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Endocrinology Vol. 138, No. 4 1592-1603
Copyright © 1997 by The Endocrine Society

ARTICLES

Prolactin Regulation of Islet-Derived INS-1 Cells: Characteristics and Immunocytochemical Analysis of STAT5 Translocation1

L. E. Stout, A. M. Svensson2 and R. L. Sorenson

Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Address all correspondence and requests for reprints to: Robert L. Sorenson, Ph.D., Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, 4–157 Jackson Hall, 321 Church Street SE, Minneapolis, Minnesota 55455. E-mail: soren{at}lenti.med.umn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The major changes in pancreatic islet function during pregnancy and after exposure to lactogens are an increase in ß-cell proliferation and enhanced insulin secretion. In this study we examined INS-1 cells as a potential model for further inquiry into PRL signaling in ß-cells. Proliferation of ß-cells, insulin secretion, and quantitative immunocytochemical analysis of STAT5 translocation were studied.

PRL treatment of INS-1 cells resulted in a 2- to 4-fold increase in cell proliferation compared to that in the control group. In contrast, there was no effect of PRL treatment on HIT cell proliferation and only a very small effect on RIN cell proliferation. A significant effect on INS-1 cell proliferation was observed at 10 ng/ml and reached a maximum at 200 ng/ml.

PRL treatment resulted in enhanced insulin secretion from INS-1 cells. There was a time-dependent increase in insulin secretion, which when corrected for cell number was 1.5-fold greater in the PRL-treated cells. The effects of PRL on cell division and insulin secretion were glucose dependent.

The presence of the JAK family of tyrosine kinases and the transcription factor STAT5 in INS-1 cells was examined by immunocytochemical techniques. Although all members of the JAK family of kinases were detected, the staining intensity of JAK-2 was noticeably more intense. Initial studies of STAT5 translocation were performed using PRL-dependent Nb2 lymphoma cells, in which PRL treatment resulted in a nearly complete translocation of cytoplasmic STAT5 to the nucleus. Under control conditions there was a near-equal fluorescence intensity of STAT5 staining in the nucleus and cytoplasm of INS-1 cells. PRL treatment resulted in a time-dependent increase in STAT5 staining in the nucleus, with a corresponding decrease in the cytoplasm. The STAT5 staining intensity in the nucleus remained elevated for the duration of PRL treatment. This effect was reversible upon removal of PRL from the medium. Besides PRL, both GH and FBS induced a similar translocation of STAT5 to the nucleus. Although present in RIN cells, no detectable changes in STAT5 were observed in RIN cells after exposure to PRL, GH, or FBS.

INS-1 cells should provide a good model for further inquiry into the intracellular signaling pathways used by PRL and how these events alter islet function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BECAUSE of the evidence in support of a role for placental lactogen in the adaptation of islets to pregnancy, it has become increasingly important to identify an islet ß-cell line that is responsive to lactogenic hormones. Adaptive changes in islets that occur during pregnancy include 1) enhancement of glucose-stimulated insulin secretion and a decreased glucose stimulation threshold (1, 2, 3), 2) increased ß-cell proliferation and islet volume (2, 4, 5, 6), 3) increased insulin synthesis (7, 8), 4) increased gap junctional coupling among ß-cells (9), 5) increased amount of PRL receptor and its messenger RNA (10, 11), and 6) increased glucose utilization and oxidation and cAMP metabolism (12, 13).

Similarly, in vitro and in vivo experiments examining the effects of the homologous PRL or placental lactogen on islets indicate that hormones of lactogenic specificity induce the same changes in islets observed during pregnancy. These changes include 1) enhancement of glucose-stimulated insulin secretion and a decreased glucose stimulation threshold (14, 15), 2) increased ß-cell proliferation and islet volume (15, 16, 17, 18, 19), 3) increased insulin synthesis (20), 4) increased gap junctional coupling among ß-cells (14, 15), 5) increased PRL receptor messenger RNA (11), and 6) increased glucose utilization and oxidation (13, 16). Based on these observations, we have proposed that placental lactogen is the key regulatory hormone for adaptation of islets to pregnancy in rat islets, and placental lactogen and/or PRL are the principal hormones involved during human pregnancy (17, 18).

