1 Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94141-1900,
USA
2 Cardiovascular Research Institute, University of California, San Francisco, CA
94143, USA
3 Department of Anatomy, University of California, San Francisco, CA 94143,
USA
4 Department of Medicine, University of California, San Francisco, CA 94143,
USA
5 Laboratory of Genetics and Physiology, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
20892,USA
Author for correspondence (e-mail:
scases{at}gladstone.ucsf.edu)
Accepted 8 March 2004
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SUMMARY |
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Key words: Mammary gland, Acyl CoA:diacylglycerol acyl transferase, Lipid, Triacylglycerol
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Introduction |
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There is increasing evidence that interactions between the mammary
epithelium and surrounding stroma are crucial for normal mammary gland
development. The stroma is a complex tissue composed of myoepithelial cells,
extracellular matrix components within the basement membrane, fibroblasts, and
adipocytes. Several studies showed that extracellular matrix components
(Wiseman and Werb, 2002) and
stromal fibroblasts (Darcy et al.,
2000
; Lukashev and Werb,
1998
) can influence epithelial development. However, the role of
adipocytes, the most abundant cell type in the stroma, in mammary gland
development is poorly understood. Mammary gland adipocytes have been
hypothesized to serve as a source of growth factors and lipids that may
influence mammary epithelium development and function
(Hovey et al., 1999
).
Co-culture of mammary epithelial cells with 3T3L1-derived adipocytes
(Levine and Stockdale, 1985
;
Wiens et al., 1987
), or with
isolated rat mammary adipocytes (Zangani
et al., 1999
), enhances differentiation and milk protein
expression. In mice that lack adipose tissue, the proliferation of mammary
epithelium is impaired, although differentiation proceeds normally
(Couldrey et al., 2002
).
Stromal adipocytes synthesize and store large amounts of triglycerides,
which may serve as reservoirs of substrates for milk production by the mammary
epithelium. During this process, adipocyte triglycerides must be hydrolyzed
and the fatty acids transferred to the epithelial cells for re-esterification.
Triglyceride synthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase
(DGAT) enzymes, which covalently join diacylglycerol with fatty acyl CoA
(Bell and Coleman, 1980;
Brindley, 1991
;
Lehner and Kuksis, 1996
).
Studies from our group have identified genes encoding two mammalian DGAT
enzymes, DGAT1 and DGAT2 (Cases et al.,
1998
; Cases et al.,
2001
). The Dgat1 gene is expressed in nearly all tissues,
including the mammary glands (Farese et
al., 2000
). Mice lacking DGAT1
(Dgat1/) have decreased triglyceride content
in tissues, and are resistant to diet-induced obesity and diabetes mellitus
(Chen et al., 2002
;
Smith et al., 2000
). In
addition, female Dgat1/ mice are unable to
lactate (Smith et al., 2000
).
This result was surprising because other mouse knockout models that are
deficient in protein components of milk are capable of producing milk with an
altered composition. For example,
-lactalbumin-deficient mice
(Stacey et al., 1994
;
Stinnakre et al., 1994
)
produce highly viscous milk containing little or no lactose, and
ß-casein-deficient mice (Kumar et
al., 1994
) produce milk with a reduced total protein
concentration. This suggests that the lactation defect in
Dgat1/ mice might result from impaired
mammary gland development.
In the present study, we characterized mammary gland development in Dgat1/ mice at different stages, and used tissue transplantation techniques to determine whether the expression of DGAT1 was required in extraglandular, stromal and epithelial compartments. Our results indicate that DGAT1 plays important functional roles in both the mammary epithelial and stromal compartments during pregnancy.
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Materials and methods |
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Antibodies
Mouse monoclonal antibodies for E-cadherin, smooth muscle actin and
ß-catenin were from Transduction Laboratories and Pharmingen (San Diego,
CA). Rabbit polyclonal antibody for STAT5A (L-20) and mouse monoclonal
antibody for phosphotyrosine (PY99) were from Santa Cruz Biotechnology (Santa
Cruz, CA). The polyclonal antibodies for NKCC1 (sodiumpotassium-chloride
co-transporter characteristic for ductal epithelium), NPT2B (sodium-phosphate
co-transporter isoform characteristic for lactating epithelium) and WAP have
been described (Miyoshi et al.,
2001; Shamay et al.,
1992
).
