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Yale University School of Medicine (S.E.P., P.D., R.C.V.), New Haven, Connecticut 06520; The Department of Cell Biology (R.L.S.), The University of Minnesota, Minneapolis, Minnesota 55455; The Division of Endocrinology (A.G.-O., A.F.S.), The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213; and the Department of Veterans Affairs, Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Andrew F. Stewart M.D., Division of Endocrinology, E-1140 BST, University of Pittsburgh Medical Center, 3550 Terrace Street, Pittsburgh, Pennsylvania 15213. E-mail: stewart{at}med1.dept-med.pitt.edu
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Abstract |
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In the current report, we demonstrate that PTHrP overexpression induces a progressive increase in islet mass over the life of the RIP-PTHrP mouse, and that, in contrast to some other models of targeted PTHrP overexpression, the phenotype is not developmental, but occurs postnatally. The marked increase in islet mass is not associated with a measurable increase in ß cell replication rates. A further slowing in the normally low islet apoptosis rate could not be demonstrated in the RIP-PTHrP islet. Thus, the marked increase in islet mass in the RIP-PTHrP mouse is unexplained in mechanistic terms. Finally, RIP-PTHrP mice are resistant to the diabetogenic effects of streptozotocin.
The mechanisms responsible for the increase in islet mass in the RIP-PTHrP mouse likely lie in either very subtle changes in islet turnover or in early steps in islet differentiation and development. The ability of PTHrP to increase islet mass and function, as well as its ability to attenuate the diabetogenic effects of streptozotocin, indicate that further study of PTHrP on islet development and function are important and may lead to therapeutic strategies in diabetes mellitus.
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Introduction |
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Recently, we have begun to explore the function of PTH-related protein (PTHrP) in the islet. PTHrP was originally identified through its causal role in hypercalcemia in patients with cancer, but it is now clear that PTHrP is produced by virtually every tissue in the body. PTHrP most often appears to act in a paracrine or autocrine fashion. The physiologic roles of PTHrP in the various tissues that produce the peptide have been the subject of recent reviews (19, 20). One dominant theme in PTHrP physiology is that it serves as a growth factor, an apoptosis-inhibiting factor, a differentiation factor, and/or a developmental factor, in virtually every tissue in which these effects have been sought (19, 20, 21, 22, 23, 24, 25).
PTHrP is produced in the pancreatic islet in all four islet cell types (26, 27, 28). It is packaged in secretory vesicles with insulin and is secreted in response to insulin secretagogues (29). A receptor for PTHrP is present on ß cells as well (26), suggesting that in the islet, as in other tissues, PTHrP may play a paracrine or autocrine role. We have previously reported that targeted overexpression of PTHrP in the ß cell under the influence of the rat insulin II promoter (RIP) results in RIP-PTHrP mice that have a rather striking increase in islet mass, and this is accompanied by hyperinsulinemia and hypoglycemia (28). In that prior report, we had limited our studies to animals of 8–12 weeks of age.
In the current study, we had three goals. First, we wanted to determine the expression pattern of the RIP-PTHrP transgene as a function of age. Second, we wanted to determine whether the increase in islet mass we had observed in RIP-PTHrP mice at 8–12 weeks of age was the result of a developmental "decision" to increase islet mass, or whether PTHrP played predominantly a postnatal role in increasing islet mass throughout life. Finally, we wanted to explore the mechanisms responsible for the increase in islet mass. The studies reported herein indicate that the increase in islet mass in the RIP-PTHrP mouse is not a "developmental" or embryologic phenotype, but one that is acquired postnatally. Remarkably, the increase in islet mass cannot be clearly linked to either an acceleration of islet cell proliferation or a reduction in islet cell apoptotic rates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA preparation and RNAse protection assays
RNA was prepared as previously reported (28) using the cesium
chloride method to minimize the effects of pancreatic RNases. RNAse
protection analysis was performed using RNA probes (28) generated from
the following three DNA sequences: 1) a
PvuII-SacI cDNA fragment of the human PTHrP gene
corresponding to a 307 bp protected fragment; 2) a Sau 3A-Sau 3A cDNA
fragment of the mouse cyclophilin gene resulting in a 220 bp protected
fragment; and 3) a PstI-AvaI mouse insulin
genomic fragment resulting in a 230 bp protected band.
Serum and plasma biochemistries
Glucose was measured using an Accu-Check III glucometer
(Boehringer-Mannheim, Indianapolis, IN). Insulin was measured using the
Linco RIA (28). Calcium was measured using atomic absorption
spectroscopy.
