the only hormone with intact
staining in the 2- to 3-wk-old wasting PTP
(-/-) cohort.
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DISCUSSION |
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It is well established that Pit-1, while necessary, is not sufficient for the terminal differentiation of thyrotrophs, mammosomatotrophs, or lactotrophs. For example, coexpression of the transcription factor GATA-2 along with Pit-1 is required for differentiation of the thyrotroph (28, 29). In cooperation with Pit-1, estrogen binding to its receptor leads to weak activation of the PRL gene and induces differentiation of the mammosomatotroph (30). Members of the ETS transcription factor family are also involved in the synergistic activation of the PRL promoter when coexpressed with Pit-1 (31, 32). The current model of cytodifferentiation hypothesizes that additional repressor(s) acting on the mammosomatotroph down-regulate GH gene transcription, thus permitting terminal differentiation of the mature lactotroph (12).
GH and PRL immunostaining was significantly diminished in PTP(-/-)
newborns and 2- to 3-wk-old mice, respectively. All animals revealed
normal Pit-1 and TSH immunoreactivity. These results suggest
appropriate adenohypophyseal development to the emergence of the
somatotroph stem cell in PTP
(-/-) mice. Possible explanations for
the subsequent downstream abnormalities of the somatotroph and
lactotroph include the divergence of the majority of Pit-1-positive
cells to a thyrotroph phenotype, leaving a diminished number to retain
the somatotroph phenotype, and none to proceed to a mature lactotroph.
However, coimmunostaining for Pit-1 and TSH proved this hypothesis
wrong by demonstrating a population of cells immunoreactive for Pit-1
but not for TSH. Selective depletion of the Pit-1-positive TSH-negative
cells via apoptosis was not evident, as the TUNEL assay was negative.
Appropriate specification of the somatotrophs and lactotrophs, but
delayed expansion, could account for the observed phenotype. Normally,
somatotroph terminal differentiation is complete by the end of
gestation, and proliferation begins before birth. Terminal
differentiation, maturation, and expansion of the lactotroph population
all occur postnatally over hours to days. In the PTP
(-/-) mouse,
developmental delay of the adenohypophysis could result in terminal
differentiation and expansion of both somatotrophs and lactotrophs
postnatally. However, eventually all adenohypophyseal cell lines would
emerge. This hypothesis is supported by the persistence of a prominent
embryonic cleft in the newborn PTP
(-/-) mice, normal
immunoreactivity for all anterior pituitary hormones in the
PTP
(-/-) mice that survive to adulthood, and the presence of
developmental delay in the peripheral nervous system of PTP
(-/-)
mice (10). In addition, all surviving GH-treated neonates
revealed normal pituitary GH immunoreactivity at 5 d of age,
implying that maturation and proliferation of the somatotrophs will
occur postnatally if the animal survives.
An alternative explanation that must be considered, however, is that
the terminal cytodifferentiation of the somatotroph and lactotroph is
indeed intrinsically impaired in the newborn and wasting 2- to 3-wk-old
PTP(-/-) mice. We do not know the reason for the presence of three
distinct phenotypic cohorts of the PTP
(-/-) mice. Possible
explanations for this phenotypic diversity include variability of
PTP
penetrance, effects of modifier genes, and varying levels of
compensatory contribution from other related genes (e.g. the
PTP
close relative LAR, which is also expressed in the pituitary)
(8). Any of these events could potentially alter
development of the somatotroph/lactotroph lineage in the affected
cohorts.
A second PTP knockout mouse, generated by the targeted disruption of
the intracellular catalytic domains by Tremblay and colleagues
(11), demonstrated similar adenohypophyseal hypoplasia.
While GH immunoreactivity or serum levels were not measured in these
mice, IGF-I levels were found to be significantly decreased by 2540%
in the PTP
(-/-) animals compared with PTP
(+/+ and +/-)
controls. This result is in agreement with the decreases in GH
immunoreactivity noted in our PTP
(-/-) animals. Similarly, the
Tremblay knockout mice demonstrated a decrease in PRL immunoreactivity.
