Pennington Biomedical Research Center, Baton Rouge 70808; and Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agouti is a
secreted paracrine factor that regulates pigmentation in hair follicle
melanocytes. Several dominant mutations cause ectopic expression of
agouti, resulting in a phenotype characterized by yellow
fur, adult-onset obesity and diabetes, increased linear growth and
skeletal mass, and increased susceptibility to tumors. Humans also
produce agouti protein, but the highest levels of agouti in humans are
found in adipose tissue. To mimic the human agouti
expression pattern in mice, transgenic mice (aP2-agouti) that express
agouti in adipose tissue were generated. The transgenic mice develop a mild form of obesity, and they are sensitized to the
action of insulin. We correlated the levels of specific regulators of
insulin signaling and adipocyte differentiation with these phenotypic
changes in adipose tissue. Signal transducers and activators of
transcription (STAT)1, STAT3, and peroxisome proliferator-activated receptor (PPAR)- protein levels were elevated in the transgenic mice. Treatment of mature 3T3-L1 adipocytes recapitulated these effects. These data demonstrate that agouti has potent effects on
adipose tissue. We hypothesize that agouti increases adiposity and
promotes insulin sensitivity by acting directly on adipocytes via
PPAR-
.
adipose tissue; signal transducers and activators of transcription; peroxisome proliferator-activated receptor; melanocortins
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SEVERAL dominant
agouti mutations in mice cause a phenotype characterized by
yellow fur, mild hyperphagia, decreased thermogenesis, increased
body-fat content, insulin resistance, impaired glucose tolerance,
hyperglycemia, and increased susceptibility to tumors (48). The yellow obese syndrome seems to be caused by a
paracrine factor because heterogenic parabiotic union between obese
mutant mice and litter mates after 28 wk did not produce any changes in
body weight or body composition compared with homogenic pairs (44). Cloning and sequence analysis revealed that the
mouse agouti gene is located in the distal region of
chromosome 2 and encodes for a small secreted protein (5).
Agouti expression analysis of wild-type mice demonstrated
that agouti is only expressed in the skin during the hair
growth cycle (5). Examination of agouti mRNA from several
dominant agouti mutations revealed structural changes in the
promoter region of agouti that cause it to be expressed ubiquitously (5, 12, 24-26). However, because each of
these dominant mutant alleles contains a major structural change in the
agouti locus, it was unclear whether the ubiquitous
expression of agouti per se causes the syndrome or whether there is an
additional gene located in the vicinity of agouti that is
altered. It was later demonstrated that agouti was responsible for the
syndrome by ubiquitously expressing the wild-type agouti cDNA under
control of -actin and phosphoglycerate kinase-1 promoters in
transgenic mice (21). These results demonstrated
conclusively that the ectopic expression of agouti is solely
responsible for the mutant traits in these animals.
The human homolog of the agouti gene is 85% identical to the mouse gene and encodes a protein of 132 amino acids with a consensus signal peptide (22). The major difference between mouse and human agouti is the expression pattern. Whereas mouse agouti is only transiently expressed in the hair follicle (5), human agouti is expressed in diverse tissues; primarily adipose tissue followed by the testis, heart, liver, kidney, ovary, and foreskin (22, 43).
The understanding of the mechanisms of agouti within the hair follicle
has served as a paradigm for agouti-induced obesity. Within the hair
follicle, -melanocyte-stimulating hormone (
-MSH) binds to its
receptor (MC1-R), which is coupled to the heterotrimeric guanine
nucleotide-binding proteins that activate adenlyate cyclase (9). The resulting increase in intracellular cAMP levels
leads to the activation of the rate-limiting enzyme in melanogenesis, tyrosinase (16). Agouti decreases the overall rate of
melanogenesis by antagonizing the binding of
-MSH to MC1-R and
increases the incorporation of sulfhydryl compounds into dopaquinone to
produce yellow pigment (17, 18).
