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Department of Zoology, North Carolina State University, Raleigh, North Carolina 27695-7617
Address all correspondence and requests for reprints to: Russell Borski, Ph.D., Department of Zoology, North Carolina State University, Box 7617, Raleigh, North Carolina 27695-7617. E-mail: russell_borski{at}ncsu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although IGF-I’s regulation of GH secretion has been well documented in humans, rodents, and nonmammalian species to a lesser degree (12), its regulation of PRL secretion in vertebrates is unclear. In human decidual cells, PRL release and synthesis are stimulated by IGF-I in vitro (13). Goodyer et al. (14) showed that IGF-I inhibited PRL release from rat pituitary explants, whereas others found either a stimulation (15, 16, 17) or no change (9, 18) in PRL release from rat pituitary cells or tumor cell lines. Furthermore, depending on the cell-type studied, IGF-I inhibited, stimulated, or exerted no effect on PRL gene expression (11, 17, 19). These contradictory findings of IGF-I on PRL cell function may be explained by differences in IGF-I preparations, the use of varying ranges of IGF-I concentrations, and/or the addition of serum-supplemented medium necessary for maintaining mammalian cell cultures. Growth factors or other undefined agents present in serum, whether applied before or during cell incubations, could result in varied PRL responses.
The pituitary gland of many teleost fish, including the temperate bass (Genus Morone), provides a valuable model system for studying in vitro control of PRL and GH by IGF-I or other regulators of pituitary cell function. Fish anterior pituitary glands, unlike those of other vertebrates, are segregated into distinct regions containing discrete cell types (20, 21). PRL cells are located in the rostral pars distalis (RPD), whereas GH-secreting cells are confined to the proximal pars distalis (PPD) region. By dissecting away the anterior-most portion of the RPD, a nearly homogenous population of PRL cells (>95%) can be isolated (22). Furthermore, pituitary tissues can be incubated in a completely defined medium, one lacking components that may themselves regulate the release of the hormones examined. Finally, this model allows the study of pituitary cells in their normal, aggregated configuration rather than in a dispersed state where cellular connections are disrupted.
The pituitary model system of a teleost fish, the hybrid striped bass, was used to more clearly define whether IGF-I directly regulates PRL release and synthesis. The effects of IGF-II and insulin were also assessed to determine the specificity of IGF-I on PRL and GH release. Furthermore, IGF-I’s action on PRL or GH production using a direct measure of protein synthesis has not been previously examined. Therefore, we also determined whether IGF-I alters PRL and GH synthesis, as measured by the incorporation of [35S]methionine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hormones and antisera
Recombinant human (rh) IGF-I was provided by Genentech, Inc. (San Francisco, CA). rhIGF-II was purchased from
GroPep Pty. Ltd. (Adelaide, Australia), and porcine
insulin was obtained from Sigma (St. Louis, MO). Bass GH
and PRL and specific homologous antisera to these hormones were a gift
from Dr. Craig Sullivan (North Carolina State University, Raleigh,
NC).
Static incubations
Pituitary glands were removed from anesthetized fish and placed
in a modified Krebs-Ringer bicarbonate solution (2.35 mM
KCl, 1.25 mM
KH2PO4, 1.4 mM
MgSO4, 25 mM
NaHCO3, 2.1 mM
CaCl2, and 140 mM NaCl) containing
glucose (0.5 mg/ml), L-glutamine (0.29 mg/ml), and 1 x MEM
essential amino acids (without L-glutamine; Life Technologies, Inc., Grand Island, NY). The control medium was
adjusted to 325 mosmol, which represents the blood osmotic pressure of
freshwater bass (Morone species) and tilapia. This
isoosmotic medium induces relatively low basal release of PRL and high
basal release of GH. Under aseptic conditions, pituitaries were
dissected into RPD (PRL-containing cells) and PPD/PI (GH and other
pituitary cell types) and placed in 100 µl control medium for 2
h. After this preincubation period, medium was removed and replaced
with fresh experimental medium. Hormones were dissolved directly into
the medium to produce varying IGF-I concentrations ranging from 1–1000
ng/ml. Incubations were maintained in an air-tight culture chamber for
18–20 h at 25 ± 1 C under a humidified atmosphere containing
95% O2-5% CO2. The
culture chamber was continuously agitated at 60 rpm on a gyratory
platform. At the termination of the experiment, media and tissue were
collected separately and sonicated in SDS-2-mercaptoethanol buffer.
