0013-7227/91/1281-0045$02.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 1 Printed in U.S.A.
Role of Prolactin Versus Growth Hormone on Islet BCell Proliferation in Vitro: Implications for Pregnancy* T. CLARK BRELJE AND ROBERT L. SORENSON Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55455
proliferation and increases in islet volumes, doubling times of 23-24 days for rPRL and 89-91 days for rGH can be estimated. Of other proposed islet growth factors, cholecystokinin, epidermal growth factor, platelet-derived growth factor, and 2aminoisobutyric acid, an increase in insulin secretion and islet cell proliferation was only observed with cholecystokinin in neonatal rat islets. However, both effects were less than 20% of those observed with rPRL. In adult rat islets, rPRL was also more effective than rGH in increasing insulin secretion (1.6-fold us. 1.2-fold) during the 9 days of culture. The number of BrdU-labeled nuclei per islet was increased from 2.7 ± 0.5 (n = 96) in control islets to 9.5 ± 0.6 (n = 175) with rPRL (3.5-fold). In contrast to the neonatal islets, rGH had no effect on the number of BrdU-labeled nuclei per islet in adult rat islets (2.4 ± 0.3, n = 194). This study demonstrates that rPRL and rGH have direct effects on the growth of neonatal and adult rat islets in vitro. However, for all parameters examined the effects of rPRL were much larger than those observed with rGH. This suggests that lactogenic, rather than sommatotropic, activity is a potent regulator of islet function and supports our hypothesis that lactogens, either as PRL or placenta! lactogen, are regulators of islet function during pregnancy. {Endocrinology 128: 45-57, 1991)
ABSTRACT. This study investigated the effects of homologous rat PRL (rPRL) and rat GH (rGH) on islet growth as indicated by modifications in insulin secretion, islet cell proliferation, and islet volume with neonatal and adult rat islets in vitro. The number of proliferating cells was determined by immunocytochemical staining for 5-bromo-2'-deoxyuridine (BrdU) incorporated into replicating DNA during the final 24 h of culture. When neonatal rat islets were examined by laser scanning confocal microscopy, more than 90% of the BrdUlabeled nuclei were observed in B-cells with insulin immunoreactitivity. In neonatal rat islets, rPRL was much more effective than rGH in increasing insulin secretion (3.7-fold vs. 1.4-fold) during the 10 days of culture. The number of BrdU-labeled nuclei per islet was increased from 2.9 ± 0.4 (n = 77) in control islets to 47.3 ± 2.7 (n = 95) with rPRL (16.3-fold) and 9.7 ± 0.8 (n = 84) with rGH (3.3-fold). The effects of rPRL and rGH on both insulin secretion and BrdU labeling were approximately additive. After 10 days, an increased average islet volume was only observed with rPRL. When followed for 36 days, the total amount of islet tissue was unchanged for control islets (1.1-fold), more than doubled with rPRL (2.5-fold), and only slightly increased with rGH (1.4-fold). From the observed rates of islet cell
T
show activities in other species that they do not show in their own species (12-14). Recently, we avoided this issue by examining the effect of homologous rat PRL (rPRL) and rat GH (rGH) on neonatal and adult rat islets in vitro (15). In both cases, rPRL was observed to be more effective than rGH in increasing insulin secretion. To further investigate possible differences between rPRL and rGH on islet function, the present study was undertaken to examine their effects on islet growth. The incorporation of [3H]thymidine during the Sphase of the cell cycle has been widely used as an index of DNA synthesis and cell proliferation in islets of Langerhans (16,17). The incorporated radioactivity is quantified by liquid scintillation spectrometry of extracts (18, 19) or detected by autoradiography in sections (20, 21). Since extracts only give relative differences between experimental groups, autoradiography is required to obtain quantitative information on the frequency and spatial distribution of proliferating cells. In addition, auto-
O ACCOMODATE the increased insulin demand during pregnancy, the islets of Langerhans undergo major increases in insulin secretory response and B-cell growth (1-6). These alterations have been attributed to a direct effect of increased lactogenic or somatotropic activity on the islet during pregnancy (7-11). Unfortunately, most studies examining the biological activities of GHs, PRLs, or placental lactogens on islets have been done in heterologous systems (i.e. the hormone of one species is used with tissue from another). Interpreting data from heterologous systems is difficult since the structural similarities of these hormones allow them to Received July 9, 1990. Address correspondence and reprint requests to: Dr. Robert L. Sorenson, University of Minnesota, Department of Cell Biology and Neuroanatomy, 4-135 Jackson Hall, 321 Church St. SE, Minneapolis, Minnesota 55455. "This work was supported by NIH Grant DK-33655, Juvenile Diabetes Foundation Grant 188713, and the Minnesota Affiliate of the American Diabetes Association.
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
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radiography is more sensitive than using extracts in detecting changes in proliferation in tissues with a small proportion of dividing cells. However, autoradiography has several disadvantages: First, since the radiation from tritium labels is quenched within a distance of few microns, the tissue must be sectioned. Second, long delays are required for the exposure of autoradiographs. Third, the scoring of each section is quite laborious and timeconsuming when the proliferating cells are infrequent. Also, the radioactivity of [3H]thymidine requires protective measures during the entire procedure. These considerations limit the ability of these methods to rapidly and accurately assess the level of cell proliferation in slow growing tissues such as the islet. Recently, an alternative nonradioactive method for examining thymidine incorporation has been developed in which the thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU), is incorporated into replicating DNA and immunohistochemically detected by a specific anti-BrdU monoclonal antibody (22). Compared to autoradiography, this method is quicker and readily provides a clear distinction between positive and negative cells. Although this method was originally applied to cell monolayers in vitro (22), it has been modified for use with whole mount preparations and for in vivo studies (23-25). While previous studies have used BrdU immunocytochemistry to examine islet cell monolayers (11) and pancreas sections from in vivo labeled pregnant hamsters (25), for the present study we developed a method for examining cell proliferation in intact, isolated islets in vitro. The aim of this study was to further characterize the effects of lactogenic and sommatotrophic activity on islet growth and function. Specifically, the modulation of insulin secretion, islet cell proliferation using BrdU immunocytochemistry, and islet volume by rPRL and rGH was studied in neonatal and adult rat islets in vitro. In addition, the morphology and identity of the proliferating cells were determined by double staining for islet hormones. Materials and Methods Islet isolation and culture For each experiment, neonatal rat islets were isolated from 3 to 5-day-old neonatal rats pooled from two or more litters (Sprague-Dawley, Holtzman, Madison, WI) by a nonenzymatic culture method previously described (26). Briefly, the pancreases were finely minced, distributed in dishes which allow cell attachment (Falcon 3001, Oxnard, CA), and cultured in Ham's F-12 containing 10 mM glucose (GIBCO Laboratories, Grand Island, NY). The culture medium was supplemented with 5% fetal calf serum and 1% penicillin-streptomycin fungizone (PSF) antibiotic-antimycotic (GIBCO). The cultures were continued for 7 days at 37 C in a humidified atmosphere containing 5% CO2 with the culture medium changed after 4 days. After
Endo • 1991 Voll28«Nol