Histology, immunohistochemistry, and histomorphometry
Pancreata were removed and placed immediately in Bouin’s
solution and fixed for 12–16 h. Sections were prepared and stained
with hematoxylin and eosin using standard techniques. For insulin and
glucagon staining, immunohistochemistry was performed using primary and
secondary antisera from BioGenex, Inc. (San Ramon, CA) (28). For PTHrP
immunohistochemistry, staining was performed using an
affinity-purified rabbit antiserum raised against PTHrP(37–74)
(26, 28). In the case of insulin, glucagon and PTHrP, specificity was
confirmed by omitting the primary antiserum from controls. In addition,
for PTHrP staining, specificity was further confirmed by competition
using 10-6 M PTHrP(37–74) as described
previously (28). Quantitative histomorphometry was performed as
described previously (28) using a Nikon Labphot microscope coupled to
an Osteotablet package (Osteometrics, Atlanta, GA).
Islet cell size calculations
These measurements were performed as described in rats by
Montana et al. (30) and Scaglia et al. (31).
Briefly, pancreata of 1-yr-old normal (n = 3) and 1-yr-old
RIP-PTHrP (n = 3) mice were sectioned and stained with hematoxylin
and eosin. Total islet area was measured by planimetry as described in
the preceding paragraph. Islet cell number in a given islet was
estimated by counting the total number of individual islet cell nuclei
within that islet. Six islets were measured and counted from each
mouse, such that a total of 36 separate islets were counted, 18 normal
and 18 RIP-PTHrP. Mean islet cell area was calculated by dividing the
total islet area in a given islet by the total number of islet cells
(i.e. islet cell nuclei) within that islet. Results are
expressed in microns squared (u2).
In vivo proliferation studies
Proliferation was measured as described by Montana et
al. (30). Briefly, mice were injected ip with bromodeoxyuridine
(BrdU) (Cell Proliferation Kit, Amersham Pharmacia Biotech, Arlington
Heights, IL) and killed 6 h later. Pancreata were promptly
fixed in Bouin’s solution for 12–16 h, and then embedded, sectioned,
deparaffinized, and stained using a primary antiserum against BrdU
(Amersham) as well as with an antiglucagon antibody to aid in the
identification of islets. Sections were lightly counterstained using
hematoxylin. Sections were counted in a blinded fashion and results
expressed as the number of BrdU-labeled islet nuclei per total number
of islet nuclei. At least 2000 nuclei were counted per pancreas.
In vitro islet proliferation studies
Neonatal rat islets were isolated from 3- to 5-day-old rats
(Sprague-Dawley, Sasco, Omaha, NE) pooled from two or more litters by a
nonenzymatic culture method previously described (32). Groups of 30
islets were transferred to 24-well plates (Costar, Cambridge, MA) and
cultured free-floating in 2 ml RPMI 1640 (Gibco BRL, Life Technologies,
Grand Island, NY) containing 180 mg/dl glucose supplemented with 10%
horse serum, 25 mM HEPES, and 1% penicillin, streptomycin,
fungizone. The multiwells were incubated at 37 C in a humidified
atmosphere of 95% air and 5% CO2 for the duration of the
experiment. The medium was changed daily.
ß cell proliferation was determined using the previously described method for examining BrdU incorporation into dividing cells within cultured islets (33). PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), PTHrP(38–94)amide, and PTHrP(107–139) were prepared by solid phase synthesis as described previously (34, 35), and were added to the cultures in the concentrations shown for 6 days. BrdU (10 uM) was added to the culture medium for the final 24 h of culture. The islets were washed, fixed in 4% paraformaldehyde for 30 min at 25 C, and the islet DNA denatured by acid hydrolysis in 0.5 N HCl for 20 min at 25 C. After rinsing in PBS, the islets were immunostained with a 1:500 dilution of a mouse monoclonal anti-BrdU antibody (Clone IU-4, Cal-Tag, San Francisco, CA) and a 1:50 dilution of a fluorescein isothiocyanate-conjugated goat antimouse monoclonal anti-BrdU antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). The number of BrdU-labeled nuclei per islet was determined by direct observation with conventional epifluorescence microscopy. The number of BrdU-positive nuclei was counted in 50 islets. Each experiment was repeated twice, and the results are expressed as mean ± SEM. Statistical analysis was performed using ANOVA and Dunnett’s posthoc test for determining differences between the groups.