GH and PRL measurements
PRL and GH release were measured according to our previously
described method (23). Hormones in tissue and medium samples were
separated by SDS-PAGE with a 4% acrylamide/bis-acrylamide (37.5:1)
stacking gel and a 15% acrylamide/bis-acrylamide (37.5:1) separating
gel (24). Gels were stained with Coomassie blue R-250 in a 45%
methanol/10% acetic acid solution and then destained in a solution
containing 10% methanol/7% acetic acid until GH and PRL bands were
discernible. These two hormones are clearly distinguishable from one
another, and their representative bands constitute the predominant
proteins seen on gels. PRL and GH bands were quantified by laser
densitometry (E.C. Apparatus, St. Petersburg, FL). The peak area was
integrated using an electronic digitizer (Hewlett-Packard Co., Avondale, PA). The optical densities of stained PRL and GH
bands were linearly related to the amounts of GH and PRL loaded onto
the gel over a range extending from 0.10–4 times the amount of hormone
typically encountered. Data were calculated as a percentage of the
total hormone released or the amount of hormone released in medium
divided by total hormone (medium plus tissue) in the incubation.
Immunoblot analysis
For Western blotting, reduced proteins were separated by
SDS-PAGE and immediately transferred to Immobilon polyvinylidene
difluoride membranes (Millipore Corp., Bedford, MA)
according to the method of Towbin et al. (25). Membranes
were blocked in PBS (0.01 M
NaPO4 and 0.15 M NaCl, pH
7.3) with 5% serum albumin overnight and incubated for 2 h at
room temperature with antisera to hybrid striped bass GH and PRL (26)
diluted 1:50,000 in PBS with 0.5% Tween-20 wash buffer. Membranes were
incubated for 30 min with a secondary antibody (1:5,000 in wash buffer)
and then for 30 min with avidin-biotin complex (ABC Elite kit,
Vector Laboratories, Inc., Burlingame, CA) in wash buffer.
Antibody binding was visualized with diaminobenzidine (DAB kit,
Vector Laboratories, Inc.).
GH and PRL synthesis
Hormone synthesis was measured by
[35S]methionine incorporation with slight
modifications of a previously described procedure (27). The RPD and
rest of the pituitary were preincubated separately for a period of
2 h in control medium (325 mosmol). Medium was then replaced with
methionine-deficient medium for 30 min. Tissues were incubated in
experimental medium containing 5 µCi
[35S]methionine (SA, 1000 Ci/mmol;
Amersham Pharmacia Biotech, Arlington Heights, IL) with or
without 100 or 1000 ng/ml IGF-I for 18–20 h. At the end of the
experiment, medium and tissue were sonicated and subjected separately
to SDS-PAGE. Gels were stained and destained, and Coomassie
blue-stained PRL and GH bands were quantified by densitometry.
The gels were then dried and exposed to Kodak XAR autoradiography film (Eastman Kodak Co., Rochester, NY) for 7 days at -80 C. Autoradiographic bands corresponding to [35S]PRL and [35S]GH were quantified by densitometry. For comparison with a more sensitive technique, the dried gels were also exposed to a phosphorimager screen for 4–5 h. The radioactivity in each band was then measured by a PhosphorImager (The Storm, Molecular Dynamics, Inc., Sunnyvale, CA) and quantified using ImageQuant 4.2 (Molecular Dynamics, Inc.). As the two techniques yielded similar results, only the [35S]methionine data measured by phosphorimaging are presented.
The densities of the Coomassie blue-stained bands gave a measure of total hormone, whereas the bands from phosphorimaging represent only newly synthesized hormone. The percent release of each hormone was calculated for both total (sum of radiolabeled and nonradiolabeled) and newly synthesized (radiolabeled) hormone as described above. PRL and GH synthesis was calculated by adding values of tissue and medium bands from whole pituitaries on the phosphorimage. Total pituitary content (radiolabeled and nonradiolabeled hormone) was measured in a similar manner on Coomassie-stained gels.