this initial culture period, the adherent tissue was dislodged by vigorously aspirating the media in each dish. The media was pooled, centrifuged, and the tissue fragments cultured for another 24 h. At this time, most of the acinar tissue and debris had attached to the bottom of the dishes allowing the islets to be harvested with the aid of a fine pasteur pipette. Subsequently, groups of 30 islets were transferred to 24-well plates (Costar, Cambridge, MA) and cultured free floating in 2 ml Ham's F-12 supplemented with 25% horse serum and 1% PSF antibiotic-antimycotic. Adult rat islets were isolated from female Sprague-Dawley rats, weighing 200-250 g by pancreatic distension with a collagenase solution followed by stationary in vitro digestion (27). Islets were then purified on a discontinuous dextran gradient of 27%, 23%, and 11% solutions of 60,000-90,000 mol wt dextran (50 x g for 5 min, then 400 X g for 15 min). Subsequently, groups of 30 islets were transferred to 24-well plates and cultured free floating in 2 ml RPMI 1640 containing 10 mM glucose (GIBCO) supplemented with 25% horse serum, 25 mM HEPES, and 1% PSF antibiotic-antimycotic. For each experiment, the multiwells were incubated at 37 C in a humidified atmosphere containing 5% CO2. The hormones and growth factors were added to the cultures as concentrated sterile aqueous solutions. Depending on the length of the experiment, the culture medium was replaced with fresh medium every 1-3 days of culture, usually every 24 h, and stored frozen for subsequent assay. Insulin concentrations were measured by RIA using rat insulin standards (Novo, Danbury, CT). The mammosomatotropic hormones used in this study were obtained from the National Hormone and Pituitary Program of the NIDDK. The rPRL (NIDDK-rPRL-B-6, 25.0 IU/mg) had 0.35% GH contamination by weight. The rGH (NIDDKrGH-B-11,1.8 IU/mg) had less than 0.09% PRL contamination by weight. The [Tyr-SO3H27]cholecystokinin, fragment 26-33 amide (CCK8S), mouse epidermal cell growth factor (EGF), and human platelet derived growth factor (PDGF) were obtained from Sigma (St. Louis, MO). BrdU immunocytochemistry To estimate islet cell proliferation, BrdU was added to the culture medium to a final concentration of 10 nM for the final 24 h of culture. When the cultures were discontinued, the islets were washed with PBS and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 30 min at room temperature. Excess fixative was removed by washing several times with PBS. The BrdU-labeled islets were then immunostained by a modification of a whole-mount procedure previously developed for the immunofluorescent staining of isolated islets (28). First, the islet DNA was denatured by acid hydrolysis in 2 M HC1 for 1 h at room temperature. After several brief washes with PBS, the islets were incubated in PBS for an additional 1 h to neutralize any residual acid. All of the following steps were done at 4 C. The islets were incubated with a 1:25 dilution of a mouse monoclonal anti-BrdU antibody (Becton-Dickinson, Mountain View, CA) in PBS with 0.3% Triton X-100 (PBS/ T) for 18 h on a rotating table. Excess primary antibody was removed by washing with Sorenson's phosphate buffer with
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
,
0.1% Triton X-100 (SPB/T, 4 x 30 min). The islets were then incubated with a 1:25 dilution of fluorescein isothiocyanate(FITC) -conjugated goat anti-mouse immunoglobulin G (Jackson Laboratories, West Grove, PA) in PBS/T for 18 h on a rotating table. Excess secondary antibody was removed by washing with SPB/T (4 X 30 min). Islets were stored in SPB/ T until being mounted in a glycerol-based medium containing the anti-fade agent p-phenylenediamine (29) and glass beads, 50-60 fim maximum diameter, to support the coverslips. For double-labeling experiments, islets were immunostained for BrdU as described with Texas red-conjugated goat antimouse IgG (Jackson Laboratories) as the secondary antibody. The staining procedure was then repeated using a 1:100 dilution of either a mouse monoclonal anti-insulin antibody (HUI-018, Novo Biolabs, Danbury, CT), a mouse monoclonal anti-glucagon antibody (GLU-001, Novo Biolabs), or rabbit anti-SRIF antiserum as primary antibodies, and the appropriate FITCconjugated IgGs as secondary antibodies. Fluorescence microscopy and volume determination
f
'
i
Immunostained islets were examined with a MRC-500 Confocal Imaging System (Bio-Rad Microscience, Cambridge, MA) mounted on an Olympus BH-2 microscope equipped for epifluorescence (Lake Success, NY). As previously described (28), this system allows the three-dimensional structure of intact isolated islets to be investigated without the need for physical sectioning. For double-labeled specimens, this system uses the 514 nm emission of the argon ion laser to simultaneously excite fluorescein and Texas red with the resulting fluorescence emission separated into two detectors. As such, the poor excitation of Texas red at 514 nm and the significant fluorescein emission above 600 nm requires an image processing step to remove the contaminating fluorescein emission from the Texas red images (30). The two images can then be merged into a single twocolor image for comparing the distribution of positive immunoreactivity for each antibody. For experiments examining differences in islet cell proliferation, the number of BrdU-labeled nuclei/islet was determined by direct observation with conventional epifluorescence microscopy. Slides were coded so that the evaluator was unaware of the treatment groups. When individual islets were examined with a 40x objective (0.85 NA), the intense positive and low background staining allowed the number of nuclei to be rapidly counted while focusing through the islet. In several experiments, the islet volumes were calculated after BrdU immunostaining to determine whether the observed changes in islet cell proliferation could also be detected as growth of the islets. For each islet examined, an image through its maximum diameter was acquired with the confocal microscope. From the background staining of the islet, the length of the major and minor axes were measured. With these values and the maximum diameter of the glass beads supporting the coverslip, the islet volume was calculated as an ellipsoid as previously described (26). For the islets cultured with rPRL and rGH for 36 days, the islets of each well were photographed on days 1, 18, and 36, and prints made at a known magnification. Using a digitizing
47
tablet, the major and minor axes were measured for each islet. The volumes were calculated as above, except an average of the major and minor axes was used for the thickness of the islet. Data analysis and presentation of results All results are expressed as the mean ± SEM of n observations. Statistical differences between means were assessed by analysis of variance with Newman-Keuls post-hoc test for multiple comparisons. Results BrdU immunocytochemistry of islets When islets were cultured with 10 fiM BrdU during the final 24 h of culture and immunohistochemically stained for BrdU, the BrdU immunoreactivity was only observed within the nuclei of islet cells, other cellular organelles were devoid of staining (Fig. 1A). No staining was observed with islets not incubated with BrdU or when the DNA denaturation step was omitted. The DNA denaturation is required since the anti-BrdU antibody only recognizes incorporated BrdU in single stranded DNA (22-24). Also, no staining was detectable when the antiBrdU primary antibody or the fluorescein isothiocyanate-conjugated secondary antibody were replaced with normal serum. When BrdU-labeled islets were examined using laser scanning confocal microscopy, nuclei with BrdU immunoreactivity were observed with different morphologies. The majority of the labeled nuclei had a diameter of 6.57.0 um with the BrdU immunoreactivity distributed at the periphery of the nucleus (Fig. 1, A and B). Typically, these nuclei occurred as pairs, suggesting they are the daughter cells of a labeled cell that completed cell division during the 24-h labeling period. Less common were single large, 8.5-9.0 /im diameter, BrdU-labeled nuclei with immunoreactivity distributed throughout the nuclei observed (Fig. 1A). Since the volume of the larger nuclei would be twice the volume of the smaller paired nuclei and there exists a positive correlation between nuclear volume and islet cell DNA content (31), these nuclei probably represent cells which entered or completed Sphase near the end of the labeling period. Although, it cannot be excluded that these cells were blocked in the G2 phase and represent polyploid islet cells (31, 32). Less frequently, prophase nuclei with condensing DNA (Fig. 1A), early anaphase nuclei with segregating chromosomes (Fig. 1C), and late anaphase nuclei (Fig. ID) could be identified. The identity of the proliferating cells was investigated by using laser scanning confocal microscopy to obtain serial optical sections from BrdU-labeled islets double stained for insulin, glucagon, or somatostatin. In neonatal rat islets cultured with 1000 /tg/liter rPRL for 8
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
Endo • 1991 Voll28»Nol
FlG. 1. BrdU Immunostaining of Cultured Neonatal Rat Islets. The islets were cultured with 1000 Mg/liter rPRL for 8 days with 10 /zM BrdU present during the final 24 h of culture, immunohistochemically stained for BrdU, and examined with a laser scanning confocal microscope. A, Projection through an entire islet (40 optical sections acquired at 1.4 *tm intervals). Note the nucleus with condensing DNA (arrow). Bar, 25 um. B, Reconstruction of one nucleus from a typical pair of BrdU-labeled nuclei (eight optical sections acquired at 1.0 um intervals). Bar, 5 um. C, Reconstruction of an early anaphase BrdU-labeled nucleus with segregating chromosomes (seven optical sections acquired at 1.0 fitn intervals). Bar, 5 um. D, Reconstruction of a late anaphase BrdU-labeled cell (eight optkal sections acquired at 1.0 /tm intervals). Bar, 5 um.