Early experiments demonstrated that mouse adipocytes express high-affinity binding sites for melanocortin peptides (31). Boston and Cone (3) demonstrated that both MC2 and MC5 receptors are expressed in differentiated 3T3-L1 adipocytes and mouse adipose tissues but not in preadipocytes. Mountjoy and Wong (29) have shown expression of MC1, MC2, and MC5 receptor mRNA in differentiated 3T3F442A adipocytes using RT-PCR and in both white adipose tissue (MC1-R and MC2-R) and brown adipose tissue (MC2-R and MC5-R) in mice. The mRNA for all five melanocortin receptors was detected by RT-PCR in human subcutaneous adipose tissue (7). Expression of agouti protein and melanocortin receptors in human fat raises questions as to their normal functions in maintaining energy balance and whether a defect in the functioning of these receptors in fat might contribute to an obese, insulin-resistant, or diabetic phenotype.
ACTH, -MSH, and
-lipotropin are potent lipolytic hormones
(2). However, considerable species variability exists in
the lipolytic response to melanocortins. The mouse adipocyte MC2
receptor exhibits properties similar to the ACTH receptor characterized in adrenocortical cells, coupling to activation of adenylyl cyclase with an EC50 of ~1 nM. Both ACTH and
-MSH bind to
mouse adipocytes, but only ACTH elevates cAMP and stimulates lypolysis
(2). Therefore, agouti antagonism of ACTH to adipocytes
may lead to an inhibition of lipolysis and/or stimulation of
lipogenesis. Data from Moustaid and colleagues (19)
suggest that the agouti protein can increase lipogenesis in adipocytes.
The mRNA levels of fatty acid synthase (FAS) and stearoyl-CoA
desaturase (SCD), two key enzymes in de novo fatty acid synthesis and
desaturation, respectively, were dramatically increased in obese
(Avy) mice relative to lean (a/a) controls
(19). Treatment of 3T3-L1 adipocytes with recombinant
agouti protein increased FAS and SCD mRNA levels 1.5- and 4-fold,
respectively (19). In addition, FAS activity and
triglyceride content were threefold higher in agouti-treated 3T3-L1
cells relative to controls.
Experiments on the lipolytic effects of melanocortins in humans are in
disagreement. Variables between the experiments included different fat
depots, different fat cell isolation procedure and culture conditions,
and different time courses. Experiments using adipocytes isolated from
human intra-abdominal fat show no effects of -MSH and ACTH on
lipolysis in human adipose tissue (4). Xue et al.
(47) investigated the role of agouti and ACTH in regulating lipolysis in primary cultures of adipocytes isolated from
subcutaneous depots. Short-term (1 h) exposure to recombinant agouti
protein had no effect on basal lipolysis, although longer term
treatment (24 h) caused a 60% decrease in basal lipolysis. Short-term agouti treatment inhibited ACTH-induced lipolysis. This effect, combined with agouti-induced lipogenesis, may represent a
coordinate control of adipocyte lipid metabolism.
The 3T3-L1 adipocytes are comparable to native adipocytes as they have
the ability to accumulate lipid, respond to insulin and secrete leptin.
The major transcription factors involved in adipocyte gene regulation
include peroxisome proliferator-activated receptor (PPAR)-, proteins
belonging to the CCAAT/enhancer-binding protein (CEBP) family, and
adipocyte determination and differentiation dependent factor 1, also
known as sterol regulatory element binding protein (reviewed in Refs.
27 and 33). Recent studies have also suggested that the
signal transducers and activators of transcription (STAT) family of
transcription factors may also be important in fat cells. A STAT family
member shows a distinct pattern of activation by cytokines and, upon
nuclear translocation, can regulate the transcription of particular
genes in cell- or tissue-specific manners (10). In fat
cells, the expression of STAT1, STAT5A, and STAT5B is highly induced
during differentiation and correlates with lipid accumulation
(38, 40). The regulation of STAT expression has also been
investigated in NIH/3T3 cells ectopically overexpressing CEBP-
and
CEBP-
, a condition that results in adipogenesis
(45). In these studies, the expression of STAT1,
STAT5A, and STAT5B was induced in a PPAR-
ligand-dependent fashion
during adipogenesis (39). STAT3 and STAT6 are also
expressed in adipocytes, but the expression of these proteins does not
change during differentiation. However, the tyrosine phosphorylation of
STAT3 occurs after the induction of differentiation, and a study
(11) with antisense STAT3 suggest that this protein may be
important in adipogenesis. Although the functions of STATs in fat cells
have not been identified, numerous studies suggest that these
transcription factors are important regulators of adipocyte gene expression.