Histological and whole-mount analyses
For histology, inguinal mammary glands were removed from wild-type and
Dgat1/ mice on day 18 of pregnancy (P18) or
day 1 of lactation (L1), fixed in 4% paraformaldehyde/phosphate-buffered
saline (PBS) at 4°C overnight, washed for 15 minutes in PBS and embedded
in paraffin. Tissue sections (6 µm) were stained with Hematoxylin and
Eosin, and mounted on pre-treated glass slides (Superfrost Plus Micro Slides,
VWR).
For whole-mount analyses, mammary glands (3 or 4) were removed at different times during pregnancy and immediately spread onto glass slides. The glands were then immersed in Carnoys fixative (3:1, ethanol:glacial acetic acid) overnight, washed in 70% ethanol for 15 minutes, washed under running tap water for 5 minutes, drained and then stained overnight with Alum Carmine stain [2 g/l carmine dye (C6152, Sigma, St Louis, MO) and 5 g/l aluminum potassium sulfate in distilled water]. The glands were then dehydrated by three 15-minute immersions in 70%, 90% and 100% ethanol, and the lipids were removed by a 15-minute immersion in xylenes. The tissues were stored in methyl salicylate (Sigma).
For neutral lipid staining, inguinal mammary glands were fixed with 2% glutaraldehyde/2% paraformaldehyde, stained in 1% osmium tetroxide in sodium phosphate buffer (0.1 M, pH 7.4), dehydrated in a series of ethanols, transitioned into propylene oxide, and embedded in Epon resin. Sections (1 µm) were cut with a glass knife and stained in warm Toluidine Blue.
Image analysis
For quantification of epithelial areas, images were captured with a SPOT
digital camera (Diagnostic Instruments, Sterling Heights, MI) and converted
into a binary format with Adobe Photoshop 5.0.1 (Adobe Systems, San Jose, CA)
and the Image Processing Tool Kit (Reindeer Games, Gainesville, FL). The
binary black and white images were compared with the original images to ensure
an accurate conversion. Areas (mm2) were calculated with the
command Measure All, and epithelial and lumen areas were then
expressed as percentage of total area.
Immunohistochemistry and analysis of proliferation and apoptosis
NKCC1, WAP and NPT2B were detected as described
(Shillingford et al., 2003;
Shillingford et al., 2002
).
Briefly, paraffin-embedded mammary tissue sections were cleared in xylene and
rehydrated. Antigen was retrieved by heat treatment using an antigen-masking
solution (1:100 dilution; Vector Laboratories, Burlingame, CA), and tissue
sections were blocked for 30 minutes in PBS with 0.05% Tween-20 (PBST)
containing 3% goat serum.
Sections were incubated with solutions containing various combinations of diluted primary antibodies for smooth muscle actin (1:1000), NKCC1 (1:1000), ß-catenin (1:100), WAP (1:600), NPT2B (1:200) and E-cadherin (1:100). After 30 minutes at 37°C, the slides were rinsed in PBST to remove nonspecific antibody binding. The primary antibodies were detected by incubation with either anti-mouse FITC-conjugated (1:400) or anti-rabbit Texas Red-conjugated (1:400) secondary antibodies (Molecular Probes, Eugene, OR) for 30 minutes at room temperature in the dark. Sections were then washed twice in PBST and mounted in Vectashield (Vector Laboratories), and fluorescence was visualized with an Olympus BX51 microscope equipped with DAPI, FITC, TRITC and FITC:TRITC filters. Images were captured with a Nikon 1200DXM digital camera (Nikon) and composed in Adobe PhotoShop. Three mice of each genotype were analyzed.
For analysis of cellular proliferation, mice at P15 were injected with bromodeoxyuridine (BrdU) (Amersham Biosciences, Piscataway, NJ) (1 mg/10 g body weight) 2 hours before dissection of mammary glands (4). After paraffin embedding and sectioning of glands, BrdU was detected with a biotinylated mouse anti-BrdU antibody (Zymed Laboratories, South San Francisco, CA) according to the manufacturers instructions. Images of the sections were captured, and epithelial areas were determined as described earlier in Image analysis. Positive nuclei were counted in five random fields per mammary gland and their number was expressed per unit of epithelial area. Four to five mice of each genotype were analyzed.