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
days, labeled with 10 fxM BrdU during the final 24 h of culture, more than 90% of the cells with BrdU-labeled nuclei contained clearly discernable insulin immunoreactivity (Fig. 2, A and B). Many of the remaining BrdUlabeled cells may contain low levels of insulin immunoreactivity, but the difficulty in distinguishing between light positive and background staining in the deeper optical sections prevents an unequivocal determination. In contrast, no BrdU-labeled cells with glucagon immunoreactivity (Fig. 2C), and only one pair of BrdU-labeled cells with SRIF immunoreactivity (Fig. 2F) were observed in all the islets examined during this study. For unknown reasons, the BrdU-labeled nuclei in insulin containing cells appeared to be preferentially distributed towards the surface of the islets and frequently adjacent to, or just beneath, A- or D-cells at the periphery of the islets (Fig. 2, D and E). Effect of rPRL and rGH on neonatal rat islets To examine the effects of rPRL and rGH on the function of cultured islets, neonatal rat islets were cultured for 10 days in the presence of 1000 jtg/liter rPRL, rGH, or the combination of rPRL and rGH. Although insulin secretion for the control islets was relatively constant during this period, both rPRL and rGH induced a gradual increase in the 24-h insulin secretion values (Fig. 3). However, rGH was much less effective than rPRL or the combination of rPRL and rGH. Therefore, the total amount of insulin secreted during the 10 days of culture was considerably larger with rPRL than with rGH (3.7-fold vs. 1.4-fold), with their effects being approximately additive for the combination of rPRL and rGH (4.3-fold). To determine whether islet cell proliferation was similarily affected by rPRL and rGH, BrdU was added to the culture medium to a final concentration of 10 fiM for the final 24 h of culture (i.e. day 10). The islets were then immunohistochemically stained for BrdU and the number of labeled nuclei/islet determined (Fig. 4). In the control islets, the number of BrdU-labeled nuclei per islet were distributed with decreasing frequency from 0 to 21 with an average of 2.9 ± 0.4 (n = 77). rPRL resulted in a dramatic increase to 47.3 ± 2.7 BrdU-labeled nuclei/ islet (n = 96, P < 0.01). Even though the number of BrdU-labeled nuclei per islet were normally distributed, nearly 90% of the islets had values greater than the largest control value. In contrast, rGH resulted in a distribution more similar to that for the control islets, but was still significantly increased to 9.7 ± 0.8 BrdUlabeled nuclei per islet (n = 84, P < 0.05). Similar to insulin secretion, the effects of rPRL and rGH on BrdU labeling were approximately additive. After the islets were examined for BrdU labeling, the
49
volume of islets were calculated to determine whether similar changes could be observed. As previously observed for neonatal rat islets, the distribution of islet volumes were positively skewed to smaller volumes for all conditions examined (Fig. 5). Although the mean islet volumes for both rPRL and rGH were greater than that for control islets, only islets cultured with rPRL or the combination of rPRL and rGH resulted in significant increases in islet volume (P < 0.01). From the observed increases in islet volume during the 10 days with either rPRL, rGH, or the combination of rPRL and rGH, the average islet volume would be expected to double in approximately 24, 89, and 28 days, respectively. Dose response of the effect of rPRL and rGH on neonatal rat islets
To determine the dose response of the effects of rPRL and rGH on cultured islets, neonatal rat islets were cultured for 9 days in the presence of 0, 50,100, 250, 500, and 1000 /Kg/liter rPRL or rGH. Insulin secretion was significantly increased for islets cultured with 100 ng/ liter or more rPRL and a maximal effect with 500 ng/ liter (Fig. 6, P < 0.01). In contrast to rPRL, only rGH concentrations of 500 ^tg/liter or more significantly increased insulin secretion (Fig. 6, P < 0.01). Similar to the dose response relationship observed for insulin secretion, the number of BrdU-labeled nuclei per islet were significantly increased for islets cultured with 100 ^g/liter or more rPRL and showed a maximal effect of 500 Mg/liter rPRL (Fig. 7, P < 0.01). Unlike the insulin secretion dose response for rGH, a trend to increased numbers of BrdU-labeled nuclei/islet was apparent for the rGH concentrations examined (Fig. 8). Although 500 /ig/liter rGH was necessary to increase insulin secretion, 250 /ig/liter rGH was sufficient to significantly increase BrdU labeling (P < 0.01) to 70% of that observed for 1000 Mg/Hter rGH. When islet volumes were examined, rPRL concentrations of 250 /ig/liter or more were able to increase in islet volume (Fig. 9, P < 0.01). In contrast, none of the rGH concentrations examined had a detectable affect on islet volume (Fig. 10). rPRL induced mitosis of BrdU-labeled cells
Since previous studies have found a prominent second wave of mitosis after the initial mitosis of [3H]thymidine pulse labeled cells (21, 33), islets were cultured for 7 days with rPRL, incubated with BrdU for 24 h, extensively rinsed, and then cultured for an additional 24 h before being processed for BrdU immunocytochemistry. For the control islets, neither the average nor maximum number of BrdU-labeled nuclei per islet increased after an additional 24 h of culture. This suggests the rinsing was
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION sufficient to remove and/or deplete the islets of enough BrdU to prevent further labeling. In contrast, the number of BrdU-labeled nuclei/islet for islets cultured with rPRL almost doubled after an additional 24 h of culture (Fig. 11). These results imply that many of the BrdU-labeled cells proceeded into a second cell division during the additional 24 h of culture. Long-term effect of rPRL and rGH on neonatal rat islets To determine if the observed effects of rPRL and rGH on islet growth were maintained beyond 10 days, neonatal rat islets were cultured with 500 /ig/liter rPRL and rGH for 36 days (Fig. 12). The effects of rPRL and rGH on insulin secretion were maintained throughout the 36 days (4.1-fold for rPRL vs. 1.5-fold for rGH). The islets were photographed after 1, 18, and 36 days in culture and their volumes determined to more closely follow islet growth (Table 1). Between days 1 and 18, the aggregation of smaller islets resulted in a reduction of the number of islets for each group with a corresponding increase in average islet volume. Apparently, rPRL and rGH had little effect on this aggregation since the reduction in the number of islets was similar for each group. Because of this aggregation, the islet growth could only be followed by comparing the average islet volumes observed for each group on the same day or by following changes in the total amount of tissue present for each group. On both day 18 and 36, the average islet volumes were still significantly increased with rPRL (P < 0.01) and unchanged with rGH when compared to controls observed on the same day. Similarily, when the amount of tissue present for each group was estimated by totalling all of the islet volumes observed for that group, the total islet volume was relatively unchanged during the 36 days for the control islets, it more than doubled with rPRL, and only slightly increased with rGH. From these observed increases, a doubling time for the volume of islet tissue of 23 days for rPRL and 81 days for rGH could be calculated. To determine whether islet cell proliferation was still elevated after 36 days, BrdU labeling was examined during the final 24 h of culture (Fig. 12). For all groups,
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the number of BrdU-labeled nuclei/islet was increased compared to those previously observed after 10 days. This, in part, may reflect the larger islet volumes and the aggregation of the smaller islets during the culture period. Nonetheless, rPRL was still more effective than rGH at increasing islet cell proliferation. However, the increase with rPRL was less than that previously observed after 10 days (5.0-fold vs. 16.6-fold). Whether this difference results from a down-regulation of the rPRL response, lack of diffusion of oxygen and nutrients into the central cells of the much larger islets, and/or a depletion of cells capable of proliferation is unknown. Proposed islet growth factors To assess the relative potency of rPRL on regulating islet function, the effect of CCK8S (34), EGF (35), PDGF (36), and two-aminoisobutyric acid (AIB), an activator of ornithine decarboxylase (37), on islet cell proliferation was examined. Of these factors, only CCK8S was able to significantly increase insulin secretion (Fig. 13) and the number of BrdU-labeled nuclei/islet (Fig. 14). However, CCK8S effects were still much less than those observed with rPRL. Effect of rPRL and rGH on adult rat islets To determine whether adult rat islets respond to rPRL and rGH in the same manner as neonatal rat islets, collagenase-isolated adult rat islets were cultured for 9 days in the presence of 500 ^g/liter rPRL, rGH, or the combination of rPRL and rGH (Fig. 15). Although the increases in insulin secretion were less than that observed with neonatal rat islets, rPRL was still more effective than rGH (1.6-fold vs. 1.2-fold, P < 0.05). Even though the adult rat islets are considerably larger than the neonatal islets, the number of BrdU-labeled nuclei/islet was similar for both control neonatal and adult rat islets (Fig. 16). Even though rPRL increased the number of BrdU-labeled nuclei with adult rat islets, the increase was less than that observed with neonatal rat islets (3.5-fold vs. 16.6-fold). However, in contrast to the neonatal rat islets, rGH had no effect on islet cell proliferation in the adult rat islets.
