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Department of Laboratory Medicine and Pathology (L.J., S.Z., M.E.C., E.K., R.V.L.), and Endocrine Research Unit (B.G.B.), Mayo Clinic, Rochester, Minnesota 55905; and Tokai University School of Medicine (R.Y.O.), Isehara City, Japan
Address all correspondence and requests for reprints to: Dr. Ricardo V. Lloyd, Mayo Clinic, Department of Laboratory Medicine and Pathology, 200 First Street SW, Rochester, Minnesota 55905.
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Abstract |
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Leptin was present in a small percentage of rat (4.8 ± 0.7%) and
mouse (7 ± 2%) pituitary cells. Colocalization studies with
leptin and pituitary hormones showed leptin expression mainly in TSH
cells (24 ± 2% of TSH cells in the rat pituitary and 31 ±
1% of TSH cells in the mouse pituitary). A folliculo-stellate (FS)
cell line, TtT/GF, also expressed leptin. The long isoform of leptin
receptor (OB-Rb) was present in normal pituitary and in various
pituitary cell lines, including FS, GH3, and
T3-1 cells. Treatment of GH3 and FS cells
with leptin (1 x 10-8 M) inhibited cell
proliferation assessed by [3H]thymidine incorporation in
GH3, but not in FS, cells.
These findings show for the first time that leptin is expressed in rat and mouse anterior pituitaries mainly by TSH cells and by a mouse FS cell line. The finding of leptin and of the long isoform of leptin receptor in normal rat and mouse pituitaries and in various cell lines implicates an autocrine/paracrine loop in the production and regulation of leptin and leptin receptor in the rodent pituitary.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have also implicated leptin in anterior pituitary function (7, 11, 12, 13, 14, 15, 16). Yu et al. showed that leptin controlled anterior pituitary hormone secretion (11) and stimulated nitric oxide release from the anterior pituitary (12). Zamorano et al. (13) showed that leptin receptor (OB-R) was expressed in the rat anterior pituitary and hypothalamus, whereas other investigators reported that the OB-R gene expression was increased by GH and/or GHRH (14). Leptin deficiency in humans due to a mutation associated with a truncated leptin receptor lacking both the transmembrane and intracellular domains has been associated with pituitary dysfunction (15), emphasizing the importance of this protein in pituitary function.
The leptin receptor gene is highly expressed in many tissues (16, 17, 18, 19) and is a member of the class I cytokine receptor superfamily (18). There are various isoforms of the receptor, including the full-length form, which has a cytosolic domain of 302 amino acids that acts as a STAT-signaling competent receptor (20, 21).
Our recent study in human pituitary indicated that leptin inhibited the proliferation of the HP75 human pituitary cell line and the rat GH3 cell line, both of which expressed leptin receptors (7). Various cytokines, such as interleukin-6 (IL-6) and IL-2, have been shown to regulate pituitary cell growth (21). In the present study we examined leptin and leptin receptor expression in the mouse folliculo-stellate (FS) cell line, which has been shown to be similar to normal folliculo-stellate cells, which express various cytokines (22, 23, 24, 25, 26). Because of the effects of cytokines on pituitary cell growth, we examined the role of leptin in pituitary cell proliferation in the present study.
In this report we show that leptin is produced by normal rat and mouse anterior pituitary cells and by the mouse FS cell line and that OB-Rb is also expressed in these tissues. We also show that the TSH cell is the principal cell type producing leptin in the rat and mouse pituitary and that leptin inhibits GH3, but not FS, cell proliferation.
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Materials and Methods |
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T3–1 was a gift from Dr. P. Mellon
(University of California, San Diego, CA). The mouse FS cell line was
developed in the laboratory of Dr. K. Inoue (Hiroshima,
Japan).
Cull culture
The rodent pituitary cell lines were maintained in DMEM
(Life Technologies, Inc., Gaithersburg, MD) supplemented
with 15% horse serum, 2.5% FCS, 1 µg/ml insulin, and 1%
antibiotics in a 37 C, 5% CO2 atmosphere, as
previously reported (22, 23). The cells were grown on plastic dishes,
harvested by trypsinization, and used for RNA and protein extraction.
Aliquots of cells were used for cytocentrifugation and subsequent
immunostaining.
Immunohistochemistry
Normal rat and mouse pituitaries were fixed in neutral buffered
formalin, pH 7.2, embedded in paraffin, and used for
immunohistochemistry analysis. Three antileptin antibodies were used in
this study, including a polyclonal (used at a 1:500 dilution) and a
monoclonal (1:250) antibody, both from Sigma (St. Louis,
MO), and a polyclonal antibody (1:500) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal leptin
antibody was produced using recombinant human leptin as an immunogen.
