Somatostatin Receptor Subtype 2 Knockout Mice Are Refractory to Growth Hormone-Negative Feedback on Arcuate Neurons
Hui Zheng,
Alex Bailey,
Ming-Hao Jiang,
Kazufumi Honda,
Howard Y. Chen,
Myrna E. Trumbauer,
Lex H.T. Van der Ploeg,
James M. Schaeffer,
Gareth Leng and
Roy G. Smith
Department of Biochemistry & Physiology (H.Z., M-H.J., H.Y.C.,
M.E.T., V.d.P., J.M.S., R.G.S.), Merck Research Laboratories,
Rahway, New Jersey 07065,
Department of Physiology (A.B.,
K.H., G.L.), Edinburgh University Medical School, Edinburgh,
Scotland EH8 9AG
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ABSTRACT
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The pulsatile nature of GH release is apparently
regulated by alternating sequential changes in two hypothalamic
hormones, GH releasing hormone (GHRH) and somatostatin. Entrainment of
this pulsatility appears to involve GH-mediated negative feedback.
Recently a new receptor involved in GH release was cloned. Activation
of this receptor by GH-releasing peptides and MK-0677 initiates and
amplifies GH pulsatility and is associated with increased Fos
immunoreactivity and electrical activity in GHRH containing arcuate
neurons. We show that pretreating mice with GH blocks activation of
these neurons by MK-0677. Similarly, octreotide inhibited the action of
MK-0677. To determine whether this GH-mediated negative feedback on
GHRH neurons was direct, or by GH stimulation of somatostatin release
from periventricular neurons, we selectively inactivated the gene for
one of the five specific somatostatin receptor subtypes (subtype 2). In
the knockout mice, both GH and octreotide failed to inhibit MK-0677
activation of arcuate neurons. GH did, however, increase Fos
immunoreactivity in the periventricular nucleus, consistent with GH
stimulation of somatostatin release from periventricular neurons. Thus,
GH-mediated negative feedback involves signaling between
periventricular and arcuate neurons with the signal being transduced
specifically through somatostatin subtype 2 receptors.
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INTRODUCTION
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In mammals, GH is released in a distinctive pulsatile pattern that
has a profound importance for its biological activity (1). The release
of GH is controlled by two hypothalamic hormones: GH-releasing hormone
(GHRH) and somatostatin, and the pulsatile pattern is thought to arise
from alternating episodes of stimulation by GHRH and inhibition by
somatostatin. In addition, the pulse generator mechanism appears to be
"timed" by GH-mediated negative feedback. For example, when rats
are treated with exogenous GH at 3-h intervals (similar to the interval
normally observed between spontaneous GH pulses), then the endogenous
rhythm of GH secretion becomes entrained to the imposed rhythm of
exogenous GH administration (2). GH receptors are present in both the
arcuate nucleus, where the GHRH neurons are located, and in the
periventricular nucleus (3), where the neurosecretory somatostatin
neurons are located. These results suggest that feedback regulation of
GH pulsatility is controlled at the level of the hypothalamus, but it
is not clear whether this negative feedback is modulated directly
through GH receptors on arcuate neurons or indirectly by GH stimulating
somatostatin release from periventricular neurons.
An important class of GH secretagogues, which includes GHRP-6,
L-692,429, L-692585, and MK-0677 (4, 5, 6, 7, 8), stimulates GH release
in vivo by acting on the anterior pituitary and
hypothalamus. In particular, these GH secretagogues activate a subset
of the neurosecretory cells of the hypothalamic arcuate nucleus (9).
These activated cells include a significant proportion of the
GHRH-synthesizing cells (10), suggesting that the GH secretagogues
stimulate GH secretion in part through an increase in GHRH secretion.
This concept has been supported by measurements of GHRH in the
hypothalamo-pituitary portal circulation (11, 12). Recently a new
receptor has been cloned and characterized (13, 14, 15), which mediates the
actions of the synthetic GH secretagogues (8). This receptor is
distinct from the GHRH receptor, is present in both the hypothalamus
and pituitary, and is highly conserved across species (14, 15, 16); these
characteristics suggest that the GH secretagogues mimic an unknown
natural hormone. Interestingly, continuous exposure to elevated levels
of GH secretagogues is accompanied by a striking and prolonged
enhancement of the pulsatile patterning of GH secretion (6, 17, 18).
The actions of these secretagogues, and in particular their
interactions with GHRH neurons and somatostatin neurons in the
hypothalamus, thus take on a major significance for a full
understanding of the physiological regulation of GH release.