FIG. 2. Double Staining of Cultured Neonatal Rat Islets. The islets were cultured with 1000 fig/liter rPRL for 8 days with 10 nM BrdU present during the final 24 h of culture, immunohistochemically stained for BrdU and the indicated islet hormone, and examined with a laser scanning confocal microscope. A, Single optical section through an islet immunostained for insulin (green) and BrdU (red). All BrdU-labeled cells contain insulin immunoreactivity except one located at the periphery. Bar, 50 fim. B, Higher magnification optical section demonstrating insulin immunoreactivity (green) in cells containing BrdU-labeled nuclei (red). Bar, 10 ^m. C, Projection across the top of an islet (10 optical sections acquired at 2-^m intervals) with no glucagon immunoreactivity (green) in cells containing BrdU-labeled nuclei (red). Bar, 25 ^m. D, Higher magnification projection (10 optical sections acquired at 1-^m intervals across the top edge of an islet) of a cluster of BrdU-labeled nuclei (red) associated with A-cells (green). Bar, 10 fim. E, Projection across the top of an islet (eight optical sections acquired at 2-fim intervals) with no SRIF immunoreactivity (green) in cells containing BrdU-labeled nuclei (red). Bar, 25 fim. F, Projection across the top edge of an islet (eight optical sections acquired at l-/an intervals) of the only pair of BrdU-labeled nuclei (red) in cells containing SRIF immunoreactivity (green) observed in this study. Bar, 10 fim.
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
52 Control rPRL rCH rPRL/rCH
x10~
xlO 300 -
15.0 -
Endo • 1991 Voll28«Nol
Control rPRL rGH rPRL\rGH
4.29±0.28 6.09±0.36 4.77±0.27 5.81 ±0.30
(93) (93)" (87) (99)"
*P
20 -
Q>
10.0 -
±
100
10
^
1 2 3 4 5 6 7 8 9 10 Dov Day FIG. 3. Effect of 1000 ^g/liter rPRL and rGH on insulin secretion 12
i *
3 4 5 6 7 8 9 10
from cultured neonatal rat islets (n = 6 for each point). The left panel gives the 24-h secretion values, and the right panel the cumulative insulin secretion up to each day of the experiment. The culture medium was changed every 24 h except on day 6. For the cumulative insulin secretion, all experimental groups were significantly different from each other on day 10 (P < 0.05).
150 -
5.0 -
Control 2.9±0.4 (77) rPRL 47.3±2.7 (95)* rGH 9.7±0.8 ( 8 4 ) " rPRL/rGH 58.9±3.B (75)*
0.0 Control rPRL
rGH rPRL/rGH Control rPRL
rGH rPRL/rGH
FIG. 5. Effect of 1000 fig/liter rPRL and rGH on islet volume of cultured neonatal rat islets. The islets were cultured with the indicated hormones for 10 days, processed for BrdU immunohistochemistry, and then the volumes calculated. The left panel gives the average ± SEM (n) volume for each group, and the right panel a scatter plot of the volumes obtained for each islet. The rPRL and combination of rPRL and rGH were significantly different from the control value (P < 0.01) and rGH alone (P < 0.05). 400
n
*P 2 0 -
i 100 -
m 10 •
0 5
10
15
20
25 30
35
J
Control rPRL
rGH
Day
FIG. 12. Effect of 500 jug/liter rPRL or rGH for 36 days on insulin secretion and BrdU labeling of cultured neonatal rat islets. The left panel gives the cumulative insulin secretion up to each day of the experiment, and the right panel the average ± SEM BrdU-labeled nuclei per islet for each group. For both cumulative insulin secretion on day 36 and BrdU labeling, the rPRL and rGH groups were significantly different from the control values (P < 0.05).
BrdU is not cytotoxic at the dose and duration used in cell proliferation studies, it can subsequently alter gene expression in unexpected ways after incorporation into DNA (38). For example, culturing embryonic pancreatic rudiments for several days with BrdU results in a blocking of further differentiation (39, 40). The proliferative response of islets depends not only on the growth stimulus and its recognition by islet cells,
1 2 3 4 5
6
7
Day
FIG. 13. Effect of proposed islet growth factors on insulin secretion from cultured neonatal rat islets. The effect of the following concentrations was examined: 500 /ig/liter rPRL, 100 nM CCK8S, 2 mM AIB, 100 Mg/liter EGF, and 2 fig/liter PDGF. The left panel gives the 24-h secretion values, and the right panel the cumulative insulin secretion up to each day of the experiment. For the cumulative insulin secretion, only rPRL and CCK8S were significantly different from the control value on day 8 (P < 0.05).
but also on the number of cells capable of entering the cell division cycle and undergoing mitosis (17, 33). The majority of B-cells have entered an irreversible Go phase and only a minor fraction of the B-cell population is capable of proliferation. With increasing age, this fraction of cells decreases from about 10% of the total cell population during the perinatal period to less than 3% in adult rats (33). It remains to be determined whether rPRL induces a recruitment of B-cells from a nonreplicative state or if only the subpopulation of B-cells capable of proliferation respond to the mitogenic effect of rPRL. Since many of the BrdU-labeled cells were observed to proceed into a second cell division during an additional 24 h of culture, the pool of dividing cells could
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION Control 2.7±0.4 57.0*3.6 125 - rPRL CCK 6.310.9 2.8*0.4 AIB 3.6±0.5 ECF 2.7*0.4 100 - PDGF
(68) (42)
50 •
(68) (52) (76) (69)
40
Control rPRL rGH rPRL/rCH
2.7±0.5 9.5*0.6 2.4*0.3 12.1*0.8
55
(96) (175)' (194) (103)'
*P
Vol. 128, No. 1 Printed in U.S.A.