However, in contrast to our findings, decreases in the percentage of
TSH, LH, and FSH and increases in the percentage of ACTH-immunopositive
cells were noted in their PTP
(-/-) mice. These differences in
phenotype may be due to residual expression of the extracellular domain
of PTP
in those mice. Analysis of the pancreas, adrenals, NEBs, and
the enteroendocrine gut was not reported in the Tremblay mice.
The substantial decrease in GH immunoreactivity in newborn
PTP(-/-) animals led us to speculate that the high neonatal
mortality rate may be secondary to hypoglycemia. GH is one of the
counterregulatory hormones involved in glucose homeostasis. In times of
fasting, GH, in concert with glucagon, epinephrine, and ACTH, maintains
BG levels via stimulation of glycogenolysis, ketogenesis, and
gluconeogenesis. The gluconeogenic and ketogenic pathways do not mature
in the human newborn liver until 1224 h of age (33).
Thus, the neonate is susceptible to transient hypoglycemia. Further
challenge resulting from deficiencies in one of the counterregulatory
hormones can result in profound, sustained hypoglycemia, as seen in
panhypopituitarism and isolated GH deficiency in the human newborn. BG
levels in PTP
(-/-) newborns with severely diminished GH
immunostaining were less than 1.0 mmol/liter which, if sustained, is
incompatible with life. ACTH immunoreactivity was normal in all mice.
Intraperitoneal administration of GH to newborn PTP
(-/-) mice
significantly decreased the neonatal mortality rate. Treated
PTP
(-/-) mice killed at 5 d of age demonstrated normal GH
immunoreactivity and BG levels. This suggests that the neonatal
mortality was due to profound hypoglycemia induced, at least in part,
by GH insufficiency. The rescue achieved with GH administration,
however, was not 100%. Those neonates that were administered GH but
still died may have received inadequate GH supplementation, or a second
phenomenon may have contributed to their death. Unfortunately, the
pituitaries and pancreases of the GH-treated PTP
(-/-) newborns
that failed therapy were unavailable for postmortem analysis, due to
the fact that the mother expediently disposes of the body.
Histological assessment of the endocrine pancreas revealed islet
hypoplasia in the newborn and wasting 2- to 3-wk-old PTP(-/-)
animals. While insulin immunostaining was intact in the small islets of
the neonates, there was a marked decrease in insulin immunoreactivity
in the islets of the 2- to 3-wk-old PTP
(-/-) animals. Measurement
of serum insulin levels supported the immunohistopathology.
The 2- to 3-wk-old knockout animals had extremely low insulin
levels that were significantly decreased compared with controls. In
the healthy adult PTP
(-/-) animals, islet morphology
and insulin immunostaining were normal. Throughout all three
cohorts of PTP(-/-) animals, marked variability of PP and
somatostatin immunostaining was evident. Glucagon immunoreactivity was
intact in all animals.
While the islet is composed of four cell types, which include
insulin-producing B cells, glucagon-producing A cells,
somatostatin-producing D cells, and pancreatic polypeptide-producing PP
cells, its principal component is the B cell (34).
Rapid growth of the endocrine pancreas occurs in the late fetal and
early weeks of life, after which growth of the exocrine pancreas ensues
(35). This makes the finding of hypoplasia of the islets
at 23 wk of age even more striking, as the endocrine/exocrine tissue
ratio should be at its peak at that time. TUNEL assay revealed that
loss of islet mass was not secondary to apoptosis, but was indeed due
to true hypoplasia. Numerous proteins, including differentiation
factors such as activin, classic growth factors such as hepatocyte
growth factor, and hormones such as GH and PRL, have been variably
implicated in islet cell differentiation and proliferation, as well as
activation of expression of insulin by the B cell
(35, 36, 37, 38, 39, 40). In rats bearing GH- and PRL-producing tumors,
and human GH-expressing transgenic mice, B cell mass increases and
insulin synthesis and secretion are enhanced (40, 41).
Thus, while there are many possible candidates to explain the islet
hypoplasia and decreased insulin immunoreactivity observed in the
PTP(-/-) mice, the most likely contributory cause is GH
deficiency. Indeed, islet morphology and insulin immunoreactivity
significantly improved in the PTP
(-/-) mice treated with GH.
Therefore, islet hypoplasia and decreased insulin immunoreactivity are
likely secondary to the pituitary abnormalities noted in the
PTP
(-/-) animals.