We (30) reported that transgenic mice that have the
aP2-promoter driving agouti expression are sensitized to insulin. In this paper, we further describe the phenotype of the aP2-agouti transgenic mice and identified a potential mechanism of increased insulin sensitivity and obesity. These studies clearly indicate that
agouti treatment of adipocytes results in the increased expression of
three transcription factors. The most prominent effect of agouti on
adipocyte transcription factors is the substantial increase in PPAR-
expression in the fat pads of the aP2-agouti mice and in culture
adipocytes that have been exposed to recombinant agouti protein. In
addition, we observed an increase in both STAT1 and STAT3 in these
conditions. Although the function of STATs in adipocytes has not been
identified, it is known that these transcription factors can be
activated in adipocytes and play a role in adipocyte differentiation
(11, 37, 38). We hypothesize that the agouti-induced regulation of STAT1, STAT3, and PPAR-
expression results in the regulation of various genes associated with the adipocyte phenotype.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice. Transgenic mice were generated at Oak Ridge National Laboratory (ORNL) as previously described (30). The aP212 and aP273 lines were rederived at Charles River Laboratory and maintained on the FVB/N background at Pennington Biomedical Research Center (PBRC; Baton Rouge, LA). All mice were fed a diet containing 11% fat by weight (Mouse Diet 5015, Purina Mills) and weaned at 21-25 days of age. Food and water were provided ad libitum. All data are from mice that are hemizygous for the transgene or their nontransgenic littermates. There were four litters examined per time point at 4, 6, and 10 wk, and two litters were examined in the 8-wk-old group. Transgenics were compared with littermates in every case but one at 4 wk, in which there were two transgenic males and no wild-type males in the litter. Mice were euthanized by cervical dislocation, and adipose tissue was quickly removed, weighed, and frozen in liquid nitrogen for future analysis. PCR genotyping and Northern blot hybridization analyses were performed as previously described (30).
Materials.
Dulbecco's modified Eagle's medium (DMEM) was purchased from Life
Technologies (Grand Island, NY). Bovine and fetal bovine serum (FBS)
were obtained from Sigma (St. Louis, MO) and Life Technologies,
respectively. Murine agouti was a gift from Derril Willard of
GlaxoWellcome Pharmaceuticals (Research Triangle Park, NC). The STAT
antibodies were monoclonal IgGs purchased from Transduction Laboratories (Affiniti Research Products) or polyclonal IgGs from Santa
Cruz Biotechnology (Santa Cruz, CA). PPAR- antibody was a mouse
monoclonal from Santa Cruz.
Cell culture. Murine 3T3-L1 preadipocytes were plated and grown to 2 days postconfluence in DMEM with 10% bovine serum. Medium was changed every 48 h. Cells were induced to differentiate by changing the medium to DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1.7 µM insulin. After 48 h, this medium was replaced with DMEM supplemented with 10% FBS, and cells were maintained in this medium until utilized for experimentation.
Preparation of adipose tissue and whole cell extracts. Fat pads were homogenized in a nondenaturing buffer containing 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 1 µM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 µM leupeptin, and 2 mM sodium vanadate. Homogenates were centrifuged for 10 min at 10,000 rpm to remove any debris and insoluble material. Monolayers of fully differentiated 3T3-L1 adipocytes were rinsed with phosphate-buffered saline (PBS) and then harvested in the above nondenaturing buffer. Samples were extracted for 30 min on ice and centrifuged at 15,000 rpm at 4°C for 15 min. Supernatants containing whole cell and fat pad extracts were analyzed for protein content using a bicinchoninic acid kit from Pierce (Rockford, IL) according to the manufacturer's instructions.
