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Endocrinology Vol. 139, No. 9 3743-3751
Copyright © 1998 by The Endocrine Society

ARTICLES

Progressive Pancreatic Islet Hyperplasia in the Islet-Targeted, Parathyroid Hormone-Related Protein-Overexpressing Mouse1

Scott E. Porter, Robert L. Sorenson, Pamela Dann, Adolfo Garcia-Ocana, Andrew F. Stewart and Rupangi C. Vasavada

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH-related protein (PTHrP) is a paracrine/autocrine factor produced in most cell types in the body. Its functions include the regulation of cell cycle, of differentiation, of apoptosis, and of developmental events. One of the cells which produces PTHrP is the pancreatic ß cell. We have previously described a transgenic mouse model of targeted overexpression of PTHrP in the ß cell, the RIP-PTHrP mouse. These studies showed that PTHrP overexpression markedly increased islet mass and insulin secretion and resulted in hypoglycemia. Those studies were limited to RIP-PTHrP mice of 8–12 weeks of age.

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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS BEEN clear for many years that while the islet cells of the pancreas are highly differentiated, they are capable of replication. Indeed, in physiological situations that require augmented insulin secretion, such as pregnancy, recovery from pancreatic resection, and overfeeding, pancreatic ß cells proliferate and overall islet mass increases (1, 2, 3). Conversely, when these stimuli are withdrawn, or when fasting or insulin-induced hypoglycemia is induced experimentally, islet mass is reduced, at least in part through the activation of the apoptosis pathway (1, 2, 3, 4). Recently, much interest has focused on the factors which regulate islet proliferation and apoptosis, including glucose itself, 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). Furthermore, the pace of research in islet development has accelerated recently with the identification of a family of islet homeodomain factors which regulate the various stages of pancreatic and islet development. These factors include PDX-1/STF-1/IPF1/IDX1, ISL1, PAX-4, PAX-6, NeuroD/ß-2, and others (11, 12, 13, 14, 15, 16, 17, 18).

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RIP-PTHrP mice
The generation of RIP-PTHrP mice has been described in detail (28). Briefly, these mice were developed using the rat insulin II promoter (RIP) and the human PTHrP complementary DNA (cDNA) using standard transgenic techniques. Two lines, designated 1799 and 1807, were generated and displayed similar phenotypes. Both lines were used in this study. In each case, as described below, RIP-PTHrP mice were compared with normal littermates. Genotyping was performed using tail DNA PCR as described (24). RIP-PTHrP mice were studied at three different ages. These included mice at 1 week of age, at 8–12 weeks of age, and, at 11–14 months of age, referred to hereafter as "1-yr-old" mice. The data described here for the 8- to 12-week-old mice have been reported previously (28), and are shown for purposes of comparison.

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The PTHrP transgene is expressed throughout postnatal life
Figure 1Go 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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  		Figure 1. Comparison of the level of transgene mRNA expression, as determined using RNAse protection analysis, in total RNA prepared from whole pancreas of RIP-PTHrP mice at 1 week of age, 8–12 weeks of age and at 1 yr of age, as indicated. "N" indicates normal littermates and "TG" indicates transgenic RIP-PTHrP mice. The location of migration of human PTHrP (i.e. the transgene-derived RNA), and of insulin and cyclophilin mRNA are indicated. The samples from the 1-week-old animals were analyzed on one gel and those from the 8–12 week and 1-yr-old animals were analyzed on another gel. As can be seen from the figure, the transgene is expressed at 1 week of age at low levels. At 8–12 weeks and at 1 yr there is clear and obvious expression of the transgene, and the level of expression appears to be increasing with time. Note also that the steady-state level of insulin expression is higher in the 1-yr-old animals than in the 8- to 12-week-old transgenics, and that at the two older ages, the level of insulin expression exceeds that of the normal littermates.