Although these studies have demonstrated the regulation of islet function by lactogens, the absence of a lactogen-responsive ß-cell line has limited studies into the intracellular signaling pathways used by PRL receptors. Although RIN cells have PRL receptors (19, 20), human GH, which binds to both rat (r) GH and PRL receptors, has a relatively small effect on cell division, insulin secretion, and insulin content in these cells (21). Recently, INS-1 cells have been introduced as a model ß-cell line (22). These cells are well granulated and release insulin in response to glucose. Importantly, these cells appeared to be responsive to PRL (23, 24). In this report, we further characterized the effect of PRL on INS-1 cells, with particular emphasis on PRL-induced translocation of STAT5 from the cytoplasm to the nucleus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Cells were cultured at 37 C in a humidified atmosphere of 5% CO2-95% air. The rat ß-cell line RIN-1046–38 (25) was used between passages 12 and 18. The hamster ß-cell line HIT-T15 (26) was used between passages 73 and 79. The rat insulinoma cell line INS-1 (22) was used between passages 75 and 83. Growth medium for RIN, HIT, and INS-1 cells consisted of RPMI 1640 with 10 mM glucose supplemented with 10 mM HEPES, 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B. Additional supplements for INS-1 cells were 1 mM pyruvate and 50 µM ß-mercaptoethanol. The growth medium used for Nb2 cells was Fisher’s medium supplemented with 10% horse serum, 10% FBS, and 100 µM ß-mercaptoethanol. To assess the hormone effects on islet cells, they were cultured in a defined serum-free medium (27) including 0.1% human serum albumin, 10 µg/ml human transferrin, 0.1 nM T3, 50 µM ethanolamine, and 50 µM phosphoethanolamine (Sigma Chemical Co., St. Louis, MO), whereas Nb2 cells were cultured in their regular medium with serum components reduced to 1% horse serum. These basal media, to which growth factors were added, are subsequently referred to as basal medium.

Growth studies
Cells were plated in growth medium at 10–15% confluence in 24-well cluster plates (Costar, Cambridge, MA). After 24 h, the cells were washed twice with basal medium and cultured in this medium for the duration of the experiment. Hormones or vehicle control were added to each well, and the medium was changed daily. For growth curves, cells from replicate plates were trypsinized and counted in quadruplicate each day using a ZBI Coulter particle counter (Coulter Electronics, Hialeah, FL). For dose-response and growth comparison experiments, cell counts from day 5 or 6 (representing log phase growth) were used. rPRL and rGH were obtained from the National Hormone and Pituitary Program, NIH (Baltimore, MD).

Insulin secretion
Daily insulin secretion was monitored in the culture medium during cell growth experiments. Medium was collected from each well every 24 h and stored at -20 C, and mean insulin secretion was determined by RIA (28) using rat insulin standards (Linko, St. Louis, MO).

Immunocytochemistry
Attached cells were plated in growth medium on 22-mm2 glass coverslips at 10–15% confluence and changed to basal medium after 2 days. Nb2 cells were grown in T-25 culture flasks (Costar, Cambridge, MA) and maintained at a concentration below 106 cells/ml. After hormone treatment for the specified times, the cells were washed in PBS, fixed in 4% paraformaldehyde for 20 min at room temperature, and then again thoroughly rinsed in PBS. The primary antibodies were diluted in PBS containing 0.3% Triton X-100, 1% normal donkey serum, 1% BSA, and 0.02% sodium azide. The cells were incubated in the primary antibodies overnight at 4 C. After rinsing (six times) in PBS containing 0.1% Triton X-100, the cells were incubated in the secondary antibodies for 4 h at room temperature.

Primary antibodies, rabbit anti-STAT5 (1:200), rabbit anti-JAK1 (1:200), goat anti-JAK3 (1:200), and rabbit anti-Tyk2 (1:200) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The STAT5 antibody recognizes both STAT5a and STAT5b. Rabbit anti-JAK2 (1:200) was obtained from Upstate Biotechnology (Lake Placid, NY). The following secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used in these studies: FITC-labeled donkey antirabbit IgG (1:150), cyanine 3.18-labeled donkey antirabbit IgG (1:400), and cyanine 3.18-labeled donkey antigoat IgG (1:400). In the absence of the primary antisera, there was no detectable staining.

The immunostained specimens were examined using a Bio-Rad MRC-1000 confocal microscope equipped with a krypton/argon laser (Bio-Rad Life Science Group, Hercules, CA). Final image processing was performed using the Confocal Assistant program written by T. C. Brelje (Department of Cell Biology and Neuroanatomy, University of Minnesota) and Adobe Photoshop; printing was performed using a Fuji Pictrograph 3000 digital printer (Tokyo, Japan). Details of these procedures have been previously reported (29, 30).