Our objective was to determine whether the negative feedback effects of
GH in the hypothalamus were mediated by direct inhibition of arcuate
neurons or indirectly by activation of neurosecretory somatostatin
neurons. One approach in the evaluation of the role of somatostatin is
to inactivate its receptor. Five different subtypes of G
protein-coupled receptors for somatostatin (SSTR15) have been cloned
and characterized according to their pharmacology, signal transduction
pathways, and localization (19, 20, 21). Based on its pharmacology,
somatostatin receptor subtype 2 (SSTR2) appears to be important for
inhibiting GH secretion from somatotrophs in the pituitary gland
(19, 20, 21). We speculated that the same subtype might also be involved in
hypothalamic pathways regulating GH release.
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RESULTS
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Inactivation of SSTR2
To assess the role of SSTR2 we developed a line of mice in which
the SSTR2 gene was inactivated by homologous recombination in mouse
embryonic stem (ES) cells. The targeting vector (Fig. 1A
) encoded from 5' to 3', a 2.3-kb
segment preceding the SSTR2 coding sequence, a PGKneo
(neomycin-phospho-transferase) expression cassette, and a 4.6-kb
fragment located 3' of SSTR2a cDNA (SSTR2-derived sequences used for
targeting are presented as open rectangles in Fig. 1A
). A
1.2-kb sequence encoding the SSTR2a cDNA could thus be deleted by
homologous recombination and replaced with the positive selectable
marker, PGKneo (22).

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Figure 1. Inactivation of SSTR2 Gene in Mice by Homologous
Recombination
A, Targeting vector used for inactivation of SSTR2 in mouse ES cells.
r, E.CoRl; K, Kpnl. B, Southern blot analysis of targeted clones. C,
Southern blot analysis of offspring from heterozygous matings. Genomic
DNA isolated from 3-week-old pups generated from crosses of
heterozygous mice was digested with KpnI, transferred to
membranes, and hybridized with the 3'-probe. +/+, wild-type; +/-,
heterozygous; -/-, homozygous SSTR2 mutant mice.
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The linearized targeting vector was electroporated into AB2.1 ES cells
(23), and transfected cells were selected in the presence of G418.
G418-resistant colonies were screened by Southern blot analysis. Four
targeted clones were identified from a total of 147 clones analyzed
(Fig. 1B
). The 3'-probe used for Southern analysis (Fig. 1A
) flanked
the targeting sequences. The expected polymorphic KpnI
restriction enzyme fragments were detected for the wild type (11.0 kb)
and the targeted SSTR2 alleles (14.0 kb) since the KpnI site
located within the SSTR2a sequence was deleted in the targeting vector.
Gene targeting events at the SSTR2 locus were confirmed by
hybridization with the neo sequence (data not shown).
The targeted clones were injected into 3.5 days post coitus C57BL/6J
blastocysts to generate chimeric mice. A total of seven male chimeras
were produced from two of the clones (nos. 117 and 164) with ES cell
contributions ranging from 60% to 100%, as judged by the percentage
of agouti coat color in the chimeric mice. Chimeric mice from both
clones were bred with C57BL/6J females to assess their potential for
germline colonization. Successful germline transmission of ES cells was
achieved with both clones, and approximately 50% of the agouti pups
contained the disrupted SSTR2 allele (data not shown). Heterozygous
mice were indistinguishable from their normal littermates. To produce
mice homozygous for the disrupted SSTR2 gene, cross-matings between
heterozygous mice were arranged. Normal litter sizes were observed. The
resulting pups were genotyped by Southern blot analysis (Fig. 1C
). Of
78 mice analyzed at 3 weeks of age, 22 (28%) were wild type (+/+), 39
(50%) were heterozygous (+/-), and 17 (22%) were homozygous for the
deleted SSTR2 allele, representing a normal 1:2:1 Mendelian inheritance
of the targeted allele. This result ruled out an essential function of
SSTR2 in mouse embryogenesis. The homozygous SSTR2 knockout mice
appeared normal and healthy up to 15 months of age.
To determine whether the SSTR2 gene was completely inactivated,
Northern blot analysis was performed to determine SSTR2 mRNA level with
brain RNA using SSTR2 cDNA as a probe. SSTR2 mRNA could not be detected
in mice homozygous for the targeted allele (Fig. 2
). The production of a null allele was
also confirmed by in situ hybridization (data not
shown).

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Figure 2. Northern Blot Analysis of SSTR2 Expression
Total brain RNA (20 µg) from 6-week-old wild-type (+/+), heterozygous
(+/-), and homozygous (-/-) SSTR2 mice (two mice each) hybridized
with the full-length SSTR2a cDNA sequence. Lower panel,
Mouse ß-actin cDNA hybridization for loading control.