 
Hypercalcemia does not occur
Figure 2Go 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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  		Figure 2. Serum calcium concentrations in the normal and transgenic animals at 8–12 weeks of age and at 1 yr of age. The calcium concentrations at 8–12 weeks of age have been reported previously (28 ) and are shown for comparison.

 
Hypoglycemia and hyperinsulinemia persist throughout adult life
Fasting blood glucose concentrations in 1-week-old RIP-PTHrP mice were slightly, but not significantly, lower than in their normal littermates (98.0 ± 19.1 vs. 105.1 ± 23.0 mg/dl, P = 0.4). However, by the time the animals reached 8–12 weeks of age, as reported previously (28), animals display both postprandial and fasting hypoglycemia, and inappropriate hyperinsulinemia. This relative hypoglycemia persists throughout the life of the animals (Fig. 3Go). 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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  		Figure 3. Blood glucose and insulin concentrations in the RIP-PTHrP mice and their normal littermates at 8–12 weeks (panel A) and at 1 yr of age (panel B). The 8–12 week findings have been described previously (28 ) and are shown for comparison. Note that the RIP-PTHrP animals are relatively hypoglycemic as compared with their normal littermates at both ages and to a comparable extent, and that the relative hypoglycemia occurs in both the fasting and postprandial state. Note also that despite the hypoglycemia, the RIP-PTHrP animals are inappropriately hyperinsulinemic as compared with their littermates under all conditions.

 
Islet mass increases progressively throughout life
As shown in Fig. 4Go, 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. 5Go, 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. 5Go). The increase in islet mass is so striking by 1 yr, that it is apparent on casual observation in a low power photomicrograph (Fig. 6Go). When the data shown in Figs. 4Go and 5Go are normalized as a percent of normal islet volume (Fig. 7Go), the gradual progression in islet volume throughout life is appreciated.



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  		Figure 4. Islet histomorphometry in 1-week-old animals. "N" indicates normal littermates and "TG" indicates RIP-PTHrP mice. Despite expression of the transgene in the islet at 1 week of age (Fig. 1Go), there is no measurable change in islet number nor in islet mass at 1 week of age.

 


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  		Figure 5. Islet histomorphometry in 8- to 12-week animals and in 1-yr-old animals. The findings in the 8- to 12-week-old animals have been reported previously (28 ) and are shown for comparison. Note that in contrast to the findings at 1 week (Fig. 4Go), there is a progressive increase in overall islet volume as the animals age. Note also that this increase is due to an increase in the size of individual islets because the number of islets in a fixed area of pancreatic tissue remains constant with age.

 


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  		Figure 6. Islet histology in a normal (A) and a RIP-PTHrP (B) mouse at 1 yr of age. Islets are stained with insulin for ease of identification and are shown at a 20x magnification. Sections are shown from similar, periductal regions from both mice. Note that the islet mass and number are both increased. Note that the shapes of the transgenic islets are irregular.

 


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  		Figure 7. Islet histomorphometry as a function of age in normal and RIP-PTHrP mice. The data shown here are the same as those shown in Figs. 4Go and 5Go, except that islet volume in transgenic mice is expressed here as an increase over the islet volume of normal littermates, the value of which has been normalized to 100% to correct for the differences in islet volume as a function of age. This figure clearly shows the progression in islet volume in the RIP-PTHrP mice as they age.

 
The increase in islet mass is a result of islet cell hyperplasia
In theory, an increase in islet mass could result from an increase in individual islet cell size (hypertrophy), from an increase in islet cell number (hyperplasia), or from a combination of both. To address this question, we calculated islet cell size in normal and RIP-PTHrP mice at 1 yr of age. Islet cell size in normal mice (94 ± 27 u2) was indistinguishable from that in RIP-PTHrP mice (105 ± 25 u2, P = 0.2). Thus, islet cell hyperplasia, not hypertrophy, accounts for the marked increase in islet mass at 1 yr of age (Fig. 5Go).