Assessment of STAT5 translocation to the nucleus was conducted using Metamorph 2.0 image analysis software (Universal Imaging Corp., West Chester, PA). The average pixel intensity was measured in the cytoplasm and nucleus of individual cells, and the ratio was calculated for 100 cells in each treatment group.

Statistical analysis
All experiments were repeated 3–5 times, with comparable results obtained in the replicates. Within each experiment there were between 4–8 samples/group. For quantitative immunohistochemistry, 100 cells were examined for each group. The results are presented as those typical for each experiment, and the data are expressed as the mean ± SEM. Statistical analysis was performed using Student’s t test or ANOVA with post-hoc tests for determining significance among groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of PRL on cell division in INS-1, HIT, and RIN cells
Initial studies were performed to compare the effect of PRL treatment on INS-1, HIT-T15, and RIN-1046–38 cell proliferation. The cells were plated at a density of 10–15% confluence in growth medium. The cells were then washed free of FBS and cultured for 5 days in basal medium with or without 500 ng/ml rat PRL. At the end of the experiment, the cells were counted to determine the effect of PRL on cell growth. There was no detectable effect of PRL on HIT cell proliferation, and there was only a small effect on RIN cell proliferation (P < 0.01). In contrast, PRL treatment resulted in a 2.5-fold increase in INS-1 cell proliferation (P < 0.001; Fig. 1Go).



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  		Figure 1. Effect of 500 ng/ml rPRL on HIT, RIN, and INS-1 cells. The cells were counted after 5 days of culture. There was no effect of rPRL on HIT cells, a 26% increase in rPRL-treated RIN cells (P < 0.01; n = 6), and a 2.5-fold increase in the INS-1 cells (P < 0.001; n = 6).

 
Effect of PRL on cell division and insulin secretion in INS-1 cells
To further characterize the effect of PRL on INS-1 cells, the time and dose dependencies of PRL treatment were determined. For the cell growth studies, cell proliferation was determined by examining cell number on each day during the experiment. Although INS-1 cells cultured in the basal medium had a slow rate of growth, those cultured in the presence of 200 ng/ml rat PRL had a significant increase in cell proliferation by day 2 of culture (P < 0.003). After 6 days of culture, cell number for the PRL-treated INS-1 cells was 3-fold greater than that for control cells (Fig. 2Go, left panel).



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  		Figure 2. The effect of 200 ng/ml rPRL on growth kinetics of INS-1 cells is shown in the left panel. There was a significant increase in cell growth in the rPRL-treated cells after 2 days of culture (P < 0.003; n = 6). After 6 days of culture, there was a 3-fold increase in the number of cells in the rPRL-treated group compared to the control group. The dose-response effect of rPRL on INS-1 cell growth is shown in the right panel. The maximum effect of PRL on INS-1 cell growth was 200 ng/ml, and a half-maximum effect was between 5–10 ng/ml (n = 6). The error bars for some groups were less than the size of the symbol and are not apparent in the figure.

 
To determine the dose-response effect of PRL on INS-1 cell proliferation, the cells were cultured for 6 days in the presence of 0, 5, 10, 20, 50, 100, 200, or 500 ng/ml rat PRL. At the end of the culture period, the cells were counted. The maximum effect of PRL on cell proliferation was 200 ng/ml. The half-maximum effect of PRL on cell proliferation was between 5–10 ng/ml (Fig. 2Go, right panel).

To examine the effect of PRL on insulin secretion from INS-1 cells, culture media were changed daily, and the cumulative insulin secreted into the media was determined. Insulin secretion from the PRL-treated cells was significantly increased by day 2 of the experiment (P < 0.001). After 6 days of culture, there was a 2.5-fold greater amount of insulin secreted by the PRL-treated cells (Fig. 3Go, left panel). As PRL treatment results in an increased number of cells, the amount of insulin secreted was normalized by the number of cells on each day of the experiment. These data are reported as microunits of insulin secreted per 24 h/1000 cells (Fig. 3Go, right panel). Insulin secretion in the control cells was about 7 µU per 24 h/1000 cells for the first 2 days of the experiment and was reduced to approximately 2 µU per 24 h/1000 cells for days 3–6 of culture. PRL-treated cells had an insulin secretion rate similar to the control cells for the first 2 days of culture. Although there was a reduction of insulin secretion in the PRL-treated cells on days 3–6 of culture, the insulin secretion rate was about 1.5-fold greater in the PRL-treated cells than in the control cells (P < 0.005).



