Thyroid Hormone-Responsive Pituitary Hyperplasia Independent of Somatostatin Receptor 2

Michelle L. Brinkmeier, Justin H. Stahl, David F. Gordon, Brian D. Ross, Virginia D. Sarapura, Janet M. Dowding, Susan K. Kendall, Ricardo V. Lloyd, E. Chester Ridgway and Sally A. Camper

Department of Human Genetics (M.L.B., J.H.S., S.K.K., S.A.C.), Departments of Radiology and Biological Chemistry (B.D.R.), University of Michigan, Ann Arbor, Michigan 48109; Division of Endocrinology, Metabolism, and Diabetes (D.G., V.D.S., J.M.D., E.C.R.), University of Colorado Health Sciences Center, Denver, Colorado 80262; and Division of Anatomic Pathology (R.V.L.), Mayo Clinic, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Dr. Sally Camper, Department of Human Genetics, University of Michigan Medical School, 1500 West Medical Center Drive, 4301 MSRB III, Ann Arbor, Michigan 48109-0638.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice homozygous for the targeted disruption of the glycoprotein hormone {alpha}-subunit ({alpha}Gsu) display hypertrophy and hyperplasia of the anterior pituitary thyrotropes. Thyrotrope hyperplasia results in tumors in aged {alpha}Gsu-/- mice. These adenomatous pituitaries can grow independently as intrascapular transplants in hypothyroid mice, suggesting that they have progressed beyond simple hyperplasia. We used magnetic resonance imaging to follow the growth and regression of thyrotrope adenomatous hyperplasia in response to thyroid hormone treatment and discovered that the tumors retain thyroid hormone responsiveness. Somatostatin (SMST) and its diverse receptors have been implicated in cell proliferation and tumorigenesis. To test the involvement of SMST receptor 2 (SMSTR2) in pituitary tumor progression and thyroid hormone responsiveness in {alpha}Gsu-/- mutants, we generated Smstr2-/-, {alpha}Gsu-/- mice. Smstr2-/-, {alpha}Gsu-/- mice develop hyperplasia of thyrotropes, similar to {alpha}Gsu-/- mutants, demonstrating that SMSTR2 is dispensable for the development of pituitary adenomatous hyperplasia. Thyrotrope hyperplasia in Smstr2-/-, {alpha}Gsu-/- mice regresses in response to T4 treatment, suggesting that SMSTR2 is not required in the T4 feedback loop regulating TSH secretion.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MANY LINES OF evidence support a role for somatostatin (SMST) and its receptors in regulation of TSH secretion in normal and hypothyroid animals (1, 2). SMST is a hypothalamic peptide that has roles in inhibiting a variety of physiological activities in many organs including the inhibition of GH, PRL, and TSH secretion, and the inhibition of cell proliferation (3). SMST effects are controlled through five receptors, SMSTR1–5, all of which are expressed in the anterior pituitary gland (4, 5, 6, 7).1 Thyrotropes express predominantly Smstr1, Smstr2, and Smstr5, but little or no Smstr3 and Smstr4 (10, 11). Regulation of TSH secretion by somatostatin appears to be mediated by both SMSTR2 and SMSTR5 (12). Thus, thyrotrope responsiveness to SMST probably involves Smstr1, Smstr2, and/or Smstr5.

Studies on large numbers of human tumor types, including pituitary adenomas, have shown that most tumors express high levels of at least one SMSTR subtype. More than 80% of the time the subtype expressed is Smstr2 (13, 14). GH- and TSH-producing pituitary adenomas express increased levels of Smstr2 compared with the normal gland (15). The SMST analog, octreotide, has been used to treat TSH-secreting tumors when options such as surgery or radiotherapy are not feasible. Octreotide binds with the highest affinity to SMSTR2 followed by SMSTR5 and SMSTR3 (15). This is consistent with the idea that Smstr2 is important in thyrotrope adenomas.