The affinity-purified polyclonal leptin antibody from
Sigma was produced from an 18-amino acid peptide from the
N-terminal domain of human and mouse leptin (amino acid residues
22–40). The polyclonal leptin antibody from Santa Cruz Biotechnology, Inc., was produced from a peptide from the
carboxyl-terminus of human leptin. Polyclonal antisera to rat pituitary
hormones PRL (used at a 1:1000 dilution), GH (1:5000), TSH (1:1000), LH
(1:500), and FSH (1:500) were obtained from the National Pituitary
Agency (Baltimore, MD). ACTH (1:1000) and S100 (1:500) antisera were
purchased from DAKO Corp. (Carpinteria, CA). S100 protein
immunostaining was used to identify FS cells. Immunostaining and
colocalization studies were performed using the
avidin-biotin-peroxidase or alkaline phosphatase methods (Vector Kit,
Vector Laboratories, Inc., Burlingame, CA) as previously
reported (7, 28). For colocalization studies on rat pituitaries,
antileptin monoclonal antibody with an avidin-alkaline
phosphatase-nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate
kit was used, followed by immunostaining with various pituitary hormone
polyclonal antibodies with the
avidin-biotin-peroxidase-diaminobenzidine kit. Polyclonal antibody to
leptin was used for mouse pituitaries and colocalized with TSH, FSH,
and LH monoclonal antibodies (1/2000, 1/800, and 1/800, respectively)
from DAKO Corp. Before incubation with primary antibodies,
slides were microwaved for 15 min in 10 mmol/liter citric acid (pH 6.0)
for antigen retrieval. Absorption controls using purified leptin
(Eli Lilly & Co., Indianapolis, IN) at 10 and 50 µg/ml
were performed for leptin immunostaining studies.
Immunoblot analysis
Immunoblot analysis was performed as previously reported (27, 29), using proteins from rodent pituitaries and from the
GH3 and FS cell lines. One-dimensional SDS-PAGE
was performed with a ready to use 10–20% gradient gel using the
discontinuous buffer system of Laemmli (Bio-Rad Laboratories, Inc., Richmond, CA). The electrophoresed proteins were
transferred to a polyvinylidene difluoride membrane and subjected to
immunoblot analysis with a monoclonal leptin antibody (used at a 1:1000
dilution) from Sigma. A recombinant rat leptin protein (50
ng; Sigma) was used as a positive control. The separated
leptin protein was detected with enhanced chemiluminescence
(Amersham Pharmacia Biotech, Arlington Heights, IL). To
determine whether equal amounts of proteins were added to the gels, the
membranes were reblotted with a ß-actin monoclonal antibody (1:2500;
from Sigma).
RT-PCR
Total RNA from rodent pituitary cell lines was extracted with
the TRIzol reagent kit (Life Technologies, Inc.) and used
for analysis of leptin and leptin receptor isoforms, OB-Ra, and OB-Rb
(OB-R) messenger RNAs (mRNAs) by RT-PCR. The sequences of primers and
hybridization probes were made based on published sequences (1, 30, 31)
and are shown in Table 1
. Additional primers
for rat leptin were used as previously reported (4). The housekeeping
gene hypoxanthine phosphoribosyl transferase (HPRT) was used as an
internal control. The RT-PCR reaction was performed as previously
described (7, 32). Forty cycles of PCR amplification were used with 57
C annealing temperature for OB-Ra and OB-Rb and 54 C annealing
temperature for OB. The PCR products were analyzed by 2% agarose gel
electrophoresis with ethidium bromide staining and Southern
hybridization as previously reported (7, 22, 23). All primers spanned
at least one intron. Negative controls consisted of omitting the
reverse transcriptase for each sample, which resulted in no bands after
RT-PCR and Southern hybridization. At least two independent RT-PCR
experiments were performed, which resulted in similar findings. The PCR
reactions were shown to be within the linear range by using different
volumes of complementary DNA samples for PCR followed by Southern
hybridization and densitometric analysis.
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Quantitation
A minimum of 2000 cells from 10 fields of each slide were
enumerated after leptin immunostaining, and the results were expressed
as the percentage of positive cells. For leptin and pituitary hormone
colocalization by immunostaining, 200–500 hormone-positive cells were
enumerated, and leptin-positive cells were expressed as a percentage of
each type of hormone-producing cell. Student’s t test and
ANOVA were used for statistical analysis. The results are expressed as
the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cell lines
along with mouse and rat adipose tissues as a 244-bp band (Fig. 1A). Southern hybridization with an internal
probe confirmed the identity of the amplified band (Fig. 1A). Leptin
mRNA was also detected in the normal rat pituitary using previously
published primers (4), which resulted in a 420-bp band (Fig. 1B).