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  		Figure 3. The effect of rPRL on insulin secretion from INS-1 cells is shown in the left panel. Insulin secretion from PRL-treated cells was significantly increased after 2 days of culture (P < 0.001; n = 6) and was 2.5-fold greater after 6 days of culture. The effect of PRL on insulin secretion per 24 h normalized to 1000 cells is shown in the right panel. Insulin secretion per cell was increased on days 3–6 (P < 0.005) of culture (n = 6). The inset shows the same data presented as a percentage of the control value. The error bars for some groups were less than the size of the symbol and are not apparent.

 
The effect of PRL on cell division and insulin secretion from isolated islets is glucose dependent (31). Thus, it was important to determine whether a similar dependency was observed with INS-1 cells. INS-1 cells were cultured for 6 days in the presence of 4.2 mM glucose or 10 mM glucose with and without 200 ng/ml PRL. The data are reported as cell number (Fig. 4Go, left panel) and cumulative insulin secretion (Fig. 4Go, right panel) on day 6 of the experiment. Although PRL stimulated cell proliferation at both 4.2 and 10.0 mM glucose, there was a significant enhancement of PRL’s effect at the higher level of glucose. In the insulin secretion studies, PRL significantly increased insulin secretion under both low and high glucose conditions. However, there was no indication of glucose potentiation of PRL-stimulated insulin secretion.



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  		Figure 4. The effects of glucose concentration and rPRL on INS-1 cell proliferation (left panel) and cumulative insulin secretion (right panel) are shown. The cells were cultured for 6 days with 4.2 or 10 mM glucose with and without 200 ng/ml rPRL. INS-1 cell proliferation was increased by rPRL at both 4.2 and 10 mM glucose (P < 0.001; n = 4). Similarly, cumulative insulin secretion was increased by rPRL at both 4.2 and 10 mM glucose (P < 0.001; n = 4).

 
Immunohistochemistry of JAK family tyrosine kinases in INS-1 cells
As the tyrosine kinase JAK-2 is associated with PRL and GH signaling, it was of interest to determine its presence and cellular location in INS-1 cells. There was a uniform high staining intensity for JAK-2 in the nucleus and a much lower staining intensity in the cytoplasm (Fig. 5Go). To determine whether other members of the JAK family had a similar staining pattern, JAK-1, JAK-3, and Tyk-2 were also examined. All three were observed in INS-1 cells and showed a staining intensity in the nucleus as well as the cytoplasm (Fig. 5Go).



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  		Figure 5. Immunocytochemical detection of JAK family kinases in INS-1 cells. A, JAK-1; B, JAK-2; C, JAK-3; D, Tyk-2. Scale bar = 25 µm.

 
However, differences in staining intensity and the ratio of JAK kinase immunofluorescence in the nucleus vs. the cytoplasm were observed. The most intense immunofluorescence was observed with JAK-2, followed by Tyk-2, JAK-1, and JAK-3. The nuclear/cytoplasmic immunofluorescence ratios were 2.57 ± 0.06 for JAK-2, 1.67 ± 0.02 for Tyk-2, 1.16 ± 0.01 for JAK-1, and 1.84 ± 0.05 for JAK-3. Experiments examining the effect of PRL on JAK family kinases did not detect an effect of rPRL on the immunolocalization or staining intensity of these enzymes (data not shown).

Effect of PRL on STAT5 translocation in Nb2 cells and INS-1 cells
As cell proliferation in Nb2 cells is dependent on PRL, initial studies included Nb2 cells and INS-1 cells. The cells were cultured in basal medium 24 h before treatment with 200 ng/ml rPRL for INS-1 cells and with 50 ng/ml rPRL for Nb2 cells. The cells were then examined by immunocytochemistry for STAT5 at time zero and 1 h after the addition of PRL. In both Nb2 cells and INS-1 cells STAT5 was detected in the nucleus as well as the cytoplasm. After PRL treatment for 1 h, there was a large increase in STAT5 immunofluorescence in the nucleus in Nb2 cells and INS-1 cells (Fig. 6Go). The staining pattern in individual cells appeared to be particulate or vesicular, with a range of staining intensities in both the nucleus and cytoplasm (Figs. 6Go and 8Go, A and B). The vast majority of cells were of this appearance; however, there were a few cells (<0.1%) in which there was a very high level of staining intensity (Figs. 6CGo and 8CGo).