Thyrotropic tumor cells, TtT97, grown in hypothyroid mice, have been used as a model to study hormone responsiveness of thyrotropes (1). TtT97 tumors propagated in hypothyroid mice shrink in response to T4. The expression of Smstr2 is maintained while Smstr1 and Smstr5 increased in response to T4 (1). Rats rendered hypothyroid with antithyroid drugs exhibit alterations in Smstr1 and Smstr2 RNA levels in response to T3 treatment (2). These studies implicate a role for Smstr1, Smstr2, and Smstr5 in the negative feedback loop regulating TSHß transcription in response to thyroid hormone.

We have used {alpha}Gsu-/- mice as a model for hypothyroidism with increased TSHß transcription (16). The diffuse thyrotrope hyperplasia seen in {alpha}Gsu-/- mice is different in appearance from the focal hyperplasia induced in normal adult mice rendered hypothyroid by chemical or radiation treatment. This suggests that early onset hypothyroidism has the potential to recruit more pituitary precursors to the thyrotrope fate. We demonstrate progression of global thyrotrope hyperplasia in {alpha}Gsu-/- mice into transplantable adenomatous hyperplasia that retains T4 responsiveness. Using Smstr2 and {alpha}Gsu double knockout mice, we have shown that despite previous studies suggesting its involvement, SMSTR2 is not required for tumor progression or thyroid hormone responsiveness in this thyrotrope tumor model.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Progression of Thyrotrope Hyperplasia in Aging {alpha}GSU-/- Mice
At 2 months of age {alpha}Gsu-/- mice exhibited thyrotrope hypertrophy and hyperplasia with little or no significant change in pituitary size (17). We aged {alpha}Gsu-/- mice to assess whether the thyrotrope hyperplasia would progress. At approximately 1 yr of age, all mice had pituitary masses that were larger than the average wild-type pituitary, although the size of the pituitary masses varied considerably (Fig. 1Go).



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Figure 1. Progression of Thyrotrope Hyperplasia to Transplantable Tumors

Pituitary glands from approximately 1-yr-old wild-type (A) and {alpha}Gsu-/- mice (B and C) demonstrate an increase in pituitary size in the {alpha}Gsu-/- mice from 5-fold (B) to 47.1-fold (C) compared with wild-type (A). Black bar = 3 mm. D, Pituitary masses continue to grow when transplanted between the scapula of hypothyroid C57BL/6 mice.

 
The pituitaries of the mutant mice could be classified in two groups. One group had pituitaries that retained a lobular structure (Fig. 1BGo). These pituitaries exhibited a 5- to 10-fold increase in weight (Table 1Go). The other group had completely lost the lobular pituitary structure and had markedly increased vascularization and necrotic areas in the portion of the gland that had undergone the most dramatic growth (Fig. 1CGo). This group had pituitary weights that were approximately 30- to 50-fold above normal (Table 1Go). These large masses exhibited increased mitotic activity and absence of other cell types within the proliferating population, consistent with progression to adenomas (data not shown).


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Table 1. {alpha}GSU-/- Pituitaries Are Enlarged

 
Pituitaries from four 12-month-old {alpha}Gsu-/- mice were removed and transplanted into C57BL6/J male mice rendered hypothyroid 6 wk before transplant via radiothyroidectomy. After 6 months, hyperplastic pituitaries ranging initially from 34 mg to 88 mg increased in weight to 3–5 g (Fig. 1Go D), which represents a 50- to 100-fold increase in weight. This suggests that growth of the adenomatous hyperplastic masses is independent of the normal pituitary surroundings.

Thyroid Hormone Treatment of {alpha}Gsu-/- Mice Results in Regression of Hyperplasia
Thyroid hormone (T4) treatment can prevent and reverse hypertrophy and hyperplasia of thyrotropes in {alpha}Gsu-/- neonates and young adults, respectively (17). {alpha}Gsu-/- mice ranging from 7–11 months of age were treated with T4 injections for 40 d to test whether the pituitary hyperplasia would respond in a similar manner to the hormone regimen as the younger {alpha}Gsu-/- mice and the TtT97 tumors (1). Because of the variation in size of pituitary masses, the pituitary volumes of eight {alpha}Gsu-/- mice were measured by magnetic resonance imaging (MRI) and placed into two groups, control and T4 treated (Fig. 2Go). This strategy made it possible to monitor growth and regression longitudinally by carrying out repeat MRI studies after 21 and 40 days (Fig. 2Go). Longitudinal monitoring of individual mice via MRI provides an accurate assessment of mass growth and regression during the treatment regimen and is an attractive alternative to killing groups of mice at each time point.