Apoptosis
Apoptosis was measured using the DNA fragmentation (TUNEL)
method using the In Situ Cell Death kit from
Boehringer-Mannheim (Mannheim, Germany) with modifications as described
by Scaglia et al. (31).
Streptozotocin studies
Streptozotocin (SZN) (1 mg/50 µl in 10 mM sodium
citrate in 0.9% saline, pH 4.0–4.5) was administered ip to mice
between 8–12 weeks of age in two doses 100 mg/kg separated by 12
h. Tail vein blood glucose concentrations were obtained at the times
indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the expression of
hPTHrP messenger RNA (mRNA) derived from the transgene in whole
pancreas RNA in RIP-PTHrP mice at the three stages of life as compared
with age-matched littermates. As can be seen in the figure, the
transgene is expressed as early as 1 week of age, and, as expected, is
absent in the pancreatic RNA prepared from normal control mice. The
levels of insulin mRNA expression did not appear to differ in the
normal and RIP-PTHrP mice at 1 week of age, but by 8–12 weeks through
1 yr of age, the level of expression of both the transgene and of
endogenous insulin appeared to increase in the transgenic mice.
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shows the results of serum
calcium in the RIP-PTHrP mice as a function of age. As can be seen from
the figure, serum calcium concentrations are normal and are comparable
with those of their normal littermates. We have previously reported
that PTHrP concentrations in the systemic circulation are undetectable
in RIP-PTHrP mice (28). Here, serum calcium is shown to remain normal
even at 1 yr of age.
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). As is shown
in the figure, the mean blood fasting glucose in the 1-yr-old RIP-PTHrP
mice is approximately 25 mg/dl below their littermates, and their
postprandial glucose is approximately 15 mg/dl below their littermates.
As shown in the bottom panel, this hypoglycemia is associated with
persistent hyperinsulinemia.
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, at 1 week of
age, islet number and islet mass, as determined by quantitative
histomorphometry, are indistinguishable in RIP-PTHrP mice and their
normal littermates. However, as previously reported (28) and as shown
for comparison in Fig. 5, by 8–12 weeks
of age, islet volume and islet number have increased by approximately
2-fold. This increase in islet mass continues through life, such that
by 1 yr of age, islet volume in RIP-PTHrP mice has increased to
approximately three times that observed in their normal siblings (Fig. 5). The increase in islet mass is so striking by 1 yr, that it is
apparent on casual observation in a low power photomicrograph (Fig. 6). When the data shown in Figs. 4 and 5
are normalized as a percent of normal islet volume (Fig. 7), the gradual progression in islet
volume throughout life is appreciated.
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).
The increase in islet mass is not associated with a measurable
increase in ß cell proliferation in vivo or in vitro, nor with a
measurable reduction in apoptosis.
Proliferation rates in islets from 1-week-old and 8- to
12-week-old animals, as assessed using BrdU incorporation, are shown in
Fig. 8. As can be seen in the figure,
while islet proliferation rates were higher in the younger animals as
compared with the older animals, as is well described (1), there was no
measurable increase in islet proliferation rate in the RIP-PTHrP mice
as compared with their normal littermates at either age.
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, whereas PRL at a dose of 25
nM dramatically stimulated islet proliferation, no such
effect was observed with any of the three PTHrP secretory peptides, at
doses that are several logarithms higher than required to exert effects
in other PTHrP-responsive systems.
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Pancreatic PTHrP mRNA levels do not change with pregnancy nor with
parturition in the mouse
Islet proliferation occurs during pregnancy in the rat, and
islet apoptosis occurs during the early postpartum period (1, 2, 3, 31).
Because PTHrP is associated with an increase in islet mass, and because
it is associated with apoptosis in other tissues (19, 20, 21, 22, 31), we
wondered whether endogenous islet PTHrP mRNA concentrations would
change during gestation or the postpartum period. As can be seen in
Fig. 10, the level of endogenous PTHrP
mRNA expression in the pancreas is low (28); it is derived exclusively
from the islet and pancreatic duct (26, 27, 28). However, no change in
endogenous pancreatic PTHrP mRNA levels were observed either during the
trimesters of pregnancy, nor in the postpartum period.