For analysis of apoptosis, mammary glands were dissected at P18, and sections were prepared as described above. Apoptotic cells were detected by terminal deoxynucleotide UTP nick-end labeling (TUNEL), using an in situ cell death detection kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturers instructions. Nuclei were counterstained with DAPI. TUNEL-positive epithelial nuclei were counted in 10 random microscopic fields, and expressed as a percentage of DAPI-stained nuclei. Four mice of each genotype were analyzed.
RNA analyses
Total RNA from wild-type and Dgat1/
mammary glands from virgin or pregnant mice was extracted with Trizol
(Invitrogen, Carlsbad, CA). For northern blots, samples of total RNA (10
µg) were separated by gel electrophoresis, transferred to membranes, and
hybridized with 1 ng of [-32P]dATP-labeled oligonucleotide
probes in 0.4 M NaCl, 1% SDS, 1xDenhardts solution, and salmon
sperm DNA. Membranes were washed twice for 15 minutes in 1xSSC and 0.1%
SDS, and twice for 15 minutes each in 0.2xSSC and 0.1% SDS. Membranes
were stripped and rehybridized with a 655-bp
[
-32P]dCTP-labeled DNA probe for mouse keratin 18 (K18). WAP
oligonucleotide was 5'-CAACGCATGGTACCGGTGTCA-3', and ß-casein
oligonucleotide was 5'-GTCTCTCTTGCAAGAGCAAGGGCC-3'. The template
for synthesizing the K18 probe was obtained by amplifying mouse mammary cDNA
with the following primers: sense, 5'-AGACCGAGAAAGAGACCATGCAAG-3';
antisense, 5'-GAGACCACACTCACGGAGCTGA-3'.
Immunoblot analysis and immunoprecipitation
Mammary glands were homogenized in lysis buffer (5 ml/g of tissue),
containing 40 mM Tris-HCl (pH 7.5), 275 mM NaCl, 20% glycerol, 2% Nonidet
P-40, 4 mM EDTA, and a cocktail of protease and phosphatase inhibitors
(Calbiochem, San Diego, CA). Lysate (500 µl) containing 1.5 mg total
protein was used for immunoprecipitation for 1 hour at 4°C with 1 µl of
rabbit polyclonal anti-STAT5A antibody. Immunocomplexes were captured
overnight with 50 µl of protein A-Sepharose beads (Pharmacia Biotech,
Piscataway, NJ), washed three times with lysis buffer, and resuspended in
2xsample buffer (Bio-Rad). Samples were boiled for 5 minutes,
centrifuged briefly, and analyzed by gel electrophoresis using 10% SDS-PAGE
pre-cast gels (Bio-Rad). Separated proteins were transferred to polyvinylidene
difluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 5%
milk in PBS for 1 hour at room temperature (RT), incubated with anti-STAT5A
(1:500) or mouse monoclonal anti-phosphotyrosine (PY99, 1:500) antibodies for
2 hours at room temperature, and washed three times for 15 minutes each with
0.05% Tween-20 in PBS. The blots were then incubated for 1 hour at room
temperature with horseradish peroxidase-conjugated rabbit IgG (1:2000), washed
three times for 5 minutes each with 0.05% Tween-20 in PBS, incubated for 1
minute with enhanced chemiluminescence substrate (Pierce Biotechnology,
Rockford, IL) and exposed to X-ray film.
Mammary tissue transplantations
For whole mammary gland transplantations, the inguinal mammary glands from
8- to 10-week-old mice were removed and transplanted into the subcutaneous
space in the interscapular region of 8- to 10-week-old recipient female mice,
in a manner similar to that described for transplantation of the reproductive
fat pad (Chen et al., 2003;
Gavrilova et al., 2000
). Two
weeks after surgery, the recipient mice were mated. Endogenous and
transplanted mammary glands were removed at L1 for histological analyses.