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Figure 2. MRI Permits Noninvasive Examination of Pituitary Volume

T2-weighted magnetic resonance images were used to document the size of pituitary glands in 9-month-old {alpha}Gsu-/- mice at 0, 21, and 40 d of thyroid hormone treatment (panels A–C) compared with aged matched wild-type littermates (panel D). Midsagittal image reveals the olfactory lobe (OL), cerebellum (CE), spinal cord (SC), and pituitary gland (between arrowheads). Each displayed image is from approximately the same region of the brain.

 
At the beginning of the experiment, the 7- to 10-month-old wild-type mice had an average pituitary volume of 3 mm3 (Table 2Go). The pituitary volumes of 7- to 9-month-old {alpha}Gsu-/- mice were larger, ranging from 5 to 8 mm3. The starting pituitary volumes of 11-month-old {alpha}Gsu-/- mice were substantially larger than normal, ranging from 8 to 18 mm3. Note that the midsagittal pituitary diameter is as large as the cerebellum at its widest point.


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Table 2. Tumor Regression in Thyroid Hormone-Treated {alpha}Gsu-/- Mice

 
Changes in pituitary volumes were observed at d 21 and d 40 in both groups (Fig. 3Go). All individuals in the T4-treated group had significant reduction in the size of the pituitary mass at both time points. Volumes decreased in T4-treated {alpha}Gsu-/- mice from 37 to 71% of the starting volume. Over the same time interval the untreated {alpha}Gsu-/- mice exhibited 21–117% increases in volume (Table 2Go). This demonstrates that the thyroid hormone treatment rapidly arrested growth and induced remarkable regression of the adenomatous hyperplasia.



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Figure 3. Thyroid Hormone Treatment Arrests Increase in Pituitary Volume and Initiates Involution

A three-dimensional picture generated from MRI was used to calculate the volume of pituitary glands. {alpha}Gsu-/- mice were chosen at 9 months to 13 months of age for daily injections of 2 µg of thyroid hormone (dashed line). Pituitary gland volumes in thyroid hormone-treated mice were calculated and compared with age-matched untreated {alpha}Gsu-/- mice. MRI of two wild-type mice was used to determine the average pituitary volume (gray bar). * Denotes mouse pictured in Fig. 2Go.

 
SMSTR2 Is Not Required for the Progression of Hyperplasia in {alpha}Gsu-/- Mice
SMSTRs have been shown to be expressed at high levels in many human tumor systems (15, 18, 19). In addition, Smstr2 is expressed at high levels in some neuroendocrine tumors (20) as well as pituitary somatotrope adenomas (21). Smstr2 transcripts were detected by Northern analysis in transplanted {alpha}Gsu-/- pituitary masses (Fig. 4Go); however, Smstr3 and Smstr4 were not detected (data not shown). We used mice with targeted disruptions in Smstr2 (22) to test whether Smstr2 is involved in the thyrotrope hyperplasia or T4-induced regression observed in {alpha}Gsu-/- mice. Mice were bred to produce double mutants with targeted disruptions to both Smstr2 and {alpha}Gsu. If SMSTR2 is necessary for the development of the thyrotrope adenomatous hyperplasia in {alpha}Gsu-/- mice, disrupting Smstr2 in the {alpha}Gsu-/- mice should prevent it. Double heterozygous matings are expected to produce Smstr2-/-, {alpha}Gsu-/- mice at a frequency of 1/16 (6.25%). We observed 3.2%, which is slightly less than what is expected, P = 0.03.



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Figure 4. Northern Blot Analysis of Smstr2 Transcripts in polyA(+) RNA Derived from an {alpha}Gsu-/- Mouse Pituitary Gland Transplanted into a Hypothyroid C57BL/6 Mouse

DNA size standards are shown on the left. The autoradiograph was exposed for 5 d at -80 C.