Role of Prolactin Versus Growth Hormone on Islet BCell Proliferation in Vitro: Implications for Pregnancy* T. CLARK BRELJE AND ROBERT L. SORENSON Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55455
proliferation and increases in islet volumes, doubling times of 23-24 days for rPRL and 89-91 days for rGH can be estimated. Of other proposed islet growth factors, cholecystokinin, epidermal growth factor, platelet-derived growth factor, and 2aminoisobutyric acid, an increase in insulin secretion and islet cell proliferation was only observed with cholecystokinin in neonatal rat islets. However, both effects were less than 20% of those observed with rPRL. In adult rat islets, rPRL was also more effective than rGH in increasing insulin secretion (1.6-fold us. 1.2-fold) during the 9 days of culture. The number of BrdU-labeled nuclei per islet was increased from 2.7 ± 0.5 (n = 96) in control islets to 9.5 ± 0.6 (n = 175) with rPRL (3.5-fold). In contrast to the neonatal islets, rGH had no effect on the number of BrdU-labeled nuclei per islet in adult rat islets (2.4 ± 0.3, n = 194). This study demonstrates that rPRL and rGH have direct effects on the growth of neonatal and adult rat islets in vitro. However, for all parameters examined the effects of rPRL were much larger than those observed with rGH. This suggests that lactogenic, rather than sommatotropic, activity is a potent regulator of islet function and supports our hypothesis that lactogens, either as PRL or placenta! lactogen, are regulators of islet function during pregnancy. {Endocrinology 128: 45-57, 1991)
ABSTRACT. This study investigated the effects of homologous rat PRL (rPRL) and rat GH (rGH) on islet growth as indicated by modifications in insulin secretion, islet cell proliferation, and islet volume with neonatal and adult rat islets in vitro. The number of proliferating cells was determined by immunocytochemical staining for 5-bromo-2'-deoxyuridine (BrdU) incorporated into replicating DNA during the final 24 h of culture. When neonatal rat islets were examined by laser scanning confocal microscopy, more than 90% of the BrdUlabeled nuclei were observed in B-cells with insulin immunoreactitivity. In neonatal rat islets, rPRL was much more effective than rGH in increasing insulin secretion (3.7-fold vs. 1.4-fold) during the 10 days of culture. The number of BrdU-labeled nuclei per islet was increased from 2.9 ± 0.4 (n = 77) in control islets to 47.3 ± 2.7 (n = 95) with rPRL (16.3-fold) and 9.7 ± 0.8 (n = 84) with rGH (3.3-fold). The effects of rPRL and rGH on both insulin secretion and BrdU labeling were approximately additive. After 10 days, an increased average islet volume was only observed with rPRL. When followed for 36 days, the total amount of islet tissue was unchanged for control islets (1.1-fold), more than doubled with rPRL (2.5-fold), and only slightly increased with rGH (1.4-fold). From the observed rates of islet cell
T
show activities in other species that they do not show in their own species (12-14). Recently, we avoided this issue by examining the effect of homologous rat PRL (rPRL) and rat GH (rGH) on neonatal and adult rat islets in vitro (15). In both cases, rPRL was observed to be more effective than rGH in increasing insulin secretion. To further investigate possible differences between rPRL and rGH on islet function, the present study was undertaken to examine their effects on islet growth. The incorporation of [3H]thymidine during the Sphase of the cell cycle has been widely used as an index of DNA synthesis and cell proliferation in islets of Langerhans (16,17). The incorporated radioactivity is quantified by liquid scintillation spectrometry of extracts (18, 19) or detected by autoradiography in sections (20, 21). Since extracts only give relative differences between experimental groups, autoradiography is required to obtain quantitative information on the frequency and spatial distribution of proliferating cells. In addition, auto-
O ACCOMODATE the increased insulin demand during pregnancy, the islets of Langerhans undergo major increases in insulin secretory response and B-cell growth (1-6). These alterations have been attributed to a direct effect of increased lactogenic or somatotropic activity on the islet during pregnancy (7-11). Unfortunately, most studies examining the biological activities of GHs, PRLs, or placental lactogens on islets have been done in heterologous systems (i.e. the hormone of one species is used with tissue from another). Interpreting data from heterologous systems is difficult since the structural similarities of these hormones allow them to Received July 9, 1990. Address correspondence and reprint requests to: Dr. Robert L. Sorenson, University of Minnesota, Department of Cell Biology and Neuroanatomy, 4-135 Jackson Hall, 321 Church St. SE, Minneapolis, Minnesota 55455. "This work was supported by NIH Grant DK-33655, Juvenile Diabetes Foundation Grant 188713, and the Minnesota Affiliate of the American Diabetes Association.
45
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
46
radiography is more sensitive than using extracts in detecting changes in proliferation in tissues with a small proportion of dividing cells. However, autoradiography has several disadvantages: First, since the radiation from tritium labels is quenched within a distance of few microns, the tissue must be sectioned. Second, long delays are required for the exposure of autoradiographs. Third, the scoring of each section is quite laborious and timeconsuming when the proliferating cells are infrequent. Also, the radioactivity of [3H]thymidine requires protective measures during the entire procedure. These considerations limit the ability of these methods to rapidly and accurately assess the level of cell proliferation in slow growing tissues such as the islet. Recently, an alternative nonradioactive method for examining thymidine incorporation has been developed in which the thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU), is incorporated into replicating DNA and immunohistochemically detected by a specific anti-BrdU monoclonal antibody (22). Compared to autoradiography, this method is quicker and readily provides a clear distinction between positive and negative cells. Although this method was originally applied to cell monolayers in vitro (22), it has been modified for use with whole mount preparations and for in vivo studies (23-25). While previous studies have used BrdU immunocytochemistry to examine islet cell monolayers (11) and pancreas sections from in vivo labeled pregnant hamsters (25), for the present study we developed a method for examining cell proliferation in intact, isolated islets in vitro. The aim of this study was to further characterize the effects of lactogenic and sommatotrophic activity on islet growth and function. Specifically, the modulation of insulin secretion, islet cell proliferation using BrdU immunocytochemistry, and islet volume by rPRL and rGH was studied in neonatal and adult rat islets in vitro. In addition, the morphology and identity of the proliferating cells were determined by double staining for islet hormones. Materials and Methods Islet isolation and culture For each experiment, neonatal rat islets were isolated from 3 to 5-day-old neonatal rats pooled from two or more litters (Sprague-Dawley, Holtzman, Madison, WI) by a nonenzymatic culture method previously described (26). Briefly, the pancreases were finely minced, distributed in dishes which allow cell attachment (Falcon 3001, Oxnard, CA), and cultured in Ham's F-12 containing 10 mM glucose (GIBCO Laboratories, Grand Island, NY). The culture medium was supplemented with 5% fetal calf serum and 1% penicillin-streptomycin fungizone (PSF) antibiotic-antimycotic (GIBCO). The cultures were continued for 7 days at 37 C in a humidified atmosphere containing 5% CO2 with the culture medium changed after 4 days. After
Endo • 1991 Voll28«Nol
