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Medical Department II, Laboratory of Endocrine Research, Klinikum Grosshadern (M.M.W., C.F., D.E.); Institute for Animal Pathology (K.M.H.B., P.S.); and Institute for Molecular Animal Breeding/Gene Center (K.G., H.L., E.W.), Ludwig-Maximilian University, Munich 81366, Germany
Address all correspondence and requests for reprints to: Matthias M. Weber, M.D., Medizinische Klinik II, Klinikum Grosshadern, Marchioninistrasse 15, 81377 München, Germany.
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Abstract |
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Introduction |
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We have established a transgenic mouse model in which a human IGF-II (hIGF-II) complementary DNA (cDNA) is placed under the control of the rat phosphoenolpyruvate carboxykinase (PEPCK) promoter (14). The PEPCK gene becomes active around birth, and its transcriptional activity increases in the postnatal period. Therefore, the PEPCK promoter has successfully been used for studies involving the transgenic overexpression of various gene constructs (15). The PEPCK-IGF-II transgenic mice investigated in our study are characterized by 4- to 6-fold elevated postnatal serum IGF-II concentrations, elevated serum IGFBP-2 levels, and subtle changes in organ growth (14, 16, 17). Because transgene expression is higher in male than in female mice of this strain, we used male PEPCK-IGF-II transgenic mice to investigate the effect of chronically elevated IGF-II levels on growth and morphology of the adrenal gland, as well as on adrenocortical function in vivo and in vitro.
Our study demonstrates expression of hIGF-II in the adrenal gland of PEPCK-IGF-II transgenic mice, which is associated with significantly increased adrenal weights, mainly caused by hyperplasia of the zona fasciculata. Furthermore, these animals show 2-fold elevated basal and ACTH-stimulated serum corticosterone levels, whereas plasma ACTH levels are unchanged, compared with normal control mice. In vitro, adrenocortical tissues from both transgenic and normal animals show a comparable basal and ACTH-induced corticosterone production when normalized for adrenal weight. This supports the hypothesis that the increased serum levels of corticosterone in the transgenic animals are mainly caused by a direct mitogenic effect of IGF-II on adrenocortical cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental procedure
Adult male animals (12 weeks old) were housed under conditions
of controlled illumination (lights on from 0700–1900 h) and
temperature (23 C), with free access to food (Altromin 1324; Altromin,
Lage, Germany) and tap water. For measurement of basal ACTH and
corticosterone levels, the animals were housed singly in opaque cages
for 2 weeks before the measurement. The animals were anesthetized
individually in a glass jar containing saturated ether vapor, and
retroorbital blood was collected within 30 sec of the initial
disturbance from the cage. This procedure has previously been shown to
yield valid basal hormone values of the hypothalamic-pituitary-adrenal
axis in rats (19). For measurements of stimulated corticosterone
levels, the anesthetized animals were treated with 1 IU/100 µg BW
ACTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) (Synacthen, Ciba-Geigy, Basel, Switzerland)
ip, and a second blood sample was obtained 60 min later. Blood was
collected in ice-chilled EDTA-coated Eppendorf tubes
containing 200 I.E. aprotinin (Trasylol, Bayer AG,
Leverkusen, Germany). Plasma samples were then stored at -80 C until
analysis, by RIA, for ACTH and corticosterone, as described (19, 20).
The inter- and intraassay coefficients of variance for the ACTH assay
(Diagnostic Product Corporation, San Diego, CA) were 6.1% and 6.8%,
respectively, with a detection limit of approximately 8 pg/ml. For the
corticosterone assay (ICN Biomedicals, Inc., Costa Mesa,
CA), the inter- and intraassay coefficients of variance were 7.2% and
6.9%, respectively, with a detection limit of approximately 25 ng/ml.
The corticosterone assay was designed for use in mice and rats; the
ACTH assays were validated for use in mice.
After autopsy, the adrenal glands from transgenic and control mice were removed, cleaned from connective tissue under a dissecting microscope, weighed, and placed in serum-free cell culture medium (M 199; Biochrom, Berlin, Germany). For in vitro stimulation experiments, intact adrenal glands were cut in half and preincubated separately in 6-well plates for 30 min in tissue culture medium (2 ml of M 199 cell culture medium) at 37 C in a humidified atmosphere with 95% air-5% CO2. The medium was then replaced by fresh serum-free medium with or without ACTH (10 nM) and incubated for 6 h, and the corticosterone secreted into the medium was determined by specific RIA, as described above. The RIA for corticosterone has been validated for use in tissue culture medium. As has been shown previously, the incubation of intact mouse adrenal glands shows a dose-dependent stimulation of corticosterone secretion by ACTH (21) that is comparable with the response in mouse adrenal cell suspensions (22, 23).