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Effects of GH on MK-0677 Activation of Arcuate Neurons in SSTR2 +/+
and -/- Mice
It was previously shown that treatment of rats or mice with
GHRP-6, L-692,429, L-692,585, and MK-0677 increased c-fos
expression in arcuate neurons and increased electrical activity in
arcuate neurons projecting to the median eminence (9, 24, 25, 26). As
discussed above, recent studies using GHRP-6 have documented that GHRH
containing arcuate neurons are activated and that activation of arcuate
neurons is inhibited by somatostatin (27). Therefore, the effects of
somatostatin on controlling the activity of arcuate neurons appear to
be pivotal in the physiological regulation of GH secretion.
To investigate the potential role of SSTR2 in GH-mediated feedback
pathways, a series of experiments to measure Fos activation in the
hypothalamus were conducted with SSTR2 +/+ and SSTR2 -/- mice.
Conscious mice were injected ip with either saline or mouse (m)GH
followed 10 min later by a further ip injection of saline or MK-0677.
Ninety minutes after the second injection, the expression of
hypothalamic Fos immunoreactivity was examined. Fos immunoreactivity
was induced in the arcuate nucleus of mice injected with MK-0677 (Figs. 3
and 4
).
In the arcuate nucleus, Fos induced by MK-0677 was completely blocked
by preadministration of mGH in normal mice but was unaffected in SSTR2
-/- mice (Figs. 3
and 4
). Systemic injections of mGH produced a small
increase in Fos expression in the arcuate nucleus of both normal and
SSTR2 knockout.

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Figure 3. Photomicrographs of the Expression of Fos
Immunoreactivity in the Arcuate Nucleus of SSTR2 +/+ and SSTR2 -/-
Mice in Response to MK-0677 and GH Pretreatment
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Figure 4. The Effect of mGH and MK-0677 Injection on the
Amount (Mean ± SEM) of Fos Immunoreactivity in the
Arcuate Nucleus of Control and SSTR2 -/- Mice
All groups contain between five and seven animals. (*,
P < 0.05 compared with same group saline/saline
injection; , P < 0.05 compared with same group
saline/MK-0677 injection; , P < 0.05 comparison
between control and transgenic groups).
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The results shown in Fig. 4
indicate that the magnitude of the increase
in Fos activation by MK-0677 is reduced by about 25% in the SSTR2
-/- compared with the +/+ mice. One potential explanation is that
mice lacking SSTR2 have reduced endogenous somatostatin tone on the
arcuate neurons; thus constitutive expression of c-fos is
likely to be increased. Based on predictions from previous studies of
c-fos regulation, some degree of constitutive expression
would be expected to cause reduced sensitivity to Fos activation by
MK-0677 (29).
Effects of Octreotide on MK-0677 Activation of Arcuate Neurons in
SSTR2 +/+ and -/- Mice
Mice injected with octreotide alone showed very low levels of Fos
expression throughout the hypothalamus, levels that were not
significantly different from those seen after vehicle injection.
However, similar to GH, pretreatment of wild type mice with the
somatostatin analog octreotide attenuated Fos activation in arcuate
neurons by more than 50% (Fig. 5
). By
contrast, in SSTR2 -/- mice, while MK-0677 induced Fos expression in
arcuate neurons, the level of induced expression was not affected by
prior injection of octreotide.

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Figure 5. The Effect of Octreotide and MK-0677 Injection on
the Amount (Mean ± SEM) of Fos Immunoreactivity in
the Arcuate Nucleus of Control and SSTR2 -/- Mice
All groups contain between five and seven animals. (*,
P < 0.05 compared with same group saline/saline
injection; , P < 0.05 compared with same group
saline/MK-0677 injection; , P < 0.05 comparison
between control and transgenic groups).
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Effects of GH and MK-0677 on Activation of Fos in Periventricular
Nucleus
Systemic injections of mGH and MK-0677 produced an increased
expression of Fos in the periventricular nucleus of the hypothalamus of
wild type mice (Figs. 6
and 7
). The effects of MK-0677 were less
pronounced than that caused by mGH (Figs. 6
and 7
). Combined
administration of mGH and MK-0677 produced enhanced Fos expression in
the periventricular nucleus (Figs. 6
and 7
). When Fos expression was
compared in the periventricular nucleus of +/+ and -/- mice, the
increased expression caused by treatment with GH or MK-0677 was
unaffected in both wild type and SSTR2 -/- mice, confirming that the
activation of periventricular neurons was unaffected by somatostatin
(Fig. 7
).