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  		Figure 6. Effect of rPRL on STAT5 translocation in Nb2 cells and INS-1 cells. Cells were examined by immunocytochemistry for STAT5 at time zero and 1 h after treatment with 200 ng/ml rPRL. A, Nb2 cells at time zero. B, Nb2 cells after 1 h of rPRL treatment. C, INS-1 cells at time zero. C, INS-1 cells after 1 h of rPRL treatment. Scale bar = 25 µm.

 


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  		Figure 8. High magnification and stereoscopic views of selected features of STAT5 in INS-1 cells. A, Stereoscopic view of STAT5 in an INS-1 cell under control conditions. B, Stereoscopic view of STAT5 in an INS-1 cell after 1 h of PRL treatment. C, Stereoscopic view of the rare INS-1 cells that have intense STAT5 immunofluorescence. Scale bar = 5 µm. Note, to obtain a three-dimensional view of the cells, focus the left eye on the left panel and the right eye on the right panel. A third image will appear in the middle, and this image provides a stereoscopic view of the cells.

 
Time-dependent effect of PRL on STAT5 translocation in INS-1 cells
To further characterize STAT5 translocation in INS-1 cells, experiments were performed to determine the time dependency for translocation of STAT5 to the nucleus. INS-1 cells were cultured in basal medium 24 h before treatment with 200 ng/ml rPRL. Cells were fixed at time zero and 1, 5, 15, 30, 60, 120, and 240 min after treatment with rPRL and processed for STAT5 immunohistochemistry. There was a detectable increase in immunofluorescence in the nucleus by 5 min, and peak fluorescence occurred by 60 min (Fig. 7Go, A–C). Immunofluorescence was determined in the nucleus and cytoplasm of 100 cells for each treatment group, and the nuclear/cytoplasmic fluorescence ratio was calculated (Fig. 10Go, left panel). The ratio was 1.42 ± 0.02 (range, 1.00–2.22) at time zero and increased to 1.64 ± 0.03 (range, 1.2 to 3.2) in the 5 min group (P < 0.001). This ratio further increased to 2.66 ± 0.05 (range, 1.66–3.34) in the 30 min group and 2.84 ± 0.06 (range, 1.56–4.41) in the 60 min group. The range of ratios is provided to show heterogeneity among the cells and also demonstrates that all cells appear to respond to the PRL treatment. In some experiments, earlier time points were examined, and STAT5 translocation was observed as early as 2 min after PRL treatment. At this early time point, STAT5 appeared to coalesce at the margin of the nucleus in some cells, whereas in others there was already an indication of STAT5 transfer into the nucleus (Fig. 9Go).



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  		Figure 7. Time-dependent effect of rPRL on STAT5 translocation in INS-1 cells. A, B, and C show the effect of 200 ng/ml rPRL on STAT5 translocation to the nucleus at 0, 5, and 60 min of treatment. D, E, and F show the time dependency for decreased STAT5 in the nucleus after removal of rPRL from the medium. D shows the immunofluorescence for STAT5 after 1 h of rPRL treatment. E shows STAT5 immunofluorescence 0.5 h after removal of rPRL, and F shows STAT5 immunofluorescence 1 h after removal of rPRL from the medium. Scale bar = 25 µm.

 


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  		Figure 10. Quantitative immunofluorescent nuclear/cytoplasmic ratios of STAT5 translocation to the nucleus (left panel) and from the nucleus (right panel). In the left panel, time zero is after the culture of INS-1 cells in basal medium and 1–240 min after the addition of 200 ng/ml rPRL to the medium. There was a significant increase in the nuclear/cytoplasmic ratio by 5 min (P < 0.001); this increase was maximal by 60 min and remained elevated to the end of the experiment. In the right panel, time zero is the nuclear/cytoplasmic immunofluorescent ratio after 1 h of rPRL treatment. The nuclear/cytoplasmic ratio was significantly decreased 0.5 h after removal of rPRL from the medium (P < 0.001) and was at a basal level by 1 h.

 


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  		Figure 9. INS-1 cells after 2 min of PRL treatment. There is already evidence of STAT5 translocation in some cells, whereas in others there is a coalescence of STAT5 at the margin of the nucleus. Also seen is a negative image of a metaphase nucleus (bottom cell in the picture). Scale bar = 10 µm.