 
Six Smstr2-/-, {alpha}Gsu-/- mice were generated and killed at 2–6 months. Thyrotrope hypertrophy and hyperplasia were analyzed by immunohistochemistry with antibodies to TSHß (Fig. 5Go). The double knockouts were compared with single mutants and double heterozygote littermate controls. In Smstr2-/-, {alpha}Gsu-/- pituitaries the immunoreactive TSHß cells were enlarged and present throughout the pituitary in great abundance. This extensive hypertrophy and hyperplasia of thyrotropes in Smstr2-/-, {alpha}Gsu-/- mice are indistinguishable from those observed in {alpha}Gsu-/- mice (Fig. 4Go). These results indicate that thyrotrope hyperplasia can develop in {alpha}Gsu-/- mice in the absence of Smstr2.



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Figure 5. Thyrotrope Hypertrophy in T4-Treated and Untreated Smstr2-/-, {alpha}Gsu-/- Pituitaries Demonstrated by Immunohistochemistry with Antibodies to TSHß

 
Thyrotrope Hyperplasia Decreases in Smstr2-/-,{alpha}Gsu-/- Mice Treated with Thyroid Hormone
Thyrotrope hyperplasia decreases in {alpha}Gsu-/- mice in response to T4 treatment (17). Increased levels of T4 stimulate SMST secretion, which negatively regulates TSHß secretion. To determine whether {alpha}Gsu-/- mutants respond appropriately to T4 in the absence of SMSTR2, Smstr2-/-, {alpha}Gsu-/- mice were treated for 40 d with T4. Immunohistochemistry with TSHß antibodies was used to assess thyrotrope hyperplasia (Fig. 5Go). Pituitaries from Smstr2-/-, {alpha}Gsu-/- mutants responded to T4 treatment in a similar manner as {alpha}Gsu-/- single mutants, as indicated by a reduction in the number and size of TSH-staining thyrotropes to wild-type levels. These results indicate that SMSTR2 is not required for T4-mediated TSHß down-regulation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyrotrope hyperplasia progresses to adenomatous hyperplasia in 1-yr-old {alpha}Gsu-/- mice. Pituitary masses from 1-yr-old {alpha}Gsu-/- mice display tumor characteristics such as increased vascularization, increased mitotic activity, and regions of proliferating thyrotropes (Fig. 5Go) devoid of other pituitary cell types. Transplantation of these pituitary adenomas into hypothyroid C57BL/6 mice has demonstrated the ability of these masses to grow independently of endogenous factors specific to the pituitary gland and immediately surrounding milieu. There was no evidence of metastasis or invasiveness.

SMSTRs have been detected at high levels in many tumor types including pituitary adenomas (12, 18). Smstr2-/-, {alpha}Gsu-/- mice exhibited thyrotrope hyperplasia by 3 months of age. Thyrotrope hyperplasia is reversed in Smstr2-/-, {alpha}Gsu-/- mice treated with T4. These results suggest that SMSTR2 is not required for thyrotrope hyperplasia or thyroid hormone responsiveness in {alpha}Gsu-/- mice. SMST may bind to a receptor other than SMSTR2 in the down-regulation of TSHß or one of the other receptors present in thyrotropes may compensate for the absence of SMSTR2 in Smstr2-/-, {alpha}Gsu-/- mice.

In humans, autopsy studies reveal that the development of pituitary adenomas is a relatively common event, with a frequency of approximately 20% in the general population (23). Although TSH-secreting tumors represent only 1–2% of pituitary adenomas, they are generally large macroadenomas at the time of diagnosis (24, 25, 26). Patients’ symptoms vary according to the hormones produced by the adenoma and can include severe hyperthyroidism, goiter, clinical acromegaly, galactorrhea, amenorrhea, visual field defects, and headaches. Treatment for adenomas also varies. TSH-secreting tumors are generally removed by transsphenoidal surgery and, when this is not sufficient for remission, radiation and/or adjuvant medical therapy follows. The SMST analog octreotide has been shown to be effective for many patients in reducing the size of the tumor as well as suppressing TSH levels. In contrast to the {alpha}Gsu-/- mice, administration of T4 is usually not beneficial for treatment of TSH-producing adenomas in humans because high levels of thyroid hormone are usually already present secondary to the elevated TSH levels (25). However, T4 treatment can reduce the size of tumor-like nodules with TSH cell hyperplasia in humans (27).