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Figure 6. Photomicrographs of the Expression of Fos
Immunoreactivity in the Periventricular Nucleus of SSTR2 +/+ and SSTR2
-/- Mice in Response to MK-0677 and GH Pretreatment
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Figure 7. The Effect of mGH and MK-0677 Injection on the
Amount (Mean ± SEM) of Fos Immunoreactivity in the
Periventricular Nucleus of Control and SSTR2 -/- Mice
All groups contain between five and seven animals. (*,
P < 0.05 compared with same group saline/saline
injection; , P < 0.05 compared with same group
saline/MK-0677 injection; , P < 0.05 comparison
between control and transgenic groups).
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DISCUSSION
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The pattern of Fos expression induced in the arcuate nucleus of
the hypothalamus by injection of MK-0677 is very similar to that
reported previously for both rats and mice after systemic or central
administration of other GH secretagogues (25, 30). This study shows for
the first time that exogenous administration of mGH is also highly
effective at blocking secretagogue-induced Fos expression and, most
importantly, shows that the suppression of Fos activation that follows
either administration of mGH or a somatostatin agonist, does not occur
in the absence of the SSTR2 receptor. This suggests strongly that
circulating levels of GH feed back centrally to inhibit the activity of
arcuate neurons by inducing the release of endogenous somatostatin,
acting selectively via SSTR2 receptors. We cannot, of course, rule out
that there may be developmental changes in animals lacking SSTR2 that
eliminate the suppressive effects of GH and somatostatin on arcuate
neurons.
There are somatostatin-containing neurons within both the arcuate and
periventricular nuclei (31) and, although GH receptors are present in
the arcuate nucleus, very few are located on somatostatin neurons (32).
However, GH receptors are present in the periventricular nucleus (3),
and somatostatin neurons at this site do appear to be sensitive to GH
feedback since administration of GH receptor antisense decreases
periventricular somatostatin expression (33), and in hypophysectomized
rats GH activates periventricular somatostatin neurons to express
c-fos (34, 35). The increase in periventricular Fos
immunoreactivity observed in all animals given mGH in this study is
consistent with previous reports, and the induction of Fos in the
periventricular nucleus after MK-0677 may be expected to follow from
the effects of endogenous GH release. Such induction, although observed
in the present study, has not been observed previously in intact rats
(24). However, in intact, rather than hypophysectomized, rats, GH
itself is relatively ineffective (36), suggesting that in intact rats
central GH receptors may be down-regulated by the feedback actions of
GH. In the arcuate nucleus, the cells activated to express
c-fos in response to GH secretagogues include a high
proportion of GHRH neurons, but other cell types, including a subset of
neuropeptide Y-expressing cells, are also activated (10), and in
situ hybridization studies have shown that in hypophysectomized
rats GH induces c-fos specifically in neuropeptide Y, and
not GHRH, neurons (35).
The most concise interpretation of the present results that potentially
explains self-entrainment of GH pulsatility is represented in Fig. 8
. GH activates periventricular
somatostatin neurons directly, and the induced secretion of
somatostatin from these neurons directly inhibits the activity of
arcuate neurons via SSTR2 receptors on nerve terminals within the
arcuate nucleus (36, 37). MK-0677, on the other hand, directly
activates arcuate neurons via the GH secretagogue receptors present on
these neurons (14) but activates periventricular somatostatin neurons
indirectly via the MK-0677-induced release of GH. Most importantly, the
fact that both GH and octreotide are ineffective in attenuating MK-0677
activation of arcuate neurons in mice lacking SSTR2 argues for an
important role of this particular somatostatin receptor subtype in the
control of pulsatile GH secretion.

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Figure 8. Proposed Model of GH-Mediated Feedback Regulation
via Somatostatin (SST) Subtype-2 in the Hypothalamus
A ligand (L) for the GH secretagogue receptor (GHSR) such as MK-0677 or
GHRP-6 activates neurons in the arcuate nucleus (ArcN). GHRH is
released into the median eminence (ME) to stimulate GH release from
somatotrophs in the anterior pituitary gland. GH then feeds back by
interacting with GH receptors on neurons in the periventricular nucleus
(PeN) to cause the release of SST. SST by interacting with SSTR2
inhibits arcuate neurons, thus decreasing GHRH release.