 
To determine the time dependency for translocation of STAT5 from the nucleus to the cytoplasm, similar experiments were performed. The cells were cultured in basal medium for 24 h and then treated for 1 h with 200 ng/ml rPRL. At the end of the PRL treatment period (time zero), the cells were washed and cultured in lactogen-free medium for 0.5, 1, 2, and 24 h. After 1 h of PRL treatment, there was intense immunofluorescence in the nucleus, which diminished after 30 min and returned to control levels by 1 h (Fig. 7Go, D–F). The nuclear/cytoplasmic STAT5 ratio for time zero was 2.84 ± 0.04 and decreased to 1.96 ± 0.03 by 0.5 h (P < 0.001). The ratio at 1 h was 1.52 ± 0.01, similar to the values of 1.40 ± 0.02 and 1.41 ± 0.01 in the 2 and 24 h groups (Fig. 10Go, right panel).

Effects of FBS, rGH, and rPRL on STAT5 translocation in INS-1 cells
As FBS promotes INS-1 cell proliferation similar to that observed with rPRL (Fig. 11Go) it was of interest to determine whether it would also stimulate STAT5 translocation. For purposes of comparison, rGH- and rPRL-treated groups were included in this STAT5 translocation experiment. INS-1 cells were cultured in basal medium for 24 h before the onset of the experiment. Then the medium was changed to include 10% FBS, 200 ng/ml rGH, or 200 ng/ml rPRL. STAT5 immunohistochemistry was performed on the cells at 0 min, 5 min, and 1 h. For all treatment groups there was a detectable increase in STAT5 immunofluorescence in the nucleus after 5 min of treatment. After 1 h of treatment, the STAT5 fluorescence intensity in the nucleus was similar among the treatment groups (Fig. 12Go). The mean nuclear/cytoplasmic STAT5 ratio ± SEM for time zero was 1.50 ± 0.03. For the FBS treatment group, the ratio increased to 2.04 ± 0.05 (P < 0.001) at 5 min and 2.96 ± 0.07 at 1 h. For the rGH treatment group, the ratio increased to 1.80 ± 0.05 (P < 0.001) at 5 min and 3.08 ± 0.08 at 1 h. For the rPRL treatment group, the ratio increased to 1.69 ± 0.04 (P < 0.001) at 5 min and 2.91 ± 0.08 at 1 h (Fig. 13Go).



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  		Figure 11. Effect of 10% FBS and 200 ng/ml rPRL on INS-1 cell proliferation. The cells were counted after 6 days of culture. FBS and rPRL treatment resulted in a 2- to 2.5-fold increase in cell number (P < 0.001; n = 4).

 


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  		Figure 12. Effect of FBS, rGH, and rPRL on STAT5 translocation to the nucleus. A shows INS-1 cells after 24 h of culture in basal medium. B, C, and D show the cells after culture for 1 h in 10% FBS, 200 ng/ml rGH, or 200 ng/ml rPRL. Scale bar = 25 µm.

 


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  		Figure 13. Quantitative immunofluorescent nuclear/cytoplasmic ratios of STAT5 translocation to the nucleus in INS-1 cells. Effect of 10% FBS, 200 ng/ml rGH, or 200 ng/ml rPRL on STAT5 translocation 0, 5, and 60 min after treatment. The ratios were significantly elevated for all treatment groups at 5 min (P < 0.001) and were maximal at 60 min.

 
Effects of rPRL, rGH, and FBS on STAT5 translocation in RIN cells
Although RIN cells have GH and PRL receptors (19, 20), the response of the cells to these hormones is quite small. Thus, it was of interest to examine STAT5 translocation in these cells. RIN cells were cultured in basal medium for 24 h and then treated with 200 ng/ml rPRL, rGH, or 10% FBS for 1 h. The cells were then examined by immunohistochemistry for STAT5 (data not shown). At time zero, the immunofluorescence image was similar to that observed in the INS-1 cell experiments. However, neither rPRL, rGH, nor FBS had a detectable effect on STAT5 translocation to the nucleus. The mean nuclear/cytoplasmic STAT5 ratio for time zero was 1.56 ± 0.03. The mean nuclear/cytoplasmic ratio of STAT5 at 1 h was 1.51 ± 0.03 for the rPRL-treated cells, 1.45 ± 0.03 for the rGH-treated cells, and 1.59 ± 0.03 for the FBS-treated cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Lactogenic hormones have a central role in the regulation of ß-cells as they adapt to pregnancy (1, 2, 4, 10, 14, 15, 16, 17, 18). The most notable changes are an increase in ß-cell division and enhanced insulin secretion with a lower glucose stimulation threshold. In rodents, the principal lactogenic hormones during pregnancy are placental lactogen I and II, which mediate their effects by binding to the PRL receptor (32). PRL receptors are members of the cytokine family of receptors that use the JAK-STAT signaling pathways (33, 34, 35). An important feature of this pathway is that members of the STAT family are translocated to the nucleus after phosphorylation by a JAK kinase. As this part of the pathway is essential and amenable to immunocytochemical analysis, we examined this aspect of the PRL signaling pathway. Also, immunocytochemical evidence for the translocation process is very limited (36, 37, 38). JAK-2 has been shown to be associated with PRL and GH receptors and is involved with phosphorylation of STAT5 (39, 40, 41, 42). Therefore, we examined the effect of PRL on cell proliferation and insulin secretion from INS-1 cells and characterized the effect of PRL on STAT5 translocation by quantitative immunocytochemical analysis.