The low insulin levels and clinical picture of the wasting 2- to
3-wk-old PTP(-/-) animal suggested that diabetic ketoacidosis or a
hyperglycemic hyperosmolar state may be the cause of death in this
cohort. However, hyperglycemia was not evident in these mice, and serum
bicarbonate levels were not significantly different from PTP
(+/+)
controls. Despite the hypoinsulinemia, adequate amounts were present
for the prevention of ketogenesis. Therefore, ketoacidosis was not
responsible for the death of our PTP
knockout mice.
The PTP(-/-) cohort lost at 23 wk of age from a wasting syndrome
revealed severe loss of serotonin-immunopositive cells in the proximal
gut, compared with control animals. With the exception of this
consistent finding, the PTP
(-/-) mice displayed a significant
heterogeneity between and within cohorts with respect to the level of
expression of the various gut peptide hormones. Differentiation of the
intestinal epithelium from a pluripotent stem cell to a terminally
committed enteroendocrine cell is a highly regulated and complex
process (42, 43, 44). In view of this complexity, it is
difficult to hypothesize the mechanism(s) by which lack of PTP
would
translate into a specific loss of serotonin-producing cells in the gut
of a cohort of animals destined to die, and a variable decrease in the
expression of other hormones in the other two cohorts of PTP
(-/-)
animals. We can speculate that the loss of serotonin expression leading
to abnormal gut motility, combined with impaired absorptive capacity
due to mild villous atrophy, may be responsible for the wasting
syndrome noted in the 2- to 3-wk-old knockout animals.
Overall, the mechanism by which the lack of PTP results in the
complex phenotype observed is not fully understood. While abl and
disabled were genetically identified as collaborative proteins in the
developing Drosophila nervous system, ligand(s) and
substrate(s) in the mammal have yet to be identified. The cell adhesion
molecule-like ectodomain and tyrosine phosphatase enzymatic activity
likely engage PTP
in a number of signaling networks that could
likely affect development and function of the endocrine system.
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MATERIALS AND METHODS |
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All animal experimentation was conducted in accordance with accepted standards of humane animal care.
Histological Analysis
Representative animals from the three cohorts of PTP(-/-)
and control PTP
(+/-),(+/+) mice were killed, and examined by
complete autopsy. Neuroendocrine tissues, including the pituitary,
pancreas, gut, adrenals, as well as lungs, were fixed and embedded in
paraffin, sectioned (5 µm), and stained with H & E for histological
evaluation. The pituitaries were also stained with the Gordon-Sweet
silver method to demonstrate the reticulin fiber network. Thyroid and
parathyroid were not systematically examined due to difficulty of
reliably harvesting these tissues from the mice.
Immunocytochemical stains to localize hormones and other cellular antigens were performed using the avidin- biotin-peroxidase complex technique and visualized using 3,3'-diaminobenzidine (DAB). For pituitary, the duration of exposure to primary antiserum was 30 min at room temperature. The primary polyclonal antisera were directed against the following antigens and were used at the specified dilutions: ACTH (DAKO Corp., Carpinteria, CA); rat GH, PRL, ßTSH, ßFSH, and LH [all donated by the National Hormone and Pituitary Program (NHPP), National Institute of Diabetes, Digestive and Kidney Disease (NIDDK), Bethesda, MD], at 1:2,000, 1:15,000, 1:3,000, 1:1,000, 1:500, and 1:400, respectively. Nuclear transcription factors Pit-1, SF-1, and ER were localized after microwave antigen retrieval and detected with the avidin-biotin-peroxidase Elite technique (Vector Laboratories, Inc., Burlingame, CA). For Pit-1 a polyclonal antiserum raised against a synthetic peptide corresponding to the epitope depicted by residues 214230 of rat pit-1 (BabCo, Berkeley Antibody Company, Richmond, CA) was used at 1:600 for 1 h at room temperature. For ER a monoclonal antisera (Novocastra, Newcastle upon Tyne, UK), was used at a 1:70 dilution for 1 h. A polyclonal antisera against SF-1 (Upstate