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MATERIALS AND METHODS
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Generation of SSTR2-Deficient Mice by Gene Targeting in Mouse ES
Cells
The deletion targeting vector contained a 2.3-kb
EcoRI-XbaI fragment upstream of the ATG
translation initiation codon and a 4.6-kb
XbaI-EcoRI fragment downstream of the SSTR2a cDNA
as homologous targeting sequences (represented by open
rectangles). A 1.2-kb XbaI fragment containing the
SSTR2a cDNA (shown as solid rectangle) was deleted and
replaced with the positive selective marker PGK-neo (represented by the
shaded rectangle). Homologous recombination between the
vector and the wild type SSTR2 locus results in the deletion of the
entire SSTR2a sequence, followed by its replacement with the neo gene.
The probe used for detecting the targeted event was a 0.7-kb
BamHI fragment downstream of the 3'-homologous sequence
(3'-probe). Digestion with the restriction endonuclease KpnI
was used to separate the wild type and the targeted SSTR2 alleles,
which generates a 11-kb and a 14-kb fragment, respectively, due to the
loss of the KpnI site by neo insertion.
Southern Blot Analysis of Targeted Clones
ES cell DNA (8 µg) from wild type AB2.1 ES cells and from four
targeted clones (46, 68, 117, and 164) were digested with restriction
enzyme KpnI, electrophoresed on a 0.6% agarose gel,
transferred onto Gene Screen Plus nylon membranes (New England Nuclear
Dupont, Boston, MA) and hybridized with the 3'-probe.
Southern Blot Analysis of Offspring from Heterozygous Matings
Genomic DNA isolated from tails of 3-week-old pups generated
from crosses of heterozygous mice was digested with KpnI,
transferred to membranes, and hybridized with the 3'-probe. +/+, wild
type; +/-, heterozygous; -/-, homozygous SSTR2 mutant mice.
Northern Blot Analysis of SSTR2 Expression
Total brain RNA (20 µg) from 6-week-old wild-type (+/+),
heterozygous (+/-), and homozygous (-/-) SSTR2 mice (two mice each)
was isolated by using the RNAzol B method (Biotecx Laboratories, Inc.,
Houston, TX) and hybridized with the full-length SSTR2a cDNA sequence
(Fig. 2
, upper panel). The lower panel of
Fig. 2
represents hybridization with mouse ß-actin cDNA as a gel for
loading control.
Evaluation of Fos Activation in the Hypothalamus
Compounds used were: MK-0677 (50 µg), octreotide (100 µg),
and mGH (30 µg). Mice were give an initial ip injection (0.1 ml) of
either saline, octreotide or mGH, followed 10 min later by an ip
injection (0.1 ml) of either saline or MK-0677. Thus, the first study
comprised of the following groups: saline/saline, saline/MK-0677,
mGH/saline, mGH/MK-0677 saline/saline, saline/MK-0677, mGH/saline,
mGH/MK-0677, and the second study of: saline/saline, saline/MK-0677,
octreotide/saline, octreotide/MK-0677. Additionally, a number of mice
were injected ip with hypertonic saline (0.2 ml, 1.5 M) to
serve as positive controls for the immunocytochemistry. Ninety minutes
after injection animals were terminally anesthetized with sodium
pentobarbitone (60 mg/kg; ip) and perfused transcardially with
heparinized saline followed by 4% paraformaldehyde in 0.1
M phosphate buffer (PB, pH 7.4). Brains were then removed
and placed in the same solution for 24 h before being stored at
-80 C until processing. Coronal sections of forebrain (40 µm) were
cut on a freezing microtome and placed in 0.1 M PB
containing Triton X-100 (PB-T, pH 7.4). Sections were incubated for
24 h at 4 C in Ab-2 Fos antibody (rabbit polyclonal; Oncogene
Science, New York, NY, 1:1000 in 1% normal sheep serum). The
antibody-antigen complex was localized with a 1-h incubation in
biotinylated anti-rabbit Ig (Vector, Peterborough, U.K., 1:100),
followed by a 1-h incubation in avidin, biotinylated horseradish
peroxidase (Vector, Peterborough, U.K., 1:50). The reaction product was
visualized using a glucose oxidase-diaminobenzidine-nickel method, and
Fos-like immunoreactivity was visualized as a dense purple-black
precipitate restricted to the nucleus. The number of c-fos
positive nuclei in the arcuate and periventricular nuclei (six to eight
sections per mouse) were counted double-blind and a group mean
calculated (mean ± SEM). Statistical analysis was
performed by a two- way ANOVA followed by an all pairwise multiple
comparison of data (Student-Newman-Keuls method) with significance
taken as P < 0.05.
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FOOTNOTES
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Address requests for reprints to: Dr. Roy G. Smith, Merck Research Laboratories (RY80K), P.O. Box 2000, Rahway, New Jersey 07065.
Received for publication June 6, 1997.
Accepted for publication July 28, 1997.
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