this initial culture period, the adherent tissue was dislodged by vigorously aspirating the media in each dish. The media was pooled, centrifuged, and the tissue fragments cultured for another 24 h. At this time, most of the acinar tissue and debris had attached to the bottom of the dishes allowing the islets to be harvested with the aid of a fine pasteur pipette. Subsequently, groups of 30 islets were transferred to 24-well plates (Costar, Cambridge, MA) and cultured free floating in 2 ml Ham's F-12 supplemented with 25% horse serum and 1% PSF antibiotic-antimycotic. Adult rat islets were isolated from female Sprague-Dawley rats, weighing 200-250 g by pancreatic distension with a collagenase solution followed by stationary in vitro digestion (27). Islets were then purified on a discontinuous dextran gradient of 27%, 23%, and 11% solutions of 60,000-90,000 mol wt dextran (50 x g for 5 min, then 400 X g for 15 min). Subsequently, groups of 30 islets were transferred to 24-well plates and cultured free floating in 2 ml RPMI 1640 containing 10 mM glucose (GIBCO) supplemented with 25% horse serum, 25 mM HEPES, and 1% PSF antibiotic-antimycotic. For each experiment, the multiwells were incubated at 37 C in a humidified atmosphere containing 5% CO2. The hormones and growth factors were added to the cultures as concentrated sterile aqueous solutions. Depending on the length of the experiment, the culture medium was replaced with fresh medium every 1-3 days of culture, usually every 24 h, and stored frozen for subsequent assay. Insulin concentrations were measured by RIA using rat insulin standards (Novo, Danbury, CT). The mammosomatotropic hormones used in this study were obtained from the National Hormone and Pituitary Program of the NIDDK. The rPRL (NIDDK-rPRL-B-6, 25.0 IU/mg) had 0.35% GH contamination by weight. The rGH (NIDDKrGH-B-11,1.8 IU/mg) had less than 0.09% PRL contamination by weight. The [Tyr-SO3H27]cholecystokinin, fragment 26-33 amide (CCK8S), mouse epidermal cell growth factor (EGF), and human platelet derived growth factor (PDGF) were obtained from Sigma (St. Louis, MO). BrdU immunocytochemistry To estimate islet cell proliferation, BrdU was added to the culture medium to a final concentration of 10 nM for the final 24 h of culture. When the cultures were discontinued, the islets were washed with PBS and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 30 min at room temperature. Excess fixative was removed by washing several times with PBS. The BrdU-labeled islets were then immunostained by a modification of a whole-mount procedure previously developed for the immunofluorescent staining of isolated islets (28). First, the islet DNA was denatured by acid hydrolysis in 2 M HC1 for 1 h at room temperature. After several brief washes with PBS, the islets were incubated in PBS for an additional 1 h to neutralize any residual acid. All of the following steps were done at 4 C. The islets were incubated with a 1:25 dilution of a mouse monoclonal anti-BrdU antibody (Becton-Dickinson, Mountain View, CA) in PBS with 0.3% Triton X-100 (PBS/ T) for 18 h on a rotating table. Excess primary antibody was removed by washing with Sorenson's phosphate buffer with
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
,
0.1% Triton X-100 (SPB/T, 4 x 30 min). The islets were then incubated with a 1:25 dilution of fluorescein isothiocyanate(FITC) -conjugated goat anti-mouse immunoglobulin G (Jackson Laboratories, West Grove, PA) in PBS/T for 18 h on a rotating table. Excess secondary antibody was removed by washing with SPB/T (4 X 30 min). Islets were stored in SPB/ T until being mounted in a glycerol-based medium containing the anti-fade agent p-phenylenediamine (29) and glass beads, 50-60 fim maximum diameter, to support the coverslips. For double-labeling experiments, islets were immunostained for BrdU as described with Texas red-conjugated goat antimouse IgG (Jackson Laboratories) as the secondary antibody. The staining procedure was then repeated using a 1:100 dilution of either a mouse monoclonal anti-insulin antibody (HUI-018, Novo Biolabs, Danbury, CT), a mouse monoclonal anti-glucagon antibody (GLU-001, Novo Biolabs), or rabbit anti-SRIF antiserum as primary antibodies, and the appropriate FITCconjugated IgGs as secondary antibodies. Fluorescence microscopy and volume determination
f
'
i
Immunostained islets were examined with a MRC-500 Confocal Imaging System (Bio-Rad Microscience, Cambridge, MA) mounted on an Olympus BH-2 microscope equipped for epifluorescence (Lake Success, NY). As previously described (28), this system allows the three-dimensional structure of intact isolated islets to be investigated without the need for physical sectioning. For double-labeled specimens, this system uses the 514 nm emission of the argon ion laser to simultaneously excite fluorescein and Texas red with the resulting fluorescence emission separated into two detectors. As such, the poor excitation of Texas red at 514 nm and the significant fluorescein emission above 600 nm requires an image processing step to remove the contaminating fluorescein emission from the Texas red images (30). The two images can then be merged into a single twocolor image for comparing the distribution of positive immunoreactivity for each antibody. For experiments examining differences in islet cell proliferation, the number of BrdU-labeled nuclei/islet was determined by direct observation with conventional epifluorescence microscopy. Slides were coded so that the evaluator was unaware of the treatment groups. When individual islets were examined with a 40x objective (0.85 NA), the intense positive and low background staining allowed the number of nuclei to be rapidly counted while focusing through the islet. In several experiments, the islet volumes were calculated after BrdU immunostaining to determine whether the observed changes in islet cell proliferation could also be detected as growth of the islets. For each islet examined, an image through its maximum diameter was acquired with the confocal microscope. From the background staining of the islet, the length of the major and minor axes were measured. With these values and the maximum diameter of the glass beads supporting the coverslip, the islet volume was calculated as an ellipsoid as previously described (26). For the islets cultured with rPRL and rGH for 36 days, the islets of each well were photographed on days 1, 18, and 36, and prints made at a known magnification. Using a digitizing
47
tablet, the major and minor axes were measured for each islet. The volumes were calculated as above, except an average of the major and minor axes was used for the thickness of the islet. Data analysis and presentation of results All results are expressed as the mean ± SEM of n observations. Statistical differences between means were assessed by analysis of variance with Newman-Keuls post-hoc test for multiple comparisons. Results BrdU immunocytochemistry of islets When islets were cultured with 10 fiM BrdU during the final 24 h of culture and immunohistochemically stained for BrdU, the BrdU immunoreactivity was only observed within the nuclei of islet cells, other cellular organelles were devoid of staining (Fig. 1A). No staining was observed with islets not incubated with BrdU or when the DNA denaturation step was omitted. The DNA denaturation is required since the anti-BrdU antibody only recognizes incorporated BrdU in single stranded DNA (22-24). Also, no staining was detectable when the antiBrdU primary antibody or the fluorescein isothiocyanate-conjugated secondary antibody were replaced with normal serum. When BrdU-labeled islets were examined using laser scanning confocal microscopy, nuclei with BrdU immunoreactivity were observed with different morphologies. The majority of the labeled nuclei had a diameter of 6.57.0 um with the BrdU immunoreactivity distributed at the periphery of the nucleus (Fig. 1, A and B). Typically, these nuclei occurred as pairs, suggesting they are the daughter cells of a labeled cell that completed cell division during the 24-h labeling period. Less common were single large, 8.5-9.0 /im diameter, BrdU-labeled nuclei with immunoreactivity distributed throughout the nuclei observed (Fig. 1A). Since the volume of the larger nuclei would be twice the volume of the smaller paired nuclei and there exists a positive correlation between nuclear volume and islet cell DNA content (31), these nuclei probably represent cells which entered or completed Sphase near the end of the labeling period. Although, it cannot be excluded that these cells were blocked in the G2 phase and represent polyploid islet cells (31, 32). Less frequently, prophase nuclei with condensing DNA (Fig. 1A), early anaphase nuclei with segregating chromosomes (Fig. 1C), and late anaphase nuclei (Fig. ID) could be identified. The identity of the proliferating cells was investigated by using laser scanning confocal microscopy to obtain serial optical sections from BrdU-labeled islets double stained for insulin, glucagon, or somatostatin. In neonatal rat islets cultured with 1000 /tg/liter rPRL for 8
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48
EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
Endo • 1991 Voll28»Nol
FlG. 1. BrdU Immunostaining of Cultured Neonatal Rat Islets. The islets were cultured with 1000 Mg/liter rPRL for 8 days with 10 /zM BrdU present during the final 24 h of culture, immunohistochemically stained for BrdU, and examined with a laser scanning confocal microscope. A, Projection through an entire islet (40 optical sections acquired at 1.4 *tm intervals). Note the nucleus with condensing DNA (arrow). Bar, 25 um. B, Reconstruction of one nucleus from a typical pair of BrdU-labeled nuclei (eight optical sections acquired at 1.0 um intervals). Bar, 5 um. C, Reconstruction of an early anaphase BrdU-labeled nucleus with segregating chromosomes (seven optical sections acquired at 1.0 fitn intervals). Bar, 5 um. D, Reconstruction of a late anaphase BrdU-labeled cell (eight optkal sections acquired at 1.0 /tm intervals). Bar, 5 um.