Mammary epithelial transplantations were performed essentially as described
(Young, 2000). The nipple
region of a mammary gland (4) containing epithelial structures was removed
under anesthesia from 21-day-old mice, and the cleared fat pad was immediately
implanted with a small piece (0.5-1.0 mm diameter) of donor mammary gland
containing epithelium from 8- to 10-week-old virgin mice. To assess complete
clearing of the recipient mammary fat pad, the surgically removed mammary
tissue was used for whole-mount staining. The contralateral mammary fat pad
served as an unoperated control. Transplanted mice were mated 5-7 weeks after
surgery. Recipient unoperated control and transplanted mammary glands were
removed at L1 for histological analyses.
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Results |
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Impaired mammary gland development during pregnancy in Dgat1/ mice
Defective milk production can result from various developmental and
functional abnormalities in the mammary gland. To investigate the cause of the
lactation defect in Dgat1/ mice, we first
examined the morphology of the mammary ductal tree in virgin mice. Whole-mount
analyses (not shown) and immunostaining for the sodium-potassiumchloride
co-transporter NKCC1 (Fig. 1A), a marker for ductal epithelial cells, revealed no differences in mammary
structures in wild-type and Dgat1/ virgin
mice. No gross abnormalities were observed in the mammary fat pads, and
Dgat1/ mammary adipocytes appeared
morphologically normal as assessed by histological techniques. The weights of
Dgat1/ mammary fat pads were similar to
those of wild-type mice during pregnancy, but were reduced at L1
(Fig. 1B), probably reflecting
their lack of milk production. These data suggest that the impairment in
development occurred during pregnancy.
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DGAT1 is required in both the mammary epithelial and extraepithelial tissues for normal development
The defect in lobuloalveolar development in pregnant
Dgat1/ mice could be due to the lack of
DGAT1 function in the mammary gland or to changes in systemic factors. To
differentiate between these possibilities, we transplanted whole mammary
glands into the interscapular region of recipient mice and analyzed the
endogenous control mammary glands and the transplants at L1
(Fig. 6). Twenty mice received
transplants during the study, and 18 had viable grafts, as judged by visual
inspection at sacrifice and by histology. In control experiments, wild-type
endogenous glands (Fig. 6A)
were indistinguishable from wild-type transplants
(Fig. 6B). The endogenous
Dgat1/ mammary glands were underdeveloped
and lacked milk secretion (Fig.
6C), whereas wild-type mammary glands transplanted into
Dgat1/ recipient mice exhibited fully
developed lobuloalveolar structures and visible milk droplets
(Fig. 6D). Conversely,
Dgat1/ mammary glands transplanted into
Dgat1+/+ recipient mice failed to develop normally
(Fig. 6F). These results
suggest that local DGAT1 expression in the mammary gland is sufficient for
normal mammary gland development.
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Discussion |
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Our whole-gland transplantation studies suggested that the requirement for DGAT1 in mammary gland development is restricted to the mammary gland. Wild-type mammary glands transplanted into Dgat1/ mice developed normally, whereas Dgat1/ mammary glands transplanted into Dgat1+/+ mice did not. These findings indicate that systemic or hormonal factors in the recipient mice did not appreciably affect the phenotype of the transplanted gland. Thus, it is unlikely that deficiencies in the plasma hormones prolactin, estrogen or progesterone contribute to the impaired mammary gland development in Dgat1/ mice. In support of this conclusion, Dgat1/ female mice have normal reproduction, which is also dependent on these hormones.
The epithelial transplantation experiments suggested that DGAT1 is required in the extraepithelial compartment for normal alveolar growth and development. The transplantation of Dgat1/ epithelium to wild-type mammary fat pads resulted in apparently normal epithelial growth, whereas the transplantation of wild-type epithelium to Dgat1/ fat pads resulted in an underdeveloped and poorly differentiated epithelium. Thus, the presence or absence of DGAT1 in the extraepithelial compartment had a profound effect on epithelial development.
