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Somatostatin Receptor Subtype 2 Knockout Mice Are Refractory to Growth Hormone-Negative Feedback on Arcuate Neurons

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
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-692–585, 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 (SSTR1–5) 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.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
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).

View larger version (30K): [in this window] [in a new window]   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.

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).

View larger version (47K): [in this window] [in a new window]   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.

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.

View larger version (77K): [in this window] [in a new window]   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

View larger version (24K): [in this window] [in a new window]   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).

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.

View larger version (26K): [in this window] [in a new window]   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).

View larger version (103K): [in this window] [in a new window]   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

View larger version (25K): [in this window] [in a new window]   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).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

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.

View larger version (31K): [in this window] [in a new window]   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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

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.

	FOOTNOTES

 
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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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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