Statistical analysis
For replicate experiments performed on different days, data were
combined across days, and differences between treatments were
determined using a two-way mixed model ANOVA where day of experiment
was the random variable and treatment was the fixed variable (28).
After the two-way ANOVA, a Tukey-Kramer post-hoc test was
run to determine differences among treatments (SAS version 6.12,
SAS Institute, Inc., Cary, NC). When experiments were run
on a single day, differences between treatments were determined using a
one-way ANOVA followed by Fisher’s protected least significant
difference test for predetermined comparisons (29). To determine the
relative potencies of rhIGF-I, rhIGF-II, and porcine insulin on GH and
PRL release, estimations of ED50 values were
generated using the computer program DeltaGraph 4.0 (DeltaPoint, Inc.,
Monterey, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In blots probed with GH
antiserum, a GH band of approximately 23 kDa was illuminated in those
preparations containing either purified GH, PPD (i.e.
GH-containing tissue), or whole pituitaries (Fig. 1, top
panel). No bands were detected in those preparations containing
purified PRL or PRL tissue (i.e. RPD). Blots probed with
antiserum to PRL reacted positively with a single band of approximately
24 kDa in those preparations containing only purified PRL, the RPD
tissue, or whole pituitaries (Fig. 1, bottom panel). There
was no cross-reactivity of PRL antibody for purified GH or any protein
separated from the PPD/PI.
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). The lowest
concentration of IGF-I that maximally inhibited GH release was 100
ng/ml. In contrast to GH, PRL release from the RPD increased in a
dose-dependent manner in response to IGF-I, becoming significantly
different from controls at 100 ng/ml (P < 0.05; Fig. 2B). Maximal stimulation of PRL release occurred in response to the 500
ng/ml IGF-I dose (P < 0.001).
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). At a 100 ng/ml concentration, IGF-I
reduced GH release as much as 50% in striped bass and its congener,
white perch (P < 0.001). A similar inhibition occurred
in tilapia at the higher 1000 ng/ml dose (P < 0.05).
In contrast to its effect on GH release, IGF-I stimulated PRL release
in a dose-dependent manner from pituitaries of striped bass and white
perch, becoming significantly different from controls at 100 and 1000
ng/ml, respectively (P < 0.05; Table 1). In the
tilapia, which possesses two PRLs (tPRL177 and
tPRL188), IGF-I significantly augmented the
release of tPRL188(P < 0.05),
and a similar trend was seen for tPRL177
release.
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). Both
hormones inhibited GH release in a dose-dependent manner, with IGF-II
having a significant effect at 500 ng/ml (P < 0.0001),
and insulin having a significant effect at 100 ng/ml (34% inhibition;
P < 0.05). However, maximal GH inhibition for IGF-I
occurred at a 100 ng/ml concentration, whereas that for insulin and
IGF-II required 10-fold higher concentrations. The estimated dosage of
IGF-I that half-maximally inhibited GH release
(ED50, 
29 ng/ml or 3.8
nM) was 10- and 11-fold less than those for
insulin (ED50, 210 ng/ml or 36.3
nM) and IGF-II (330 ng/ml or 42.9
nM), respectively.
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510
ng/ml or 88.3 nM) and 17-fold (ED50,
600 ng/ml or 78 nM) higher than that required for IGF-I
(ED50, 35 ng/ml or 4.6 nM).
Effect of IGF-I on PRL and GH synthesis and newly synthesized
hormone release
Potential actions of IGF-I on PRL and GH synthesis were
initially assessed by compiling data from Figs. 2
and 3 and examining
the relative effects of IGF-I on hormone release and the total amount
of GH and PRL present (stored and synthesized) during 18–20 h static
incubations (Tables 2 and 3). Similar to its effects on fractional
GH release (medium GH/total GH; see Figs. 2 and 3), 100-1000 ng/ml
IGF-I reduced the amount of GH released into the medium by 80% and
caused a concomitant 60–100% rise in intracellular GH stores (Table 2). The total amount of GH (medium plus intracellular stores) was
suppressed by IGF-I (P < 0.01). In contrast to effects
on GH, IGF-I stimulated PRL release in the medium (P <
0.0001) and reduced that remaining in the tissue (P <
0.0001; Table 3). There was no significant effect of IGF-I on total PRL
present in the incubations.