Morphometric studies
For planimetric analysis of the different adrenal zones, the
right adrenal glands were fixed in 7% formalin and embedded in
paraffin, and serial sections (3 µm) were prepared from the whole
organ using a rotation microtome (Ultracut, Reichert-Jung AG,
Heidelberg, Germany). Every 10th section was stained with Masson’s
trichrome and digitized using a semiautomated image analysis system
(Videoplan Image Processing, Kontron, Eching, Germany) at
a final magnification of 90x. The total adrenal volume, as well as the
volumes of the adrenal capsule, the zona glomerulosa, the zona
fasciculata, and the adrenal medulla, were calculated based on the
planimetric volume of all 3-µm sections measured. All volumes were
corrected for tissue shrinkage caused by embedding. The volume of the
fresh adrenal gland was calculated according to Swinyard (24), and the
volume of the embedded gland was determined by the Cavalieri method
(25).
For morphometric studies of the cellular structures, the left adrenal glands were fixed in 6.25% glutaraldehyde, postfixed in osmiumtetroxide, and embedded in a glycide ether mixture (no. 21045, Serva, Heidelberg, Germany), and semithin sections (1 µm) were stained with toluidine blue. The various adrenal zones were examined with a light microscope using a 100x objective and digitized at a final magnification of 3600x. For evaluation of the number and volume of each endocrine cell type in the different adrenal zones, 10 visual fields per zone were chosen by systematic random sampling, and the number and the volume of the cells were assessed. The volume fraction (VV) of the endocrine cells in each adrenal zone was estimated by the point-counting method (26) using an integrated test grid with 150 test points. The numerical density/area (NA) of these cells was estimated by counting their nuclei in systematic random sampled locations using the counting frame recommended by Gundersen (27). The numerical density/volume (NV) of the endocrine cells was estimated using the equation given by Weibel and Gomez (28), after planimetry of the counted nuclei. The coefficients of error for VV and NA were less than 5%. The total number of endocrine cells (N) was calculated by multiplication of NV by the volume of the corresponding adrenal zone, as estimated in the right adrenal gland. The mean volume of the endocrine cells was calculated as VV divided by NV. All data were corrected for the volume and number of nonendocrine cells, which were assessed separately for each adrenal zone.
RT-PCR analysis of hIGF-II expression in adrenal glands
Adrenal glands were prepared from wild-type or PEPCK-IGF-II
transgenic animals, connective tissue was thoroughly removed, and
organs were shock-frozen immediately after preparation on dry ice.
Frozen glands were homogenized in 1 ml Tri-Pure isolation reagent
(Boehringer Mannheim, Mannheim, Germany) and total RNA was
prepared according to the manufacturer’s recommendation. In parallel,
RNA from liver tissue was prepared from the same animals. Before first
strand synthesis, RNA preparations were treated with deoxyribonuclease
(DNase) I (1 U/µg RNA) (Boehringer Mannheim) for 30 min
at 37 C to digest residual genomic DNA. DNase I was inactivated by heat
treatment (10 min at 70 C). One microgram (adrenal glands of
PEPCK-IGF-II transgenic animals) or 2.5 µg (other samples) of total
RNA were used as a template for cDNA synthesis. RT was performed for 60
min at 37 C in RT buffer (50 mM Tris/HCl (pH 8.3), 75
mM KCl, 3 mM MgCl2), 10
mM dithiothreitol, deoxynucleotide triphosphates (1
mM each), random hexamer primers (30 µM), and
20 U M-MLV reverse transcriptase (Gibco, Karlsruhe,
Germany). The reaction was terminated by incubation for 10 min at 95 C.