The extraepithelial stroma consists of many different cell types. In which
type of cell is DGAT1 function required? Because Dgat1 is highly
expressed in adipocytes (Cases et al.,
1998), the most abundant cell type in the mammary stroma, it is
likely that DGAT1 deficiency in adipocytes contributes to the defect in
epithelial development. An attractive hypothesis is that DGAT1 deficiency
alters the secretion of factors from mammary adipose tissue that are essential
for epithelial development. In support of this hypothesis, previous studies
from our laboratory indicated that DGAT1 deficiency alters the endocrine
function of white adipose tissue (WAT). In these studies, the transplantation
of 500 mg of Dgat1/ WAT into wild-type
recipients conveyed partial protection from diet-induced obesity and diabetes
(Chen et al., 2003
). Although
the secreted factors responsible for these effects have not been identified, a
candidate factor is adiponectin, which is secreted at twofold higher levels
from Dgat1/ WAT in obesity models
(Chen et al., 2003
).
Adiponectin, also known as Acrp30 (Scherer
et al., 1995
) and Acdc (Mouse Genome Informatics), is abundant in
the plasma, and enhances fatty acid oxidation and insulin sensitivity in
tissues (Hu et al., 1996
;
Maeda et al., 1996
;
Scherer et al., 1995
). It is
unknown whether increased secretion of adiponectin from adjacent WAT could
impair mammary development. However, adiponectin inhibits the proliferation of
smooth muscle cells (Matsuda et al.,
2002
), and adiponectin levels normally fall during pregnancy
(Combs et al., 2003
).
Another possibility is that DGAT1 deficiency in the mammary stroma impairs
signaling of hormones or stromal factors required for epithelial development
(Hovey et al., 1999). For
example, some effects of hormones that stimulate the mammary gland, such as
prolactin (Brisken et al.,
1999
) and growth hormone
(Gallego et al., 2001
;
Walden et al., 1998
), are
mediated by actions in the surrounding fat pad instead of targeting epithelial
cells directly. In addition, several factors that regulate growth of the
mammary epithelium are synthesized by the local fat pad, including epidermal
growth factor receptor (Wiesen et al.,
1999
), inhibins (Robinson and
Hennighausen, 1997
), and insulin-like growth factors 1 and 2
(reviewed by Hovey et al.,
1999
; Plath-Gabler et al.,
2001
; Walden et al.,
1998
). We speculate that DGAT1 deficiency in stromal cells alters
levels of lipids, such as DGAT1 substrates (fatty acyl CoAs and
diacylglycerol), or their related metabolites in the epithelial environment,
which in turn results in altered signaling for cellular proliferation and
differentiation. In support of this hypothesis, several in vitro studies have
demonstrated that specific fatty acids can modulate mammary epithelial cell
growth and functional differentiation. For instance, oleate and linoleate can
modulate functional differentiation of rat mammary epithelium in vitro, as
assessed by casein accumulation (Sigurdson
and Ip, 1993
). In addition, the trace fatty acid conjugated
linoleic acid has been shown to inhibit mammary epithelial proliferation both
in vitro (Ip et al., 1999
) and
in vivo (Ip et al., 2001
).
Our studies also show that DGAT1 function is required in the mammary epithelium for it to undergo functional differentiation. Although Dgat1/ mammary glands initiated lactogenic differentiation, as shown by the induction of ß-casein expression, they were unable to develop a secretory phenotype at the end of pregnancy. The expression of the markers of functional differentiation, WAP and NPT2B, was reduced, and milk droplets were not present. In addition, the epithelial transplantation experiments demonstrated a role for DGAT1 within the mammary epithelium. Although Dgat1/ epithelium transplanted into wild-type fat pads grew normally, terminal events of differentiation did not occur. These findings suggest that DGAT1 deficiency in the mammary epithelial cells directly impairs intracellular processes that are required for functional differentiation.
The results indicate that DGAT2, a second DGAT that is also expressed in
the WAT and mammary gland (Cases et al.,
2001), is unable to compensate for the lack of DGAT1. Similarly,
DGAT1 is unable to compensate for the lack of DGAT2
(Stone et al., 2004
). Taken
together, these results suggest that although the two DGAT enzymes catalyze
the same biochemical reaction, they have distinct functions in vivo.