Gel electrophoresis and immunoblotting. Proteins were separated in 5, 7.5, or 12% polyacrylamide gels containing sodium dodecyl sulfate (SDS) according to Laemmli (23) and transferred to nitrocellulose in 25 mM Tris, 192 mM glycine, and 20% methanol. After the transfer, the membrane was blocked in 4% milk for 1 h at room temperature. Results were visualized with horseradish peroxidase-conjugated secondary antibodies from Sigma and enhanced chemiluminescence from Pierce.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In our original study (30) performed at ORNL,
we did not observe any significant differences in body weight between
transgenic mice that expressed agouti in adipose tissue (Tg/+) and
their littermates (+/+), but the transgenic mice gained more weight if
they were given insulin injections. The ORNL mice harbored a
number of pathogens and parasites. Therefore, two transgenic lines were
rederived at Charles River Laboratories and brought to the PBRC to
establish new lines. Agouti expression in adipose tissue was confirmed
in the rederived mice (Fig. 3A). Both nontransgenic and
transgenic mice at PBRC were significantly heavier than their ORNL
counterparts (Fig. 1). Additionally, the
transgenic mice at PBRC have a statistically significant increase in
body weight over the nontransgenic littermates [Fig. 1 (12 wk) and
Fig. 2]. The most probable explanation
for the discrepancies in body weight reported from mice at ORNL and the
mice at PBRC is the healthier status of the mice at PBRC. Examination
of records from the ORNL mice revealed that many of the litters
suffered from diarrhea and were given antibiotics in the drinking
water. This immunological challenge during development caused a very
large variation in body weight between mice and between litters that
reduced ability to detect a statistically significant change in body
weight.
|
|
We choose the aP212 line to closely examine the cause for the
increased body weight of the transgenic mice. Figure 2 compares both
body weight and individual fat depot weights between transgenic mice
and wild-type littermates from 4 to 10 wk of age. The transgenic mice
are heavier than littermates at all times but become statistically different by 8-10 wk. The increased body weight correlates with an
increased mass of all fat depots that becomes significant between 8 and
10 wk. Combined fat depot weight is increased 30-50% at 10 wk in
individual transgenic mice compared with littermates. DNA content per
gram of adipose tissue was reduced in the transgenic mice by 10 wk, and
histological examination of fat depots revealed fat cell hypertrophy
(data not shown). Total RNA was extracted from the combined fat depots
from 10-wk-old mice, and leptin mRNA levels were compared by Northern
blot analysis (Fig. 3). In accordance with increased fat mass and fat cell size, leptin mRNA levels were
substantially higher in the transgenic mice. These data demonstrate that the aP2-agouti transgene causes increased fat mass, resulting in
mild obesity and increased leptin synthesis.
|
Members of both the STAT and PPAR family of transcription factors are
regulated during adipogenesis and in conditions of altered insulin
sensitivity (33). Therefore, we examined the levels of
these transcription factors in retroperitoneal fat pads from transgenic
mice and wild-type littermates between 4 and 10 wk of age. In mice that
were 4 wk of age, there were no detectable differences in the
expression of STAT1, STAT5A, or PPAR- in wild-type and transgenic
mice. However, by 6 wk of age, there was a discernible, but not
statistically significant, increase in STAT1 expression in all of the
transgenic mice compared with wild-type controls. PPAR-
expression
was elevated in three of five transgenic animals by 6 wk (data not
shown). We examined the data to see whether the differences were litter
effects, and they were not. Because these mice are an inbred line, we
assume they are genetically identical, and at this point it leaves only
environmental effects to account for mouse-to-mouse variation. We have
shown that this phenotype is "environmentally sensitive" by
comparing ORNL versus PBRC mice. We can only speculate that factors
such as birth order, nesting conditions, dominance during sucking and
adulthood, and individual activity are affecting the phenotype.
Interestingly, the increases in both STAT1 and PPAR-
expression
occurred before any effects in body weight and fat mass, suggesting
that these transcription factors may be responsible for the accelerated
accumulation of adipose tissue in the transgenic animals. By 10 wk of
age, the expression of STAT1, STAT3, and PPAR-
was significantly
elevated in transgenic animals compared with nontransgenic littermates (Fig. 4A). Also, at 10 wk of
age, there was a marked increase in body weight in the transgenic mice.