Subsequent PCR analyzes were carried out in 20-µl reactions
containing 1 µl cDNA, 0.5 U Taq polymerase (MWG, Munich,
Germany), 50 mM KCl, 10 mM Tris/HCl (pH 9),
0.01% Triton-X 100, 1.5 mM MgCl2,
deoxynucleotide triphosphates (50 µM each), and 0.1
µM of both sense and antisense primers. Amplification of
IGF-II-specific transcripts was performed as follows: samples were
heated at 94 C for 4 min, followed by 36 cycles of 94 C for 1 min, 63 C
for 1 min, and 72 C for 2 min. After a final extension period of 10 min
at 72 C, amplified products were separated in 2% TAE (0.04
Tris-acetate/0.001 M EDTA) gels and visualized by
ethidium bromide staining under UV light. The following primers were
used: IGF-II no. 1, 5' ATG GGA ATC CCA ATG GGG AAG 3' (sense
primer); and IGF-II no. 2, 5' CTT GCC CAC GGG GTA TCT GGG 3' (antisense
primer), yield an amplification product of 336 bp (29). These primers
do not discriminate between human and murine IGF-II sequences. Primer
IGF-II no. 3, 5' CGG GGT CTT GGG TGG GTA GAG 3' (antisense primer),
only recognizes hIGF-II sequences. The integrity of cDNA samples was
confirmed using ß-actin-specific primers, as previously described
(30). For measurement of hIGF-II protein expression in the adrenal
glands of the transgenic mice, a RIA (Diagnostic System Laboratories, Inc., Webster, TX) was performed with tissue
extracts of two adrenal glands.
Statistics
Data are depicted as the mean ± SEM.
Differences between different groups were assessed by one-way ANOVA and
verified by nonpaired t testing. P < 0.05
was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that, in mice
transgenic for IGF-II, serum corticosterone levels were elevated
2-fold, compared with control mice, both in the morning and in the
evening (P < 0.01). Furthermore, the typical diurnal
variation of serum corticosterone levels in mice was conserved in
transgenic animals, with a strong increase of the evening
corticosterone levels, compared with the morning values (Fig. 1). These
results were confirmed in separate groups of mice (7–9 animals/group),
assayed independently at different time points (data not shown).
Similarly, the corticosterone levels were stimulated 6- to 7-fold in
both groups, 60 min after an ACTH challenge (1 IU ACTH/100 µg BW,
ip), resulting in significantly higher serum corticosterone
concentrations in transgenic than in control mice, both under basal and
ACTH-stimulated conditions (Fig. 2).
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Steroid production by adrenal glands in vitro
When incubated under serum-free conditions for 6 h, the basal
corticosterone production of adrenals from transgenic animals was
significantly higher (420.9 ± 40.1 ng/adrenal, n = 10) than
that from control animals (215.9 ± 43 ng/adrenal, n = 10).
In analogy to the in vivo stimulation, coincubation with
ACTH (10 nM) induced a significant increase in
corticosterone production, to 559.3 ± 30 and 319.8 ± 35
ng/adrenal (n = 10) in adrenal glands from transgenic and control
mice, respectively. However, when the steroid secretion was normalized
for adrenal weight, the basal and ACTH-induced corticosterone
production was comparable between incubation experiments from
transgenic animals (126.4 ± 11.8 ng/mg and 166 ± 15 ng/mg,
respectively) and control animals (113.8 ± 12.5 ng/mg and
172.8 ± 25.5 ng/mg, respectively).
Adrenal morphology
At autopsy, the weight of adrenal glands from 3-month-old male
PEPCK-IGF-II transgenic mice was significantly greater (3.31 ±
0.18 mg, n = 10) than that of control mice of the same age
(2.01 ± 0.21 mg, n = 10, P < 0.01). A
similar increase in the relative weight and volume of the adrenal gland
has been observed in older animals (Table 1).
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). There are two clearly defined
zones in the adrenal cortex of adult male mice (31). The outermost
aldosterone producing zona glomerulosa is composed of small cells
arranged in arches, whereas the zona fasciculata contains radial
columns of cells that synthesize corticosterone, the major
glucocorticoid in the mouse (32). A third region, the zona reticularis,
is only inconsistently observed in mice and was not found in the mice
of our study. Additionally, a transitory X zone is present next to the
centrally located adrenal medulla in immature males and in females
before their first pregnancy. However, because we used only mature male
mice for this study, the X zone was not found.