At this time, the etiology of pituitary adenomas is not well understood (23, 28). Studies have been performed to identify and analyze genes that play a role in the initiation and progression of these tumors. LOH at several chromosomal loci (on 10q, 11q, 13q, and 9p) has been identified in some patients, but the critical genes in these regions have not been identified (29). However, several other critical genes have been identified (30). The gene for the G protein involved in GHRF receptor signaling (G{alpha}s or GNAS1) is altered in many GH-secreting neoplasms (31), and MEN1 (multiple endocrine neoplasia type 1) mutations lead to prolactinomas (32, 33, 34). Also, the pituitary tumor transforming gene (PTTG) is associated with a variety of tumor types (35). In mice, pituitary tumors within the intermediate and anterior lobes result from deletion of the specific CdK inhibitor genes encoding p27KIP1 (36) and p18INK4c (37) that are expressed in the normal murine pituitary. Although there has been significant progress in the molecular genetics of pituitary adenomas, the generation of animal models will be important to further understand the etiology of these tumors. In addition, these models, including the {alpha}Gsu-/- mouse, may facilitate the development of improved treatment strategies for anterior pituitary adenomas.

Many cell lines exist that are representative of the five cell types present in the anterior pituitary gland. Currently, the {alpha}TSH (38) cell line, the TtT97 tumors (39), and T{alpha}T1 cell lines (40) are available as readily manipulable models of normal thyrotrope cells. The TtT97 cells were derived from hyperplastic pituitaries in hypothyroid LAF1 mice, express TSHß, and are T4 responsive. They are cumbersome, expensive, and time consuming to maintain because they must be generated from tumors that are propagated by transplantation. The {alpha}TSH cell line represents a primordial thyrotrope that does not express TSHß. While the T{alpha}T1 cell line does express TSHß and is T4 responsive, the cells express SV40 large T-antigen and are somewhat difficult to grow and transfect. As a complement to T{alpha}T1, a stable cell line that expresses TSHß and is T4 responsive would be a valuable tool for studying transcriptional control and thyroid hormone responsiveness. We have shown that thyrotrope hyperplasia in the pituitary glands of {alpha}Gsu-/- mice can result in transplantable pituitary masses of several grams. We have also demonstrated that these tumors are T4 responsive. Therefore, these mice could be used as a rich source of rapidly growing thyrotropes to generate a stable T4 responsive thyrotrope cell line. Such a cell line would be useful for studies involving fully differentiated, thyroid hormone-responsive, thyrotropes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Propagation and Genotyping Mice
Targeted disruption of {alpha}Gsu was accomplished by gene targeting, and the mice (Cgatm1, or {alpha}Gsu-) were deposited at The Jackson Laboratory (Bar Harbor, ME) (16). A colony of {alpha}Gsu+/- with mixed genetic background (129 and C57BL/6J) mice have been maintained at The University of Michigan. Due to the hypothyroid state of the {alpha}Gsu-/- mice, they remain with their parents or are housed with normal littermates to maintain the appropriate body temperature. Mice were genotyped with genomic DNA prepared from tail biopsies by PCR analysis (16). Smstr2-/- mice were obtained from Merck Research Laboratories (Rahway, NJ). Smstr2-/- were mated with {alpha}Gsu+/- mice and F1 Smstr2+/-, {alpha}Gsu+/- mice were intercrossed to generate mice heterozygous and homozygous for both Smstr2 and {alpha}Gsu. {alpha}Gsu alleles were genotyped as previously described (16). Smstr2 alleles were identified with two sets of primers amplified at 95 C for 5 min, 35 cycles of 94 C for 30 sec, 61 C for 2 min, 72 C for 2 min, followed by 72 C for 10 min. Primers used to identify the wild-type allele, for(5'-AGG TGA GGA CCA CCA CAA AG-3') and rev(5'-ATC CGG GGC TTG GTA CAC AG-3') amplified a 306-bp product. Primers used to identify the disrupted allele, for(5'-TGA CAA ATG GAA GTA GCA CGT CTC ACT AGT CT-3') and rev(5'-GAT CTC TAG GCA GCT TGG TTC T-3') amplified a 281-bp product. Thyroid hormone injections were performed for 40 d as previously described (17). All procedures using mice were approved by the University of Michigan and the University of Colorado Health Sciences Center Committee on Use and Care of Animals. All experiments were conducted in accord with the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals.