Biotechnology, Inc., Lake Placid, NY) was used at a 1:600 dilution for 1 h. For colocalization of nuclear transcription factors and cytoplasmic hormones, the nuclear stain was performed as described and detected using cobalt DAB. The cytoplasmic antigen was identified using a peroxidase-conjugated secondary antibody method to avoid cross-reaction and visualized with DAB. For gut and pancreas, primary antisera and antibodies were directed against the following antigens and were used at the specified dilutions and incubation durations: insulin (monoclonal antibody from BioGenex Laboratories, Inc., San Ramon, CA) 1:40 for 30 min; glucagon (polyclonal antiserum from Immunon, Pittsburg, PA) 1:200 for 30 min: GLP-1 (polyclonal antiserum donated by Dr. D. J. Drucker, Toronto, Ontario, Canada) 1:1,500 for 30 min; GLP-2 (polyclonal antiserum donated by Dr. D. J. Drucker) 1:2,500 for 30 min; peptide YY (PYY) (polyclonal antiserum from Peninsula Laboratories, Inc., Belmont, CA) 1:2,000 for 30 min; somatostatin (polyclonal antiserum from DAKO Corp.) prediluted preparation further diluted 1:40 overnight; pancreatic polypeptide (PP) (polyclonal antiserum from DAKO Corp.) 1:6,000 for 30 min, CCK (polyclonal antiserum from Serotec, Oxford, UK) 1:1,000 for 30 min; serotonin (monoclonal antibody from DAKO Corp.) 1:50 for 60 min; secretin (polyclonal antiserum from Biogenex Laboratories, Inc.) prediluted preparation further diluted 1:5 overnight; gastrin (polyclonal antiserum from Zymed, San Francisco, CA) 1:150 for 60 min. For the lungs, calcitonin gene-related peptide, a polyclonal antisera (Chemicon, Temecula, CA), was used at a dilution of 1:400 overnight at 4 C. Appropriate positive and negative (omission of the primary antibody) controls were performed in each case.
Morphometric Analysis
The hormone content of representative pituitary glands
or pancreatic islets was quantified using MCID software (Imaging
Research, Inc., St. Catherines, Canada) (n = 2 to 5
mice/hormone/cohort). The area of immunopositive anterior
pituitary cells was determined and expressed as a percentage of the
total adenohypophyseal area. Representative islets were assessed for
determination of pancreatic hormone content. The area of immunopositive
cells was determined and expressed as a percentage of islet area.
An experienced endocrine pathologist, blinded to the PTP mouse
genotype and cohort, graded islet size as being normal, increased, or
decreased. Due to the heterogeneity of islet distribution throughout
the pancreas, MCID quantitation of total islet area relative to total
pancreatic area was not performed as the entire pancreas could not
realistically be scanned for analysis. The heterogeneous distribution
of islets precluded selection of a representative area and therefore
any attempts at quantitation would have been biased and inaccurate.
Similarly, due to the large lengths and surface area of small and large
bowel, immunopositivity for the enteroendocrine hormones was assessed
by an experienced pathologist, blinded to mouse genotype or cohort, and
graded as being normal, increased, or decreased.
TUNEL Assay
Nuclei of tissue sections were stripped from proteins by
incubation with 20 µg/ml proteinase K (Sigma, St. Louis,
MO) for 15 min at room temperature, and the slides were then washed in
double distilled water (DDW) for 2 min (x4). Endogenous peroxidase was
inactivated by covering the sections with 2%
H2O2 for 5 min at room
temperature. The sections were rinsed with DDW, and immersed in TDT
buffer (Oncor) (30 mM Trizma base, pH 7.2, 140
mM sodium cacodylate, 1 mM cobalt chloride).
TDT (0.3 equivalent units/µl) and biotinylated dUTP in TDT buffer
(Oncor, Gaithersburg, MD) were added to cover the sections and then
incubated in humid atmosphere at 37 C for 60 min. The reaction
was terminated by transferring the slides to TB buffer (300
mM sodium chloride, 30 mM sodium citrate) for
15 min at room temperature. The sections were rinsed with DDW, covered
with 2% aqueous solution of BSA for 10 min at room temperature, rinsed
in DDW, and immersed in PBS for 5 min. The sections were covered with
streptavidin peroxidase, diluted 1:101:20 in water, incubated for 30
min at 37 C, washed in DDW, immersed for 5 min in PBS, and stained with
DAB for about 30 min at 37 C.