Lactation failure and impaired mammary gland development during pregnancy have been observed in several knockout mouse models, and these models have enhanced our understanding of the roles of hormones and growth factors on mammary epithelium growth and differentiation. Our study adds DGAT1, a lipid-synthesis enzyme, to the list of factors that are essential for normal mammary gland development. In addition, our study is the first to show that defects in lipid metabolism in the stroma can impair normal development of the mammary epithelium.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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---|
Alexander, C. M., Howard, E. W., Bissell, M. J. and Werb, Z. (1996). Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene. J. Cell Biol. 135,1669 -1677.[Abstract]
Bell, R. M. and Coleman, R. A. (1980). Enzymes of glycerolipid synthesis in eukaryotes. Annu. Rev. Biochem. 49,459 -487.[CrossRef][Medline]
Brindley, D. N. (1991). Metabolism of triacylglycerols. In Biochemistry of Lipids, Lipoproteins and Membranes (ed. D. E. Vance and J. E. Vance), pp.171 -203. Amsterdam: Elsevier.
Brisken, C., Kaur, S., Chavarria, T. E., Binart, N., Sutherland, R. L., Weinberg, R. A., Kelly, P. A. and Ormandy, C. J. (1999). Prolactin controls mammary gland development via direct and indirect mechanisms. Dev. Biol. 210,96 -106.[CrossRef][Medline]
Cases, S., Smith, S. J., Zheng, Y.-W., Myers, H. M., Lear, S.
R., Sande, E., Novak, S., Collins, C., Welch, C. B., Lusis, A. J. et al.
(1998). Identification of a gene encoding an acyl
CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis.
Proc. Natl. Acad. Sci. USA
95,13018
-13023.
Cases, S., Stone, S. J., Zhou, P., Yen, E., Tow, B., Lardizabal,
K. D., Voelker, T. and Farese, R. V., Jr (2001). Cloning of
DGAT2, a second mammalian diacylglycerol acyltransferase, and related family
members. J. Biol. Chem.
276,38870
-38876.
Chen, H. C., Smith, S. J., Ladha, Z., Jensen, D. R., Ferreira,
L. D., Pulawa, L. K., McGuire, J. G., Pitas, R. E., Eckel, R. H. and Farese,
R. V., Jr (2002). Increased insulin and leptin sensitivity in
mice lacking acyl CoA:diacylglycerol acyltransferase 1. J. Clin.
Invest. 109,1049
-1055.
Chen, H. C., Jensen, D. R., Myers, H. M., Eckel, R. H. and
Farese, R. V., Jr (2003). Obesity resistance and enhanced
glucose metabolismin mice transplanted with white adipose tissue lacking acyl
CoA:diacylglycerol acyltransferase 1. J. Clin. Invest.
111,1715
-1722.
Combs, T. P., Berg, A. H., Rajala, M. W., Klebanov, S., Iyengar,
P., Jimenez-Chillaron, J. C., Patti, M. E., Klein, S. L., Weinstein, R. S. and
Scherer, P. E. (2003). Sexual differentiation, pregnancy,
calorie restriction, and aging affect the adipocyte-specific secretory protein
adiponectin. Diabetes
52,268
-276.
Couldrey, C., Moitra, J., Vinson, C., Anver, M., Nagashima, K. and Green, J. (2002). Adipose tissue: a vital in vivo role in mammary gland development but not differentiation. Dev. Dyn. 223,459 -468.[CrossRef][Medline]
Darcy, K. M., Zangani, D., Shea-Eaton, W., Shoemaker, S. F., Lee, P. P., Mead, L. H., Mudipalli, A., Megan, R. and Ip, M. M. (2000). Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells. In Vitro Cell. Dev. Biol. Anim. 36,578 -592.[CrossRef][Medline]
Dupont, J., Renou, J. P., Shani, M., Hennighausen, L. and
LeRoith, D. (2002). PTEN overexpression suppresses
proliferation and differentiation and enhances apoptosis of the mouse mammary
epithelium. J. Clin. Invest.
110,815
-825.