Adenomatous Hyperplasia Growth in Hypothyroid Mice
Pituitary masses from 1-yr-old {alpha}Gsu-/- mice were removed, weighed, minced in 0.9% sterile NaCl, and transplanted sc between the scapulas of hypothyroid C57BL/6 male mice. After monitoring growth of the pituitary tumor for 6 months, it was removed, weighed, and analyzed further.

MRI
In vivo magnetic resonance imaging experiments were performed on a Varian MRI system equipped with a 7 tesla, 18.3-cm horizontal bore magnet (Oxford Instruments, Concord, MA) as previously described (41). In brief, for MRI examination, mice were anesthetized with sodium pentobarbital (60–70 mg/kg, ip) and positioned within a 3-cm diameter quadrature radio-frequency coil (USA Instruments, Aurora, OH). A single-slice gradient-recalled echo image was acquired with 1-mm saturation cross-hairs imprinted on the axial and coronal images to facilitate rapid and reproducible positioning on the animal. Multislice T2-weighted sagittal images were acquired by using a spin echo sequence with the following parameters: 2.5-sec repetition time, 60-ms echo time, field of view = 25 x 25 mm using a 128 x 128 matrix, slice thickness = 0.5 mm, slice separation = 0.8 mm, number of slices = 15, and eight signal averages per phase encode step.

Pituitary volumes were obtained from the multislice magnetic resonance images by electronically outlining the region of interest using image processing software (Advanced Visual Systems, Waltham, MA) as previously described (42). The number of pixels was converted to an area on all slices, multiplied by the slice separation.

Northern Analysis
RNA isolation, purification of polyA(+) RNA, and analysis of steady-state mRNA levels were performed using methods that have been previously described (1). The cDNA encoding mouse Smstr2 was kindly provided by Dr. Graeme Bell (University of Chicago, Chicago, IL). Five micrograms of poly(A)+ RNA were separated by electrophoresis through a 0.8% agarose-6% formaldehyde gel, transferred to a Nytran filter and hybridized to a radiolabeled mouse Smstr2 cDNA probe. The autoradiograph was exposed for 5 d at -80 C with an intensifying screen.

Immunohistochemistry
Pituitary glands were removed, fixed overnight in 4% buffered paraformaldehyde, embedded in paraffin, and sectioned at 4 µm. Immunohistochemistry was performed with polyclonal antisera against rat TSHß (1:1000, no. AFP1274789, National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Biotinylated secondary antibodies were used in conjunction with avidin and biotinylated peroxidase (Vectastain rabbit kit, Vector Laboratories, Inc., Burlingame, CA) with diaminobenzidine as the chromogen. Normal serum was substituted for the primary antibody in negative controls. Sections were counterstained with Gill’s triple strength hematoxylin (Fisher Scientific, Pittsburgh, PA).


    ACKNOWLEDGMENTS
 
We would like to thank Dr. R. Andrew James, Department of Medicine, University of Newcastle (Newcastle-upon-Tyne, UK), and Novartis Pharma AG (Newcastle-upon-Tyne, UK), for their contributions during the early stages of this work; Dr. Hilary A. Wilkinson from Merck Research Laboratories (Rahway, NJ) for the Smstr2-/- mice; Tennore Ramesh, Amy Radak, Autumn Wenglikowski, and Mary Anne Potok for their help with the maintenance and genotyping of the mice; and the University of Michigan Morphology Core (Ann Arbor, MI), especially Kay Brabec, for the use of their equipment and helpful discussions.


    FOOTNOTES
 
This work was supported by NIH Grants HD-34283 (to S.A.C.), HD-30428 (to S.A.C.), R24-CA83099 (to B.D.R.), CA-47411 (to E.C.R.), and DK-36843 (to E.C.R.).

Abbreviations: {alpha}GSU, glycoprotein hormone {alpha}-subunit; MRI, magnetic resonance imaging; SMST, somatostatin; SMSTR, SMST receptor.

Received for publication April 26, 2001. Accepted for publication August 27, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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