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and 3)
as well as [35S]GH (Fig. 4) at both concentrations tested
(P < 0.0001). By contrast, the release of total PRL
and [35S]PRL were significantly stimulated by
IGF-I (P < 0.05; Fig. 4). Treatment with 100 and 1000
ng/ml IGF-I reduced [35S]GH content
(P < 0.01) as well as total GH (radiolabeled and
nonradiolabeled GH; P = 0.0876) in the 18 h incubation
(Fig. 5). Neither total PRL content nor
newly synthesized PRL was affected by IGF-I throughout this period.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The unique morphological characteristics of the teleost pituitary allow for the in vitro study of aggregated populations of distinct cell types under culture conditions lacking serum- or hormone-supplemented medium. Using these advantageous characteristics, we show that IGF-I alone is capable of altering PRL and GH release from cells in their in situ, aggregated state. The half-maximal concentration that effectively reduces GH release was 3.8 nM, which is similar to that reported in rat (9) and rainbow trout (12) primary cultures. The reduction in fractional GH release (medium/total hormone) probably reflects a lower rate of GH secretion, as IGF-I caused a dose-dependent decline in the absolute amount of hormone released in the medium and a reciprocal increase in the amount remaining in the tissue. The present findings support previous in vitro and in vivo studies in rats (5, 6, 9, 16) and humans (7) and further demonstrate that the inhibition of GH release by IGF-I occurs independent of other potentially confounding factors present in many cell culture systems. Indeed, all previous studies examining IGF-I regulation of GH and PRL used serum- or hormone-supplemented medium, the latter containing insulin, thyroid hormone, cortisol, and other components. Considering the structural similarity and overlapping actions with IGF-I, insulin’s inclusion in culture could have led to effects that are not entirely mediated by the growth factor, but are also caused by the permissive actions of insulin. Likewise, thyroid hormone and cortisol have been shown to alter PRL and GH secretion and/or gene transcription alone, and both affect the sensitivity of the somatotroph to IGF-I (16, 23, 30).
In contrast to the well characterized inhibitory effect on GH
secretion, IGF-I regulation of PRL release is poorly understood. Using
a semipurified IGF-I preparation that contained IGF-II, Goodyer
et al. (14) showed an inhibition of PRL release from rat
pituitary explants. In clonal GH3 cells, a
stimulation of basal PRL release was seen, but only with high
concentrations (70 nM) of semipurified IGF-I
(15). In normal rat primary cells, Lamberts et al. (16)
found that IGF-I stimulates PRL release, at least in the presence of
FCS, whereas others showed no effect on basal secretion in cultures
containing hormone-supplemented medium (18). In this study we clearly
show that recombinant IGF-I stimulates the release of fractional,
total, and newly synthesized PRL from normal cells maintained under
completely defined, hormone-free culture conditions. This effect is
dose dependent and is likely not due to a general stimulation of cell
function, because comparable IGF-I concentrations were shown to reduce
GH cell function. It also appears that IGF’s stimulatory action on PRL
release may be dependent in part on the length of exposure. We found
that IGF-I increases PRL release over an 18–20 h incubation period,
whereas a study with rat cells showed an inhibition at 4 h and a
paradoxical stimulation after prolonged (48 h) IGF-I treatment (17).
Thus, although we did not examine PRL release over a 4 h period, it
seems that exposure times of at least 18 h (shortest time shown to
stimulate PRL) may be necessary for IGF-I to exert its stimulatory
action on lactotrophs, a condition that differs for GH release, which
is acutely inhibited by IGF-I (9, 17). This idea is further supported
by studies of human pituitary adenomas, where IGF-I was shown to
stimulate PRL release during chronic (96 h), but not acute (4 h)
treatment. Interestingly, in this latter study a stimulation of PRL
release was seen in only those tumors derived from individuals
exhibiting low circulating PRL levels and secreting low levels of PRL
in vitro (31). The lack of PRL stimulation by IGF-I seen in
some rodent studies may therefore result in part from the inability to
elevate PRL release above the constitutively high release rates
observed under basal conditions. In contrast, the relatively low
baseline release of PRL from bass pituitaries (see striped bass, Table 1; data not shown) greatly facilitates elucidation of a stimulatory
effect for IGF-I on PRL release.