Farese, R. V., Jr, Cases, S. and Smith, S. J. (2000). Triglyceride synthesis: insights from the cloning of diacylglycerol acyltransferase. Curr. Opin. Lipidol. 11,229 -234.[CrossRef][Medline]
Gallego, M. I., Binart, N., Robinson, G. W., Okagaki, R., Coschigano, K. T., Perry, J., Kopchick, J. J., Oka, T., Kelly, P. A. and Hennighausen, L. (2001). Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects. Dev. Biol. Stand. 229,163 -175.[CrossRef]
Gavrilova, O., Marcus-Samuels, B., Graham, D., Kim, J. K.,
Shulman, G. I., Castle, A. L., Vinson, C., Eckhaus, M. and Reitman, M. L.
(2000). Surgical implantation of adipose tissue reverses diabetes
in lipoatrophic mice. J. Clin. Invest.
105,271
-278.
Hovey, R. C., McFadden, T. B. and Akers, R. M. (1999). Regulation of mammary gland growth and morphogenesis by the mammary fat pad: a species comparison. J. Mammary Gland Biol. Neoplasia 4,53 -68.[CrossRef][Medline]
Hu, E., Liang, P. and Spiegelman, B. M. (1996).
AdipoQ is a novel adipose-specific gene dysregulated in obesity. J.
Biol. Chem. 271,10697
-10703.
Ip, C., Dong, Y., Thompson, H. J., Bauman, D. E. and Ip, M. M. (2001). Control of rat mammary epithelium proliferation by conjugated linoleic acid. Nutr. Cancer 39,233 -238.[CrossRef][Medline]
Ip, M. M., Masso-Welch, P. A., Shoemaker, S. F., Shea-Eaton, W. K. and Ip, C. (1999). Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture. Exp. Cell Res. 250, 22-34.[CrossRef][Medline]
Kumar, S., Clarke, A. R., Hooper, M. L., Horne, D. S., Law, A. J. R., Leaver, J., Springbett, A., Stevenson, E. and Simons, J. P. (1994). Milk composition and lactation of ß-casein-deficient mice. Proc. Natl. Acad. Sci. USA 91,6138 -6142.[Abstract]
Lehner, R. and Kuksis, A. (1996). Biosynthesis of triacylglycerols. Prog. Lipid Res. 35,169 -201.[CrossRef][Medline]
Levine, J. F. and Stockdale, F. E. (1985). Cell-cell interactions promote mammary epithelial cell differentiation. J. Cell Biol. 100,1415 -1422.[Abstract]
Liu, X., Robinson, G. W., Wagner, K.-U., Garrett, L., Wynshaw-Boris, A. and Hennighausen, L. (1997). Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 11,179 -186.[Abstract]
Lukashev, M. E. and Werb, Z. (1998). ECM signalling: orchestrating cell behaviour and misbehaviour. Trends Cell Biol. 8,437 -441.[CrossRef][Medline]
Maeda, K., Okubo, K., Shimomura, I., Funahashi, T., Matsuzawa, Y. and Matsubara, K. (1996). cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1). Biochem. Biophys. Res. Commun. 221,286 -289.[CrossRef][Medline]
Matsuda, M., Shimomura, I., Sata, M., Arita, Y., Nishida, M.,
Maeda, N., Kumada, M., Okamoto, Y., Nagaretani, H., Nishizawa, H. et al.
(2002). Role of adiponectin in preventing vascular stenosis. The
missing link of adipo-vascular axis. J. Biol. Chem.
277,37487
-37491.
Miyoshi, K., Shillingford, J. M., Smith, G. H., Grimm, S. L.,
Wagner, K. U., Oka, T., Rosen, J. M., Robinson, G. W. and Hennighausen, L.
(2001). Signal transducer and activator of transcription (Stat) 5
controls the proliferation and differentiation of mammary alveolar epithelium.
J. Cell Biol. 155,531
-542.
Plath-Gabler, A., Gabler, C., Sinowatz, F., Berisha, B. and
Schams, D. (2001). The expression of the IGF family and GH
receptor in the bovine mammary gland. J. Endocrinol.
168, 39-48.
Robinson, G. W. and Hennighausen, L. (1997).