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
days, labeled with 10 fxM BrdU during the final 24 h of culture, more than 90% of the cells with BrdU-labeled nuclei contained clearly discernable insulin immunoreactivity (Fig. 2, A and B). Many of the remaining BrdUlabeled cells may contain low levels of insulin immunoreactivity, but the difficulty in distinguishing between light positive and background staining in the deeper optical sections prevents an unequivocal determination. In contrast, no BrdU-labeled cells with glucagon immunoreactivity (Fig. 2C), and only one pair of BrdU-labeled cells with SRIF immunoreactivity (Fig. 2F) were observed in all the islets examined during this study. For unknown reasons, the BrdU-labeled nuclei in insulin containing cells appeared to be preferentially distributed towards the surface of the islets and frequently adjacent to, or just beneath, A- or D-cells at the periphery of the islets (Fig. 2, D and E). Effect of rPRL and rGH on neonatal rat islets To examine the effects of rPRL and rGH on the function of cultured islets, neonatal rat islets were cultured for 10 days in the presence of 1000 jtg/liter rPRL, rGH, or the combination of rPRL and rGH. Although insulin secretion for the control islets was relatively constant during this period, both rPRL and rGH induced a gradual increase in the 24-h insulin secretion values (Fig. 3). However, rGH was much less effective than rPRL or the combination of rPRL and rGH. Therefore, the total amount of insulin secreted during the 10 days of culture was considerably larger with rPRL than with rGH (3.7-fold vs. 1.4-fold), with their effects being approximately additive for the combination of rPRL and rGH (4.3-fold). To determine whether islet cell proliferation was similarily affected by rPRL and rGH, BrdU was added to the culture medium to a final concentration of 10 fiM for the final 24 h of culture (i.e. day 10). The islets were then immunohistochemically stained for BrdU and the number of labeled nuclei/islet determined (Fig. 4). In the control islets, the number of BrdU-labeled nuclei per islet were distributed with decreasing frequency from 0 to 21 with an average of 2.9 ± 0.4 (n = 77). rPRL resulted in a dramatic increase to 47.3 ± 2.7 BrdU-labeled nuclei/ islet (n = 96, P < 0.01). Even though the number of BrdU-labeled nuclei per islet were normally distributed, nearly 90% of the islets had values greater than the largest control value. In contrast, rGH resulted in a distribution more similar to that for the control islets, but was still significantly increased to 9.7 ± 0.8 BrdUlabeled nuclei per islet (n = 84, P < 0.05). Similar to insulin secretion, the effects of rPRL and rGH on BrdU labeling were approximately additive. After the islets were examined for BrdU labeling, the
49
volume of islets were calculated to determine whether similar changes could be observed. As previously observed for neonatal rat islets, the distribution of islet volumes were positively skewed to smaller volumes for all conditions examined (Fig. 5). Although the mean islet volumes for both rPRL and rGH were greater than that for control islets, only islets cultured with rPRL or the combination of rPRL and rGH resulted in significant increases in islet volume (P < 0.01). From the observed increases in islet volume during the 10 days with either rPRL, rGH, or the combination of rPRL and rGH, the average islet volume would be expected to double in approximately 24, 89, and 28 days, respectively. Dose response of the effect of rPRL and rGH on neonatal rat islets
To determine the dose response of the effects of rPRL and rGH on cultured islets, neonatal rat islets were cultured for 9 days in the presence of 0, 50,100, 250, 500, and 1000 /Kg/liter rPRL or rGH. Insulin secretion was significantly increased for islets cultured with 100 ng/ liter or more rPRL and a maximal effect with 500 ng/ liter (Fig. 6, P < 0.01). In contrast to rPRL, only rGH concentrations of 500 ^tg/liter or more significantly increased insulin secretion (Fig. 6, P < 0.01). Similar to the dose response relationship observed for insulin secretion, the number of BrdU-labeled nuclei per islet were significantly increased for islets cultured with 100 ^g/liter or more rPRL and showed a maximal effect of 500 Mg/liter rPRL (Fig. 7, P < 0.01). Unlike the insulin secretion dose response for rGH, a trend to increased numbers of BrdU-labeled nuclei/islet was apparent for the rGH concentrations examined (Fig. 8). Although 500 /ig/liter rGH was necessary to increase insulin secretion, 250 /ig/liter rGH was sufficient to significantly increase BrdU labeling (P < 0.01) to 70% of that observed for 1000 Mg/Hter rGH. When islet volumes were examined, rPRL concentrations of 250 /ig/liter or more were able to increase in islet volume (Fig. 9, P < 0.01). In contrast, none of the rGH concentrations examined had a detectable affect on islet volume (Fig. 10). rPRL induced mitosis of BrdU-labeled cells
Since previous studies have found a prominent second wave of mitosis after the initial mitosis of [3H]thymidine pulse labeled cells (21, 33), islets were cultured for 7 days with rPRL, incubated with BrdU for 24 h, extensively rinsed, and then cultured for an additional 24 h before being processed for BrdU immunocytochemistry. For the control islets, neither the average nor maximum number of BrdU-labeled nuclei per islet increased after an additional 24 h of culture. This suggests the rinsing was
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION sufficient to remove and/or deplete the islets of enough BrdU to prevent further labeling. In contrast, the number of BrdU-labeled nuclei/islet for islets cultured with rPRL almost doubled after an additional 24 h of culture (Fig. 11). These results imply that many of the BrdU-labeled cells proceeded into a second cell division during the additional 24 h of culture. Long-term effect of rPRL and rGH on neonatal rat islets To determine if the observed effects of rPRL and rGH on islet growth were maintained beyond 10 days, neonatal rat islets were cultured with 500 /ig/liter rPRL and rGH for 36 days (Fig. 12). The effects of rPRL and rGH on insulin secretion were maintained throughout the 36 days (4.1-fold for rPRL vs. 1.5-fold for rGH). The islets were photographed after 1, 18, and 36 days in culture and their volumes determined to more closely follow islet growth (Table 1). Between days 1 and 18, the aggregation of smaller islets resulted in a reduction of the number of islets for each group with a corresponding increase in average islet volume. Apparently, rPRL and rGH had little effect on this aggregation since the reduction in the number of islets was similar for each group. Because of this aggregation, the islet growth could only be followed by comparing the average islet volumes observed for each group on the same day or by following changes in the total amount of tissue present for each group. On both day 18 and 36, the average islet volumes were still significantly increased with rPRL (P < 0.01) and unchanged with rGH when compared to controls observed on the same day. Similarily, when the amount of tissue present for each group was estimated by totalling all of the islet volumes observed for that group, the total islet volume was relatively unchanged during the 36 days for the control islets, it more than doubled with rPRL, and only slightly increased with rGH. From these observed increases, a doubling time for the volume of islet tissue of 23 days for rPRL and 81 days for rGH could be calculated. To determine whether islet cell proliferation was still elevated after 36 days, BrdU labeling was examined during the final 24 h of culture (Fig. 12). For all groups,
51
the number of BrdU-labeled nuclei/islet was increased compared to those previously observed after 10 days. This, in part, may reflect the larger islet volumes and the aggregation of the smaller islets during the culture period. Nonetheless, rPRL was still more effective than rGH at increasing islet cell proliferation. However, the increase with rPRL was less than that previously observed after 10 days (5.0-fold vs. 16.6-fold). Whether this difference results from a down-regulation of the rPRL response, lack of diffusion of oxygen and nutrients into the central cells of the much larger islets, and/or a depletion of cells capable of proliferation is unknown. Proposed islet growth factors To assess the relative potency of rPRL on regulating islet function, the effect of CCK8S (34), EGF (35), PDGF (36), and two-aminoisobutyric acid (AIB), an activator of ornithine decarboxylase (37), on islet cell proliferation was examined. Of these factors, only CCK8S was able to significantly increase insulin secretion (Fig. 13) and the number of BrdU-labeled nuclei/islet (Fig. 14). However, CCK8S effects were still much less than those observed with rPRL. Effect of rPRL and rGH on adult rat islets To determine whether adult rat islets respond to rPRL and rGH in the same manner as neonatal rat islets, collagenase-isolated adult rat islets were cultured for 9 days in the presence of 500 ^g/liter rPRL, rGH, or the combination of rPRL and rGH (Fig. 15). Although the increases in insulin secretion were less than that observed with neonatal rat islets, rPRL was still more effective than rGH (1.6-fold vs. 1.2-fold, P < 0.05). Even though the adult rat islets are considerably larger than the neonatal islets, the number of BrdU-labeled nuclei/islet was similar for both control neonatal and adult rat islets (Fig. 16). Even though rPRL increased the number of BrdU-labeled nuclei with adult rat islets, the increase was less than that observed with neonatal rat islets (3.5-fold vs. 16.6-fold). However, in contrast to the neonatal rat islets, rGH had no effect on islet cell proliferation in the adult rat islets.