Several lines of evidence indicate that the IGF-I concentrations used in the present study are physiological. First, the concentration of IGF-I that half-maximally stimulates PRL (35 ng/ml or 4.6 nM) and inhibits GH (29 ng/ml or 3.8 nM) is well within the range of plasma levels measured in fish, including coho salmon (117.4 ± 19.1 ng/ml or 15.3 ± 2.5 nM) (32). Moreover, IGF-I is produced locally in multiple organs, including the pituitary (33, 34). If this paracrine source of IGF-I were taken into account, pituitary cells would probably encounter substantially higher IGF-I concentrations than that solely measured in the circulation. Finally, the ED50 values of IGF-I for GH and PRL release correlate very well with the displacement curve for IGF-I binding to receptor sites on bass pituitaries (35) (our unpublished results). Fifty percent displacement of [125I]IGF-I was achieved by 3 nM IGF-I.
To our knowledge, there have been no studies that concurrently evaluated the effects of IGF-I and its related peptides, IGF-II and insulin, on PRL and GH secretion. The concentration of IGF-I that half-maximally alters PRL and GH release was 10- to 19-fold lower than that required for insulin and IGF-II, suggesting that the actions of IGF-I are specific. In the rat pituitary and other cell types, both insulin and IGF-II bind with lower affinity than IGF-I to the IGF-I receptor (36, 37). Therefore, it is possible that at high concentrations, IGF-II and insulin mimic the effects of IGF-I by acting through the IGF-I receptor. Despite being less effective than IGF-I in regulating PRL and GH release, insulin may nonetheless be an independent inhibitor of GH secretion, acting via the insulin receptor. Insulin significantly reduces GH release at concentrations ranging from 100-1000 ng/ml (17–170 nM). This effect is specific to GH, as PRL release was unaltered by physiological hormone concentrations during parallel incubations. These findings are consistent with a previous study in normal rat pituitary cells where insulin was shown to suppress GH release, but with less effectiveness than IGF-I (9). Although the physiological significance of insulin’s suppression of GH release is unclear, it does not appear to be related to glucose availability or utilization (38).
Evidence indicates that a decline in GH synthesis accompanies IGF-I’s inhibition of GH release. We found that IGF-I significantly reduces the net rate of methionine incorporation into the GH molecule, which to our knowledge is the only study examining IGF-I’s effect on GH production using direct measures of protein synthesis. This action is confirmed by indirect measures of synthesis as well. The total GH content (medium plus intracellular) present during similar incubation periods declined in the presence of increasing concentrations of IGF-I, supporting earlier observations with rat primary cell cultures (9, 16, 17). The reduced rate of GH synthesis observed with IGF-I exposure seen here probably reflects an overall attenuation of gene expression, as previous findings in rat and a preliminary study in fish have shown that IGF-I inhibits GH gene transcription, GH mRNA accumulation, or both (9, 11, 39, 40). We cannot rule out the possibility, however, that reductions in GH may also reflect a decrease in translation or an increase in hormone degradation.
In contrast to GH, PRL synthesis was not directly altered by IGF-I as measured by methionine incorporation or total hormone production. This result is probably not due to a general lack of lactotroph responsiveness, because tissues released PRL (both newly synthesized and total) in the presence of IGF-I. Our findings confirm earlier reports in mammalian cells where exposure to IGF-I was ineffective or possibly attenuated PRL mRNA levels (11, 17). Although IGF-I may not alter PRL synthesis in vitro, a recent study in mice suggests that it may increase PRL mRNA levels in vivo, raising the possibility that IGF-I could induce PRL gene expression and synthesis through indirect mechanisms involving hypothalamic or gonadal factors (41).