Southern hybridization with an internal probe confirmed the identity of
the amplified band (Fig. 1B). A weak band of leptin was detected in
GH3 cells, which was considerably weaker than
that in adipose tissue or normal rat pituitary (Fig. 1, A and B).
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). Colocalization
studies were performed to determine the anterior pituitary cell types
expressing leptin (Fig. 2). In the rat pituitary, TSH cells (24 ±
2%) were the major cell type colocalized with leptin, whereas a
smaller percentage of LH (4 ± 1%) and FSH (3 ± 0.5%)
cells expressed leptin. Less than 1% of ACTH, GH, PRL, and FS cells
were positive by colocalization studies. In the mouse pituitary gland,
31 ± 1% of TSH cells expressed leptin by colocalization studies,
and a small percentage of LH and FSH cells were also positive for
leptin by colocalization. Immunostaining of cell lines showed positive
staining for leptin in FS cells (Fig. 2), but this was undetectable in
GH3 or T3–1.cells.
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).
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T3–1 cell lines as 389- and 302-bp bands,
respectively. Southern hybridization confirmed the RT-PCR finding (Fig. 4).
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, A and B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The FS cell line, TtT/GF, was developed by Inoue et al. from a transplantable thyrotroph mouse tumor (22, 23, 24) and has been shown to have many features in common with the FS cells of the normal anterior pituitary gland. Our observations of leptin production by this cell line and by FS cells in human pituitary (7) support this observation.
The observation of differences in the types of anterior pituitary cells expressing leptin in human and rodent pituitary is not unique for this peptide. Although IL-6 is produced by FS cells in rat and mouse pituitary glands (25, 26), other cell types in human pituitary adenomas are the sources of IL-6 (33, 34). Another peptide, galanin, is present in PRL cells in female rats and in ACTH cells in human pituitaries (35, 36), suggesting different regulatory functions of this peptide in the pituitary during evolutionary development of the gland.
Although detection of leptin receptor in the normal rat and mouse
pituitary confirms earlier findings by others (13, 14), the leptin
receptor isoforms have not been previously analyzed in the
T3–1 and FS cell lines. The presence of OB-Rb
indicates that these cells express the functional receptor and are
capable of responding to leptin (11, 12, 13, 14).
Leptin had an inhibitory effect on GH3 cell, but not FS cell, proliferation. This difference was not related to the presence of leptin receptor, because both GH3 and FS cells expressed leptin receptor mRNA. The inhibition of pituitary cell growth by leptin may be related in part to the secretory activity of some pituitary cells. Various studies have shown that leptin regulates the secretion of pituitary hormones, including that of FSH, LH, and GH (11, 12). A recent study in which leptin was targeted to the regulated secretory pathway in pituitary AtT-20 cells showed that leptin behaved like a regulated protein in cells with a biosynthetic regulated secretory pathway (37). Because FS cells do not have secretory granules (22, 23, 24), the method of leptin secretion by these cells is probably different from that of normal anterior pituitary cells. Ultrastructural immunolocalization studies with leptin antibodies in anterior pituitary cells should provide more information about the ultrastructural localization of leptin, some of which may be associated with secretory granules based on the AtT-20 cell study.
Various studies have documented cytokine production by secretory anterior pituitary cells and by FS cells (25, 26, 33, 34). ILs, such as IL-1, IL-2, and IL-6, usually have an inhibitory effect on normal anterior pituitary cell proliferation, which is similar to the effects of leptin on GH3 cells in this study. However, some ILs can also stimulate cell growth (38). For example, IL-6 inhibits normal rat pituitary cell growth, but stimulates the growth of GH3 pituitary cells (38). The mechanisms by which leptin inhibits the growth of some pituitary cells, such as GH3, while not affecting others, such as the FS cell line, and the relationship to pituitary cell function are unknown, but can be readily investigated in vitro with the use as the GH3 and TtT/GF cell lines. We are currently undertaking these studies.
Putative roles of leptin in the pituitary gland would include paracrine and autocrine regulatory roles for hormone secretion and differentiation, because the leptin receptor as well as leptin are expressed by pituitary cells. Leptin may also function in the regulation of the hypothalamic-pituitary-endocrine end-organ-leptin axis, as a growing number of studies show an important role for leptin in the glucocorticoid, GH, and other hypothalamic-pituitary-end-organ axes (39, 40, 41, 42).
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Acknowledgments |
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T3-1 cell
line, and Dr. K. Inoue for the FS cell line. |
Footnotes |
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Received June 16, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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