FIG. 2. Double Staining of Cultured Neonatal Rat Islets. The islets were cultured with 1000 fig/liter rPRL for 8 days with 10 nM BrdU present during the final 24 h of culture, immunohistochemically stained for BrdU and the indicated islet hormone, and examined with a laser scanning confocal microscope. A, Single optical section through an islet immunostained for insulin (green) and BrdU (red). All BrdU-labeled cells contain insulin immunoreactivity except one located at the periphery. Bar, 50 fim. B, Higher magnification optical section demonstrating insulin immunoreactivity (green) in cells containing BrdU-labeled nuclei (red). Bar, 10 ^m. C, Projection across the top of an islet (10 optical sections acquired at 2-^m intervals) with no glucagon immunoreactivity (green) in cells containing BrdU-labeled nuclei (red). Bar, 25 ^m. D, Higher magnification projection (10 optical sections acquired at 1-^m intervals across the top edge of an islet) of a cluster of BrdU-labeled nuclei (red) associated with A-cells (green). Bar, 10 fim. E, Projection across the top of an islet (eight optical sections acquired at 2-fim intervals) with no SRIF immunoreactivity (green) in cells containing BrdU-labeled nuclei (red). Bar, 25 fim. F, Projection across the top edge of an islet (eight optical sections acquired at l-/an intervals) of the only pair of BrdU-labeled nuclei (red) in cells containing SRIF immunoreactivity (green) observed in this study. Bar, 10 fim.
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION
52 Control rPRL rCH rPRL/rCH
x10~
xlO 300 -
15.0 -
Endo • 1991 Voll28«Nol
Control rPRL rGH rPRL\rGH
4.29±0.28 6.09±0.36 4.77±0.27 5.81 ±0.30
(93) (93)" (87) (99)"
*P
20 -
Q>
10.0 -
±
100
10
^
1 2 3 4 5 6 7 8 9 10 Dov Day FIG. 3. Effect of 1000 ^g/liter rPRL and rGH on insulin secretion 12
i *
3 4 5 6 7 8 9 10
from cultured neonatal rat islets (n = 6 for each point). The left panel gives the 24-h secretion values, and the right panel the cumulative insulin secretion up to each day of the experiment. The culture medium was changed every 24 h except on day 6. For the cumulative insulin secretion, all experimental groups were significantly different from each other on day 10 (P < 0.05).
150 -
5.0 -
Control 2.9±0.4 (77) rPRL 47.3±2.7 (95)* rGH 9.7±0.8 ( 8 4 ) " rPRL/rGH 58.9±3.B (75)*
0.0 Control rPRL
rGH rPRL/rGH Control rPRL
rGH rPRL/rGH
FIG. 5. Effect of 1000 fig/liter rPRL and rGH on islet volume of cultured neonatal rat islets. The islets were cultured with the indicated hormones for 10 days, processed for BrdU immunohistochemistry, and then the volumes calculated. The left panel gives the average ± SEM (n) volume for each group, and the right panel a scatter plot of the volumes obtained for each islet. The rPRL and combination of rPRL and rGH were significantly different from the control value (P < 0.01) and rGH alone (P < 0.05). 400
n
*P 2 0 -
i 100 -
m 10 •
0 5
10
15
20
25 30
35
J
Control rPRL
rGH
Day
FIG. 12. Effect of 500 jug/liter rPRL or rGH for 36 days on insulin secretion and BrdU labeling of cultured neonatal rat islets. The left panel gives the cumulative insulin secretion up to each day of the experiment, and the right panel the average ± SEM BrdU-labeled nuclei per islet for each group. For both cumulative insulin secretion on day 36 and BrdU labeling, the rPRL and rGH groups were significantly different from the control values (P < 0.05).
BrdU is not cytotoxic at the dose and duration used in cell proliferation studies, it can subsequently alter gene expression in unexpected ways after incorporation into DNA (38). For example, culturing embryonic pancreatic rudiments for several days with BrdU results in a blocking of further differentiation (39, 40). The proliferative response of islets depends not only on the growth stimulus and its recognition by islet cells,
1 2 3 4 5
6
7
Day
FIG. 13. Effect of proposed islet growth factors on insulin secretion from cultured neonatal rat islets. The effect of the following concentrations was examined: 500 /ig/liter rPRL, 100 nM CCK8S, 2 mM AIB, 100 Mg/liter EGF, and 2 fig/liter PDGF. The left panel gives the 24-h secretion values, and the right panel the cumulative insulin secretion up to each day of the experiment. For the cumulative insulin secretion, only rPRL and CCK8S were significantly different from the control value on day 8 (P < 0.05).
but also on the number of cells capable of entering the cell division cycle and undergoing mitosis (17, 33). The majority of B-cells have entered an irreversible Go phase and only a minor fraction of the B-cell population is capable of proliferation. With increasing age, this fraction of cells decreases from about 10% of the total cell population during the perinatal period to less than 3% in adult rats (33). It remains to be determined whether rPRL induces a recruitment of B-cells from a nonreplicative state or if only the subpopulation of B-cells capable of proliferation respond to the mitogenic effect of rPRL. Since many of the BrdU-labeled cells were observed to proceed into a second cell division during an additional 24 h of culture, the pool of dividing cells could
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EFFECT OF rPRL AND rGH ON B CELL PROLIFERATION Control 2.7±0.4 57.0*3.6 125 - rPRL CCK 6.310.9 2.8*0.4 AIB 3.6±0.5 ECF 2.7*0.4 100 - PDGF
(68) (42)
50 •
(68) (52) (76) (69)
40
Control rPRL rGH rPRL/rCH
2.7±0.5 9.5*0.6 2.4*0.3 12.1*0.8
55
(96) (175)' (194) (103)'
*P
![[Human growth hormone and prolactin during pregnancy (author's transl)].](/assets/img/hold.png)