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. 8Go. 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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  		Figure 8. Islet cell proliferation rates in 1 week and 8- to 12-week-old animals. Note that islet cell proliferation rates as determined using bromodeoxyuridine incorporation are no different in RIP-PTHrP mice as compared with normal littermates. "n" indicates the number of pancreata counted in each category.

 
These studies were extended in vitro in studies in which all three of the biologically active forms of PTHrP, namely 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, or PTHrP(107–139) (19, 20, 34), were added to cultures of neonatal rat islets. As seen in Fig. 9Go, 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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  		Figure 9. Effects of PTHrP peptides and of PRL on the proliferation rates of neonatal rat islets. See Materials and Methods for details. Note that while PRL at small concentrations has potent mitogenic effects, PTHrP peptides, which represent the whole family of PTHrP secretory forms at larger concentrations, have little or no effect on proliferation.

 
In other systems such as the epiphyseal chondrocyte, PTHrP has been reported to inhibit apoptosis (21, 22, 36). It is possible that PTHrP could augment islet mass by reducing the rate of islet apoptosis. Studies exploring the rate of apoptosis in the islets of RIP-PTHrP mice and their normal littermates were therefore performed. Apoptosis rates, as assessed using a standard DNA fragmentation method (the TUNEL method), were found to be unmeasurably low in islets from both normal and RIP-PTHrP mice, but were easily discerned in intestinal villi from the same animals, a tissue known for its high apoptotic rate, which served as a positive control (not shown).

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. 10Go, 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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  		Figure 10. Effects of pregnancy and parturition on endogenous PTHrP expression in the pancreas of normal mice. "PP" indicates postpartum day number. Days 8–18 are days of pregnancy. Note that while the level of PTHrP expression in whole pancreas is measurable but low (28 ), there is no apparent change in the level of PTHrP steady-state mRNA during pregnancy or following delivery.

 
The RIP-PTHrP mouse shows an attenuated response to streptozotocin
Because the mass of islet tissue is increased in the RIP-PTHrP mouse, and because the driving rationale for studying the role of PTHrP within the islet is to explore any possible therapeutic role of PTHrP in diabetes mellitus, we wondered whether RIP-PTHrP mice might be resistant to experimental diabetes induced by the diabetogenic agent, streptozotocin. This proved to be the case. As shown in Fig. 11Go, 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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  		Figure 11. Effects of streptozotocin (Szn) administration on blood glucose in normal littermates and in RIP-PTHrP mice. See text for details. Note that the RIP-PTHrP mice appear to be less easily induced to become diabetic than their normal littermates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We had previously demonstrated that the RIP-PTHrP mouse develops islet hyperplasia associated with hyperinsulinemia and hypoglycemia (28). These studies were performed in mice at a single window of age, from 8–12 weeks. In other transgenic models of PTHrP overexpression in which PTHrP has been targeted to the epidermis (37) or the chondrocyte (23, 24), and in mouse models of disruption of the PTHrP gene or that of its receptor (21, 38, 39), marked abnormalities were observed developmentally in the skeleton, the epidermis, and the mammary rudiment, indicating that at least in some circumstances, in some tissues, PTHrP is an important developmental molecule. One purpose of the current studies was to determine whether overexpression of PTHrP within the islet served predominantly in a developmental fashion, programming a life-long increase in islet mass, or whether its effects were predominantly postnatal. It is clear from the studies reported that in the islet, the major effect of PTHrP overexpression is not developmental, but postnatal. This is best appreciated in Fig. 7Go. 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. 8Go, 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. 5Go), 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. 9Go 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. 11Go. 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. 1Go), 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.


   Acknowledgments
 
The authors want to thank Ms. Kathy Zawalich for her help with insulin immunoassay.


   Footnotes
 
1 Supported by a Howard Hughes Medical Student Research Fellowship, by NIH Grants DK-47168 and DK-33655, and by the Department of Veterans Affairs Back

Received January 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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