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, RIP-PTHrP mice and their normal
littermates at 8–12 weeks of age were injected ip with streptozotocin
in a dose designed to induce prompt hyperglycemia in normal mice. As
seen in the figure, normal mice developed severe diabetes. In rather
striking contrast, RIP-PTHrP mice either remained normoglycemic or
developed only mild to moderate diabetes, and the mean blood glucoses
in the RIP-PTHrP mice were markedly lower than their normal
siblings.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In
this respect, PTHrP overexpression in the islet is similar to that in
the mammary epithelium (25), which has predominantly a postnatal
phenotype. One important conclusion from these studies is therefore
that PTHrP may serve both as a critical developmental factor (PTHrP
gene disruption results in a neonatally lethal chondrodysplasia) (21),
and as a factor that regulates tissue modeling in adolescent and adult
life as well (in the mammary gland and in the islet). The "late"
expression of the phenotype is not due to failure of the transgene to
be expressed in the neonatal islet, for the insulin gene is expressed
as early as day e9.5 (40), and in the current study, the RIP-PTHrP
transgene was expressed as early as 1 week of age.
Histomorphometric measures indicate that the increase in islet cell
mass is a result of islet cell hyperplasia, not hypertrophy. One key
question relates to the mechanism whereby PTHrP might lead to islet
hyperplasia. One possible explanation for this could be that islet
proliferation rates in the RIP-PTHrP mice are higher than in their
normal littermates. This is not apparent from the findings shown in
Fig. 8, where islet proliferation rates in vivo were no
different in RIP-PTHrP mice than in their normal littermates. It is
important to point out, however, that to increase islet mass by 3-fold
over a period of 365 days, the daily increment in proliferation need
not be dramatically elevated. One could speculate, therefore, that the
proliferation rate of RIP-PTHrP islet cells might be, for example, 1%
higher than that of normal mice in a 24 h period. This would lead
to a 365% increase over the period of a year, as observed (Fig. 5),
yet this subtle daily increase in proliferation would be within the
error of the in vivo BrdU proliferation method. In line with
the in vivo proliferation data, the in vitro
islet proliferation data provide little support for this possibility,
but again, the long term effects of a modest effect on proliferation
such as that seen in Fig. 9 could conceivably prove significant over
the lifespan of the RIP-PTHrP mouse. On balance, there appears to be
only modest support for the concept that PTHrP is a growth factor in
the islet, as it appears to be in other tissues (19, 20).
It is formally possible that PTHrP might inhibit the involution of islets, or more specifically, inhibit the apoptosis of islet cells, as has been reported for PTHrP in the chondrocyte (22, 36). In this example, PTHrP is expressed in the proliferating and prehypertrophic chondrocytes and prolongs the life of these cells (i.e. inhibits their apoptosis) (22, 36), and thereby participates in an important way in the longitudinal growth of long bones. Scaglia et al. and others have reported that islet cells undergo apoptosis, and that this process appears to be important in the postnatal period in the neonatal rat, and in the postpartum period in maternal rats in reducing the expanded mass of islets during gestation to their pre-gravid level (1, 31). In the current study, we could find no evidence for delayed apoptosis within the islet as an explanation for a net increase in islet mass. On the other hand, it is important to point out that islet apoptosis rates in normal adult islets are slow (1, 31), and are below the limits of current quantitative techniques. Thus, a precise quantitative search for a further reduction will necessarily fail. It therefore remains formally possible that the lifelong increase in islet mass in the RIP-PTHrP mouse could be the result of a very subtle delay in the death rate of existing islet cells. This can perhaps be explored in vitro using islet cell lines or isolated islets.
Recently, the discovery of a family of islet homeobox genes including PDX1/STF1/IPF1/IDX1, ISL1, PAX-4, PAX-6, NeuroD/ß-2 and others (11, 12, 13, 14, 15, 16, 17, 18) and putative islet growth factors such as GH, PRL, placental lactogen, hepatocyte growth factor, the reg family of proteins, and the recently identified protein, INGAP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) has focused attention on the mechanisms responsible for pancreatic and islet development and on the mechanisms whereby islet neogenesis occurs in states of islet injury, subtotal pancreatectomy, or pregnancy, and whereby normal islet mass is sustained throughout life. If PTHrP does not have an obvious role in islet cell proliferation or in apoptosis of existing islets but is nevertheless very potent in increasing islet mass, it is possible that PTHrP may play a role in normal islet neogenesis or in differentiation from uncommitted precursor cells, such as ductular epithelial cells. In this regard, we have previously reported that PTHrP is indeed expressed in pancreatic ductular cells (26), but no information exists describing the effects of PTHrP in ductular cell differentiation or on the regulation of PTHrP expression during the process of ductular cell differentiation. This may be a fruitful area for future study.