Characteristics of PRL regulation of ß-cell division and insulin secretion in INS-1 cells
Initial experiments were performed comparing the effects of rPRL on HIT, RIN and INS-1 cell proliferation. These results showed that INS-1 cells were particularly responsive to PRL treatment and were chosen for further study. During the course of the experiments, PRL treatment for 6 days resulted in a 2.2- to 4.3-fold increase in cell number compared to the control value. These results were based on cell count and are somewhat greater than previously observed in similar experiments that were reported as DNA content per well and [3H]thymidine incorporation (22, 23).

The effect of PRL on insulin secretion from isolated islets is well characterized (14, 15, 16, 17, 18, 20). It requires PRL treatment for more than 24 h to have a discernible effect and for up to 5 days to have a maximum effect. In cultured neonatal rat islets, PRL treatment results in about a 3-fold increase in insulin secretion, whereas in adult islets there is about a 1.5-fold increase in insulin secretion. Insulin secretion was examined in INS-1 cells throughout 6 days of culture with PRL. The cumulative insulin secretion was 2.5-fold greater in rPRL-treated cells. Since the cells were growing during the experiment, the data were also analyzed on the basis of cell number for insulin secreted per 24 h/1000 cells. A characteristic finding was that insulin secretion decreases after 2 days of culture for both control and PRL-treated cells. Subsequently, insulin secretion from the PRL-treated cells was increased about 1.5-fold compared to that from the controls. Interestingly, this increase compares favorably with that observed in adult islets in culture (18). However, it should be pointed out that insulin secretion from INS-1 cells is considerably less than that observed in isolated islets. For example, cultured adult islets secrete about 100–200 µU insulin per 24 h/1000 cells, whereas INS-1 cells secrete less than 10 µU insulin per 24 h/1000 cells. The lower insulin secretion by INS-1 cells appears to be commensurate with their lower insulin content relative to that of ß-cells (22).

Another feature of PRL-treated islets is that the effect of PRL on cell division and insulin secretion is glucose dependent (31). Similar to cultured islets, PRL-stimulated INS-1 cell proliferation was observed at both 4.2 and 10.0 mM glucose. However, the increase in insulin secretion at low glucose in PRL-stimulated INS-1 cells is different from that observed in cultured islets. This difference may be the result of the abnormally lower threshold for glucose-stimulated insulin secretion observed in INS-1 cells (22).

These experiments indicate that PRL treatment of INS-1 cells results in enhanced cell proliferation and insulin secretion similar to those observed in PRL-treated islets and islets during pregnancy. Thus, as presently characterized, INS-1 cells should provide a good model for examining details of PRL/placental lactogen regulation of ß-cell division and insulin secretion.

Characteristics of the PRL signaling pathway in INS-1 cells
In a previous immunocytochemical study, we examined the cellular location of JAK-2 in Nb2 cells, islets, and several other tissues (10). In these cells JAK-2 was present in the cytoplasm in what appeared to be vesicular structures and was very prominent in the nucleus, but absent from the nucleolus. In the present study, a similar pattern of JAK-2 was observed in INS-1 cells. When JAK-1, JAK-3, and Tyk-2 cellular staining was examined in INS-1 cells, a comparable distribution of immunofluorescence was observed. These experiments show that all members of the JAK family are expressed in INS-1 cells, but also demonstrate that these enzymes are prominent features of the nucleus. The regulation and role of these kinases in the nucleus are unclear. While this manuscript was in preparation, Lobie et al. reported additional evidence for the presence of JAK kinases in the nucleus (43).