Lac Z Staining
Animals were killed and the pituitaries and pancreas were
harvested and fixed in a 0.1 M sodium phosphate buffer (pH
7.9) containing 1% formaldehyde, 0.1% glutaraldehyde, 2
mM MgCl2, and 5 mM EGTA
for 6 h. Organs were washed for 2 h with four exchanges of a
wash buffer (2 mM MgCl2, 0.01%
deoxycholate, 0.02% Nonidet P-40 in 0.1 M sodium
phosphate buffer, pH 7.9) at room temperature. The tissue was then
incubated in PBS containing 5 mM ferricyanide, 5
mM ferrocyanide, 2 mM
MgCl2 and X-gal (Roche,
Indianapolis, IN) 0.1 mg/ml overnight at 37 C. Tissues were
subsequently rinsed in 70% EtOH, paraffin embedded, and sectioned.
Blood and Serum Chemistry
BG levels were determined using the One Touch Glucometer
(Johnson & Johnson, New Brunswick, NJ) on venous blood
obtained via tail clip (older mice) and decapitation (newborn).
Arterial blood for blood gas analysis was obtained via cardiac puncture
on mice anesthetized with isoflurane 2.5%. Samples were collected in a
heparinized syringe, transported on ice, and immediately analyzed on a
Nova Stat Profile Plus 9 (Nova Medical, Wakefield, MA). Serum
insulin levels were measured with the Rat Insulin ELISA Kit (Crystal
Chem, Inc., Chicago, IL) according to manufacturer instructions, but
with two modifications. Purified mouse insulin (Crystal Chem, Inc.)
instead of rat, was used to generate the standard curve. Serum samples
with insulin levels below the sensitivity of the assay were spiked with
a known quantity of purified mouse insulin, to ensure absorbance of the
sample fell within the exponential portion of the standard curve. Serum
GH levels were assessed by Western blot analysis. Fifty micrograms of
serum protein were separated by 12% SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with anti-GH antibodies at a
dilution of 1:5,000 (polyclonal antisera donated by Dr. S. Ezzat,
Toronto, Ontario, Canada). Relative band intensities were quantified by
charge coupled device camera detection of enhanced
chemiluminescence and the serum GH levels of PTP(-/-) mice
expressed as a percentage of control PTP
(+/+) littermates.
Quantitation of serum GH levels by RIA or ELISA was not possible due to
the small and limiting volumes of PTP
(-/-) sera attainable.
All statistic analyses are represented as mean ± SD.
GH Replacement Therapy
This experiment was designed as a two-phase study using human
GH, as murine GH is not available for commercial use. In the first
phase recombinant human GH (Humatrope) was administered
daily to all animals within newborn litters for 4 d, at a dose of
0.06 mg/kg/d ip. Control litters were administered saline daily ip for
4 d or received no treatment. Innate survival of animals was
monitored and mortality was documented. Data were subjected to Kaplan
Meier Survival Analysis for determination of statistical
significance.
After completion of survival analyses (phase 1), a phase 2 study was
initiated to determine the effects of GH rescue on the pituitary and
pancreatic pathology in the PTP(-/-) mice. Litters were again
treated with Humatrope, saline, or received no treatment
as described above. A portion of PTP
(-/-) mice that completed
treatment were euthanized at 5 d of age, blood glucose was
measured as previously outlined, and pituitaries were immunostained for
GH and morphometric analysis as previously outlined. PTP
(-/-)
wasting 2- to 3-wk-old mice were euthanized, and the pancreases were
dissected for insulin immunostaining and morphometry as previously
described.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: BG, Blood glucose; CCK, cholecystokinin; DAB, 3,3'-diaminobenzidine; DDW, double distilled water; GLP, glucagon-like peptide; H & E, hematoxylin-eosin; LAR, leukocyte common antigen-related; NEBs, neuroendocrine bodies; Pit1, pituitary-specific POU homeodomain factor; PP, pancreatic polypeptide; PTP, protein tyrosine phosphatase; PYY, peptide YY; SF-1, steroidogenic factor 1; TUNEL, DNA nick end labeling of tissue sections.
Received for publication August 1, 2000. Accepted for publication September 21, 2001.
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REFERENCES |
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