Pregnancy is associated with insulin resistance and a requirement for increased insulin secretion. This is associated with an increase in islet mass (1, 2, 3, 31). Conversely, parturition is associated with a reduction in insulin resistance and therefore a decrease in islet mass, and this is accomplished by apoptosis of ß cells (1, 2, 3, 31). In the current study, we could find no evidence for a change in PTHrP expression in the islet during pregnancy nor during the postpartum period. This suggests that whatever the role of PTHrP in islet mass regulation during adult life, steady-state mRNA levels of PTHrP do not change during pregnancy or following parturition.
From a clinical vantage point, the primary rationale for studying the
mechanisms responsible for islet development, growth, and
differentiation is to facilitate the development of effective treatment
strategies for diabetes mellitus. With this interest in mind, we were
interested to determine whether RIP-PTHrP mice might be more resistant
to the development of streptozotocin-induced diabetes than their normal
littermates. This proved to be the case, as shown in Fig. 11.
Streptozotocin is a diabetogenic agent that is believed to cause ß
cell death through DNA alkylation and nitric oxide production (41),
and/or through the induction of DNA strand breaks with subsequent
activation of poly (ADP-ribose) synthetase (42). While "resistance"
to, or attenuation of, the effects of streptozotocin in RIP-PTHrP seem
unarguable from these experiments, one is left to explain the
mechanisms responsible. It is possible that RIP-PTHrP mice simply have
more ß cells and that the "dose" of streptozotocin on a "per
cell basis" is lower in the RIP-PTHrP mice. This seems unlikely given
that peak plasma streptozotocin concentrations following ip injection
would be similar and that the dose to which individual islets are
exposed would be the same in RIP-PTHrP mice and their normal
littermates. It is also possible that the dose selected was lethal to a
fixed percentage, for example 90%, of islets in a given mouse, and
that since the RIP-PTHrP mice have more islets at the outset, they
retain sufficient numbers of functional islets to prevent or attenuate
the development of diabetes mellitus. This, in our view, is the most
likely explanation. It is also possible, however, that while basal
proliferation rates are "normal" in RIP-PTHrP mice, that their
proliferative response to injury might be greater than normal, or their
apoptotic response to streptozotocin reduced. We were unable to
demonstrate either of these possibilities experimentally.
Another surprising feature of the RIP-PTHrP mouse is that despite
impressive overproduction of PTHrP, systemic hypersecretion of PTHrP
does not occur and hypercalcemia does not develop. This is surprising
because PTHrP is clearly sorted into the regulated secretory pathway
(19, 20, 29, 43), and is copackaged with insulin in islet cells (29),
and is secreted in response to insulin secretagogues (29). In the
current study, despite even higher levels of PTHrP overexpression in
the pancreas at 1 yr of age than at 8–12 weeks of age (Fig. 1), hypercalcemia did not occur. This may
reflect clearance of PTHrP by the liver after it is secreted into the
portal circulation.
In summary, PTHrP is a normal product of the pancreatic islet and the pancreatic ductular epithelium. Overexpression of PTHrP in the islet using the insulin promoter leads to lifelong PTHrP expression and a progressive increase in islet mass, and to hyperinsulinemia and hypoglycemia. The mechanisms responsible for the increase in islet mass do not appear to involve an dramatic increase in islet cell proliferation rates, although subtle increase in islet proliferation has not been completely excluded. Similarly, no evidence for a reduction in the rate of islet cell death could be demonstrated, although a further reduction in the normally low rates of islet cell death would be difficult to demonstrate. It is also possible that the effect on islet mass results not from a change in ß cell turnover rates but from differences in islet cell commitment or differentiation from islet cell precursor cells. While the increase in islet mass remains unexplained in mechanistic terms, understanding the physiologic role and mechanisms of action of PTHrP within the islet and the pancreatic duct are important, as underscored by the observation that experimental diabetes is attenuated in the RIP-PTHrP mouse. Future studies will be needed to explore the mechanisms through which PTHrP increases islet mass, to determine whether PTHrP may play a role in pancreatic ductular differentiation, to elucidate the mechanism of streptozotocin "resistance", and to develop strategies which might employ PTHrP to advantage in the treatment of diabetes.
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Acknowledgments |
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Footnotes |
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Received January 23, 1998.
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