Initial studies of STAT5 translocation were performed using Nb2 lymphoma cells. The Nb2 cells were examined because growth of these cells is dependent on PRL and is a well established model cell line for the study of PRL regulation (44). In control Nb2 cells, there was a uniform presence of STAT5 in the cytoplasm and nucleus, but not the nucleolus. In these cells, PRL treatment resulted in a nearly complete translocation of cytoplasmic STAT5 to the nucleus.

In INS-1 cells, under control conditions, there was a near-uniform distribution of STAT5 in the cytoplasm and nucleus. The staining pattern of STAT5 suggests that it may be segregated in particles or vesicles with varying staining intensities. This is especially suggestive in the rare cells in which there is a very high degree of staining and in the early stages of STAT5 translocation when STAT5 particles appear to coalesce at the nuclear margin. Whether the apparent particulate nature of STAT5 is real and of significance or is an artifact of fixation cannot be determined from the present experiments. The presence of STAT5 in the nucleus of untreated FS2 fibroblasts has been previously demonstrated. In that report the resident nuclear STAT5 appeared to be unphosphorylated, and it was only the cytoplasmic STAT5, once phosphorylated, that was translocated to the nucleus (36). It is likely that this is the case in INS-1 cells as well, but confirmation of this awaits further experimentation. At present, the function of the resident nuclear STAT5 is unknown.

PRL treatment resulted in a time-dependent translocation of STAT5 to the nucleus. In the earliest stages, STAT5 appears to coalescence at the nuclear margin. Translocation to the nucleus was observed within 5 min and was maximal by 30 min. The translocated STAT5 remained in the nucleus as long as PRL was present. The maximum translocation in INS-1 cells was less than that observed in Nb2 cells, where an almost complete translocation of STAT5 to the nucleus occurred. The nearly complete translocation of STAT5 to the nucleus was reported for interferon-{gamma}-treated fibroblasts (36). In contrast to the interferon-{gamma}-treated fibroblasts, in which STAT5 started to leave the nucleus by 30 min of treatment, STAT5 remained in the nucleus in INS-1 cells as long as PRL was present. After removal of PRL from the medium, the nuclear localization of STAT5 was substantially reduced by 30 min and was indistinguishable from controls after 1 h.

As FBS and GH are also growth factors for INS-1 cells (24), we examined their effects on STAT5 translocation. That study also showed that GH had a much greater effect on phosphorylation of JAK-2 than PRL, and FBS had no effect on JAK-2 phosphorylation. In this study, both GH and FBS treatment resulted in STAT5 translocation in a manner similar to that observed with PRL. It is of some interest that the report on JAK-2 phosphorylation of INS-1 cells indicated no effect of FBS (24), whereas the present study showed an effect of FBS on STAT5 translocation. This suggests that FBS-induced STAT5 translocation is mediated by a different member of the JAK family. In contrast to the effects of FBS, GH, and PRL on INS-1 cells, these treatments did not have an effect on STAT5 translocation in RIN cells. This correlates with the very small effect of PRL on growth of these cells.

In summary, we examined two of the principal characteristics, ß-cell growth and insulin secretion, of PRL-treated islets and islets from pregnancy in INS-1 cells. The response of INS-1 cells to PRL was similar to that of lactogen-treated islets, in that there was enhanced ß-cell proliferation and insulin secretion. Studies on the JAK-STAT pathway indicated the presence of all members of the JAK family in INS-1 cells. Analysis of STAT5 translocation was performed using quantitative immunocytochemical techniques. Under quiescent conditions there was a nearly equal fluorescence intensity of STAT5 in the nucleus and cytoplasm. PRL treatment resulted in translocation of STAT5 to the nucleus in a time-dependent manner, where it remained for the duration of PRL treatment. Removal of PRL from the medium resulted in a reversal of the translocation process. INS-1 cells should provide a good model for further inquiry into the intracellular signaling pathways used by rat lactogenic hormones and how the pathway segregates to regulate insulin secretion and cell division.


   Acknowledgments
 
We thank Nicholas Bhagroo for his excellent technical assistance, and Dr. Todd C. Brelje for his suggestions during the preparation of the manuscript.


   Footnotes
 
1 This work was supported by NIH Grant DK-33655. Back

2 Supported by grants from the Swedish Medical Society and an Olga Jonsson grant from Uppsala University Medical Faculty. Back

Received November 1, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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