Inhibins and activins regulate mammary epithelial cell differentiation through
mesenchymalepithelial interactions. Development
124,2701
-2708.
Robinson, G. W., McKnight, R. A., Smith, G. H. and Hennighausen,
L. (1995). Mammary epithelial cells undergo secretory
differentiation in cycling virgins but require pregnancy for the establishment
of terminal differentiation. Development
121,2079
-2090.
Scherer, P. E., Williams, S., Fogliano, M., Baldini, G. and
Lodish, H. F. (1995). A novel serum protein similar to C1q,
produced exclusively in adipocytes. J. Biol. Chem.
270,26746
-26749.
Shamay, A., Pursel, V. G., Wilkinson, E., Wall, R. J. and Hennighausen, L. (1992). Expression of the whey acidic protein in transgenic pigs impairs mammary development. Transgenic Res. 1,124 -132.[Medline]
Shillingford, J. M., Miyoshi, K., Robinson, G. W., Grimm, S. L.,
Rosen, J. M., Neubauer, H., Pfeffer, K. and Hennighausen, L.
(2002). Jak2 is an essential tyrosine kinase involved in
pregnancy-mediated development of mammary secretory epithelium.
Mol. Endocrinol. 16,563
-570.
Shillingford, J. M., Miyoshi, K., Robinson, G. W., Bierie, B.,
Cao, Y., Karin, M. and Hennighausen, L. (2003). Proteotyping
of mammary tissue from transgenic and gene knockout mice with
immunohistochemical markers: a tool to define developmental lesions.
J. Histochem. Cytochem.
51,555
-565.
Sigurdson, S. L. and Ip, M. M. (1993). Casein accumulation by rat mammary epithelial cells grown within a reconstituted basement membrane is modulated by fatty acids in a hormone- and time-dependent manner. Exp. Cell Res. 208,333 -343.[CrossRef][Medline]
Smith, S. J., Cases, S., Jensen, D. R., Chen, H. C., Sande, E., Tow, B., Sanan, D. A., Raber, J., Eckel, R. H. and Farese, R. V., Jr (2000). Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking DGAT. Nat. Genet. 25,87 -90.[CrossRef][Medline]
Stacey, A., Schnieke, A., McWhir, J., Cooper, J., Colman, A. and Melton, D. W. (1994). Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice. Mol. Cell. Biol. 14,1009 -1016.[Abstract]
Stinnakre, M. G., Vilotte, J. L., Soulier, S. and Mercier, J.
C. (1994). Creation and phenotypic analysis of
-lactalbumin-deficient mice. Proc. Natl. Acad. Sci.
USA 91,6544
-6548.[Abstract]
Stone, S. J., Myers, H., Watkins, S. M., Brown, B. E., Feingold, K. R., Elias, P. M. and Farese, R. V., Jr (2004). Lipopenia and skin barrier abnormalities in DGAT2-deficient mice. J. Biol. Chem. (in press).
Walden, P. D., Ruan, W., Feldman, M. and Kleinberg, D. L.
(1998). Evidence that the mammary fat pad mediates the action of
growth hormone in mammary gland development.
Endocrinology 139,659
-662.
Wiens, D., Park, C. S. and Stockdale, F. E. (1987). Milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction. Dev. Biol. Stand. 120,245 -258.
Wiesen, J. F., Young, P., Werb, Z. and Cunha, G. R.
(1999). Signaling through the stromal epidermal growth factor
receptor is necessary for mammary ductal development.
Development 126,335
-344.
Wiseman, B. S. and Werb, Z. (2002). Stromal
effects on mammary gland development and breast cancer.
Science 296,1046
-1049.
Young, L. J. T. (2000). The Cleared Mammary Fat Pad and the Transplantation of Mammary Gland Morphological Structures and Cells. In Methods in Mammary Gland Biology and Breast Cancer Research (ed. I. A. Asch), pp. New York: Kluwer Academic/Plenum Publishers.
Zangani, D., Darcy, K. M., Shoemaker, S. and Ip, M. M. (1999). Adipocyteepithelial interactions regulate the in vitro development of normal mammary epithelial cells. Exp. Cell Res. 247,399 -409.[CrossRef][Medline]