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. As shown in the younger animals, the adrenal
weight was increased in IGF-II transgenic mice, whereas total body
weight was comparable. In parallel to the increased weight of the
adrenal glands in IGF-II transgenic mice, an increase in adrenal volume
was observed by morphometric analysis, which was mainly caused by a
50% increase in the volume of the zona fasciculata. However, because
of the low number of adrenal glands available for the morphometric
studies, only the difference in the volume of the zona fasciculata
reached statistical significance (P < 0.05). The mean
cell number and cell volume of the endocrine cells were assessed for
each adrenal zone in three adrenal glands from each, the transgenic and
the control group. Data were corrected for the proportion of
nonendocrine cells, which was similar in both groups and accounted for
9–13% of the volume of the adrenal cortex, and for 5–7% in the
adrenal medulla. In the zona glomerulosa, the mean number of endocrine
cells and their volume were similar for controls (21 ± 3.2
x 104 glomerulosa cells, 1299 ± 113
µm3/glomerulosa cell) and PEPCK-IGF-II transgenic mice
(22 ± 0.6 x 104 glomerulosa cells, 1306 ±
63 µm3/glomerulosa cell). In contrast, the number of
fasciculata cells was increased in PEPCK-IGF-II mice (64 ±
2.6 x 104 cells, P < 0.05), compared
with controls (49 ± 7 x 104 cells), whereas the
mean cell volume was the same in both groups (2022 ± 250 and
2036 ± 177 µm3/cell). Medullary cells showed
comparable cell numbers (11.5 ± 1.9 x 104 and
9.9 ± 1.1 x 104 cells) and volumes (4120
± 670 and 4755 ± 169 µm3/cell) in both groups.
These data indicate that the increase in adrenal cell weight and volume
is mainly caused by a 50% increase in the number of the zona
fasciculata cells. Because the fasciculata cells exhibit the same size
in both groups, the enlargement of the zona fasciculata represents true
hyperplasia, and not hypertrophy, of this corticosterone-producing
adrenocortical zone.
Expression of IGF-II in the adrenal gland
RT-PCR analysis revealed the presence of hIGF-II-specific
transcripts in all investigated adrenal glands from PEPCK-IGF-II
transgenic mice. By contrast, no IGF-II-specific message could be
detected in adrenal glands from controls. The integrity of the
extracted RNA was shown using primers specific for ß-actin (Fig. 4). In accordance with the low amount of
adrenal hIGF-II mRNA expression, compared with the liver from
PEPCK-IGF-II transgenic mice, the amount of IGF-II protein in adrenal
tissue was below the detection limit of the RIA (5 ng/ml); whereas in
liver tissue from PEPCK-IGF-II transgenic mice, 314 ng/g tissue were
detected. As expected, no hIGF-II protein could be detected in adrenal
and liver tissue from normal control mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to GH transgenic mice, no difference in plasma ACTH levels between normal and PEPCK-IGF-II transgenic mice could be found in this study. This argues for a direct effect of elevated IGF-II at the adrenal level. However, the mechanism by which elevated levels of IGF-II increase serum corticosterone in IGF-II transgenic mice remains speculative, because IGFs have been shown to elicit both mitogenic and steroidogenic effects in adrenocortical cells of various species in vitro (5, 7, 10, 11, 12). Yet, in rat adrenocortical cells, an inhibitory effect of IGF-I on steroid biosynthesis has been described (37); and IGF-II (but not IGF-I), in combination with insulin, is mitogenic for fetal adrenal cells (38). The adrenocortical hyperplasia in IGF-II transgenic mice, and the fact that in vitro, transgenic adrenal glands do not secrete more corticosterone when normalized for their increased weight, suggest that the elevated corticosterone levels are mediated by a mitogenic effect of IGF-II at the adrenal level. However, our data do not allow us to exclude an additional direct steroidogenic effect of IGF-II in these animals, and further studies are necessary to identify the mechanisms of IGF-II action on the adrenal cortex in PEPCK-IGF-II transgenic mice.
Additionally, our study confirms and extends our previous finding that IGF-II transgenic mice have significantly larger adrenal glands than control mice. The structure and steroidogenesis of the murine adrenal cortex underlies considerable genetic variation, depending on strain, age, and sex of the animals (39, 40). Most notable, in this regard, is the juxtamedullary X-zone, which is present in both sexes at birth but soon degenerates in male animals, whereas it persists with considerable variation in adult female mice (31, 41, 42). Previous studies have shown that the reported strain- and gender-dependent differences in adrenal weight are mainly attributable to variations in X-zone degeneration, whereas the volumes of the definitve adrenocortex and medulla are relatively constant (43). Therefore, and because the physiological role and regulation of the X-zone remain unclear, the present study was confined to male animals. The parenchymal cell volume and cell number in normal adrenal glands found in our study were comparable with the results reported by Shire and Sprickett (42) in young adult mice of different strains, and this confirmed data reporting a higher volume of the fasciculata cells, compared with parenchymal cells of the other zones (44). Although the morphological zonation of the adrenal gland and the parenchymal cell volumes were similar in PEPCK-IGF-II transgenic and in normal control mice, the overexpression of IGF-II induced considerable volumetric changes in the adrenal glands of transgenic animals. Most prominent was the increased relative adrenal weight, both in young (12 weeks) and in older (36 weeks) adult IGF-II transgenic animals. The present detailed morphological investigation demonstrated that the overexpression of IGF-II resulted in a 1.5-fold larger volume of the zona fasciculata, whereas the volume of the other adrenal zones was only slightly elevated. The observed changes in the volume of the zona fasciculata are mainly caused by a hyperplasia of this corticosterone-producing adrenocortical zone, because overexpression of IGF-II was associated with an increased number of fasciculata cells, whereas the cell volume remained constant. This is in accordance with our finding of significantly elevated serum corticosterone levels in IGF-II transgenic animals.