Interestingly, in mammals estrogen stimulates PRL synthesis and release, increases pituitary IGF-I mRNA levels, and can activate molecules integral to growth factor transduction (42, 43, 44). This suggests that estrogen may induce PRL through growth factor as well as estrogen receptor-mediated pathways in vivo. Alternatively, IGF-I may activate the unliganded estrogen receptor to stimulate PRL cell activity (45). However, compared with the potent effects seen with IGF-I, estrogen was ineffective in stimulating PRL release in vitro in juvenile hybrid striped bass similar in age to those used in this study (our unpublished results). Thus, it is likely that the direct actions of IGF-I seen here do not occur through estrogen receptor induction, but, rather, via IGF-I receptor signaling mechanisms.
Recent studies using molecular approaches to selectively knockout IGF-I
gene expression in the liver suggest that the primary function of
circulating IGF-I is its inhibition of GH secretion (46). This negative
feedback control of GH is well documented in humans and nonprimate
mammals (6, 7, 8). Our findings show that IGF-I markedly reduces GH
release, clearly supporting this idea. Furthermore, as the inhibitory
actions on GH release occur in a variety of fish species (see Table 1;
47), it appears that IGF-I’s negative feedback control of GH secretion
is an ancient feature shared among vertebrates, predating the
divergence of bony fishes and tetrapods.
The physiological significance of PRL stimulation by IGF-I has yet to be determined for species in which this interaction has been described. PRL alone has over 300 reported actions in vertebrates (48). In addition, PRL and GH have been shown to work together to regulate a diverse array of physiological processes, including osmoregulation, reproduction, development and growth, and immune function (1, 3, 48). Thus, the multitude of possible functions altered by PRL and GH makes identifying a single function difficult based merely on IGF-I’s disparate regulation of these two hormones. Albeit speculative, we postulate that the discordant regulation of GH and PRL release by IGF-I may reflect divergent actions of these hormones. For example, the most universal function of PRL in vertebrates, and one that dominates in teleosts, is its action in osmoregulation (49). IGF-I may work in synergy with PRL to promote freshwater adaptation in striped bass by inducing Na+ retention (26, 50), while concomitantly reducing the secretion of GH, an important seawater adaptive hormone in certain teleosts (51, 52). This suppression of GH and increase in PRL may also reflect a need to shift energy requirements from anabolic growth processes to reproduction or osmoregulation, particularly in anadromous species such as striped bass that migrate to freshwater to spawn (21). Similarly, in rodents, PRL predominates over GH in regulating certain aspects of reproduction under the influence of IGF-I, including lactation, oocyte maturation, and fertility (53, 54). Overall, the actions of GH and PRL, in concert with those of IGF-I, may complement one another, allowing animals to shift from physiological states highly influenced by GH (e.g. skeletal growth) to those regulated more by PRL (e.g. reproduction and osmoregulation).
At a minimum, our findings clearly demonstrate that IGF-I is a specific
and direct regulator of PRL and support a new function for IGF-I as a
potent secretagogue of PRL. This feature of IGF-I is shared among
several teleosts (Table 1) and has been demonstrated in rodents as well
as human pituitary adenoma cells, suggesting that IGF-I is indeed a
physiological regulator of PRL. Recent evidence showing that IGF-I
stimulates PRL secretion in vivo in mice (42) further
supports this hypothesis. Whether the primary source of IGF-I
responsible for inducing PRL release at the level of the pituitary is
local, systemic, or both requires further investigation.
In summary, the present study clearly shows that IGF-I potently stimulates PRL release, but not synthesis, whereas it concomitantly inhibits both the synthesis and release of GH in vitro. These opposite actions are specific to IGF-I, as insulin and IGF-II were less potent than IGF-I in regulating GH and PRL cell function. The unique morphological characteristics of the teleost pituitary provide an excellent model to address the cellular and molecular pathways underlying the disparate regulation of these two closely related pituitary hormones by IGF-I.
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Acknowledgments |
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Footnotes |
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Received January 7, 2000.
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), rhIGF-II (
),
and porcine insulin (
) on hybrid striped bass GH and PRL release
during an 18–20 h static culture. Each bar represents
the mean ± SEM of 5–6 tissues/treatment from 3
separate experiments (i.e. total of 15–18
tissues/treatment). Values are normalized as a percentage of the
control. Asterisks denote significant differences from
controls: *, P < 0.05; **, P
< 0.001; ***, P < 0.0001.