It has been generally accepted that, in mice, IGF-II normally promotes growth only during embryogenesis, whereas IGF-I acts mainly after birth. This is supported by the strong decline in serum and tissue levels of IGF-II in mice in the postnatal period and by the fact that systemic IGF-II is a poor promoter of whole-body growth, when compared with IGF-I in intact mice (45, 46, 47, 48, 49). It is assumed that IGF-II mediates the ACTH-induced fetal adrenal growth, because ACTH induces IGF-II gene expression in human fetal adrenocortical cells and because IGF-II is mitogenic in these cells (2). Furthermore, IGF-II has been implicated in the regulation of fetal adrenal steroidogenesis because of a coordinate expression of IGF-II and steroidogenic enzyme mRNAs in fetal human and ovine adrenal glands (6, 50). In normal mice, IGF-II serum and tissue levels decline strongly after birth (49), and no IGF-II expression could be detected by RT-PCR in the adrenal glands of adult control mice in this study. In contrast, expression of IGF-II was detected in all investigated adrenal glands from IGF-II transgenic mice. This adrenal overexpression of IGF-II presumably is confined to the cortex, because in a previous study, PEPCK immunoreactivity was found in all layers of the adrenal cortex but not in the medulla of adult mice (51). The fact that the overexpression of IGF-II in the PEPCK-IGF-II transgenic mouse model is present only in postnatal life (14) supports the hypothesis that IGF-II is an important regulator of adrenocortical cell function, not only in the fetal but also in the adult adrenal gland, as it has been previously postulated, because of its predominant steroidogenic potency and mitogenic effect in adult human and bovine adrenocortical cells (14). So far, it remains unclear whether IGF-II acts on the adrenal gland in an endocrine or rather a paracrine/autocrine fashion. Because PEPCK-IGF-II transgenic mice exhibit strongly elevated serum IGF-II levels in addition to the overexpression of IGF-II in the adrenal gland, the presented mouse model does not allow us to conclude whether the local expression of IGF-II is required for the trophic effects on the adrenal gland or whether the observed adrenal changes in the transgenic animals are attributable to an endocrine effect of postnatally elevated IGF-II serum levels. However, several lines of evidence support the hypothesis that IGF-II mediates its growth-promoting effect primarily at a local level. First, systemic IGF-II is a poor postnatal growth promotor, as can be seen in the normal, or even reduced, size of IGF-II transgenic mice (17, 46, 47, 52). Second, no growth effect was observed after infusion of IGF-II in hypophysectomized rats (45) or in adult rodents with IGF-II-producing tumors (48). Third, in analogy to our results, a transgenic expression of IGF-II was found in all organs with local overgrowth in two other IGF-II transgenic mouse models with selective enlargement of the thymus (52) or the skin, gut, and uterus (47). Fourth, no adrenal overgrowth has been described in other IGF-II transgenic mice, although circulating IGF-II was elevated to comparable levels (49).
There is substantial evidence that IGF-II is involved in adrenal tumorigenesis. Overexpression of IGF-II has been found in human adrenocortical carcinomas and in pheochromocytomas (13, 14, 53). The mitogenic effect of IGF-II is dependent on the presence of the IGF-I receptor (54), and we have recently demonstrated an overexpression of IGF-I receptors in malignant adrenocortical tumors (8). However, the fact that the described PEPCK-IGF-II mice did not develop macroscopically obvious tumors, over an 18-month period (49), suggests that IGF-II overproduction, by itself, is not sufficient for malignant transformation, and that additional factors are required for tumorigenesis.
In conclusion, our data demonstrate that postnatal overexpression of IGF-II in the adrenal gland of adult transgenic mice is associated with adrenocortical hyperplasia and that it significantly elevated basal, as well as ACTH-induced, corticosterone serum levels and suggests an important role of IGF-II in the regulation of adult adrenocortical growth and steroidogenesis.
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Acknowledgments |
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Footnotes |
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Received September 22, 1998.
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