To confirm these findings, a separate group of 10-wk-old mice was
examined. Both body weight [32.66 ± 0.54 g (Tg/+) versus
28.21 ± 0.66 g (+/+), P = 0.002] and
retroperitoneal fat pad weight [0.58 ± 0.03 g (Tg/+) versus 0.33 ± 0.04 g (+/+), P = 0.003] were
significantly greater in these transgenic mice compared with
littermates. Western blots from both sets of mice were
quantitated for statistical analysis (Fig. 4B). These
results clearly demonstrate that STAT1, STAT3, and PPAR-
expression
was substantially elevated in transgenic mice compared with controls.
|
The 5.4-kb aP2 promoter exhibits low levels of ectopic
expression in many tissues (30, 34), and the changes that
we observed in adipose tissue may have been indirectly caused by
ectopic agouti expression. To examine the direct effects of agouti on
adipocytes, mature 3T3-L1 adipocytes were treated with recombinant
murine agouti. Very similar to the adipose tissue from transgenic mice, STAT1, STAT3, and PPAR-
were elevated in the agouti (50 nM)-treated 3T3-L1 adipocytes (Fig. 5). Yet there was
no change in STAT5A levels after agouti treatment. Dose-response curves
from 0.5 to 200 nM agouti demonstrated a near-maximum effect of agouti
at 50 nM (data not shown), which is in agreement with inhibition of
Nle4,D-Phe7-
-MSH binding to
cells stably expressing mouse melanocortin receptors (20).
The regulation of STAT1, STAT3, and PPAR-
by the addition of
recombinant agouti to cultured adipocytes unequivocally demonstrates that agouti directly influences adipocyte metabolism.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The agouti gene is expressed in adipose tissue in humans and has been shown to regulate lipid metabolism in cultured adipose cells in vitro (19, 47). Although the agouti gene is not normally expressed in adipose tissue in the mouse, we were able to induce the expression of high levels of agouti in white and brown adipose tissue by expressing the cloned mouse gene under the control of the aP2 promoter. The agouti expression in adipose tissue resulted in a phenotype quantitatively similar to middle-aged adiposity. The mice have a 10-15% increase in body weight caused by a 30-50% increase in fat mass. This would be equivalent to an extra 15 lb of body fat in a 150-lb person. These results indicate that the expression of agouti in adipose tissue has substantial metabolic effects and may be physiologically significant in humans.
To date, most models have focused on the central nervous system to
explain the development of the yellow obesity syndrome in mice.
Intracerebroventricular administration of -MSH or a melanocortin
analog, MTII, a potent agonist of the MC3 and MC4 receptors, suppresses
feeding behavior in rodents, whereas injection of SHU9119, an
antagonist of the same receptors, stimulates feeding (13).
Inactivation of MC4-R by gene targeting (15) results in
obese mice. Agouti-related peptide (Agrp) is a homolog of agouti found
in the brain and adrenal cortex that also inhibits melanocortin signaling (32, 35). Overexpression of Agrp in mice results in an obese and diabetic phenotype very much like that seen in the
AY and MC4-R knockout mice (32,
35), suggesting that that ectopic expression of
agouti in the hypothalamus mimics endogenous Agrp. The
conclusion from these knockout and transgenic mice experiments was that
the MC4-R/Agrp system is a major regulator of food intake and energy expenditure.
Recently, the MC3-R was inactivated resulting in decreased energy
expenditure and increased fat mass, despite being hypophagic (6,
8). Perhaps the most striking observation was that the MC3 and
MC4 pathways are not redundant. Mice lacking MC3-R are not heavier than
their littermates until about 26 wk of age. In contrast, 26-wk-old mice
lacking both MC3-R and MC4-R are significantly heavier than littermates
lacking only MC4-R. The data suggest that in the absence of MC3-R,
nutrients are preferentially partitioned into fat resulting in subtle
body weight changes and almost a doubling of fat mass. The subtle
change in body weight and increased fat mass phenotype in MC3-R
knockout mice is very similar to our aP2-agouti transgenic mice. We
predict that melanocortin signaling in adipose tissue is another
nonredundant mechanism for body weight homeostasis and a cross between
aP2-agouti and MC4-R/
mice will have a similar effect
to the MC3-R knockout mice.
The most striking difference between transgenic and wild-type mice was
the difference in PPAR-. Several studies (14, 27, 28, 36, 39,
42, 46) have shown that PPAR-
is an essential transcription
factor for differentiation and maturation of adipocytes. Additionally,
ectopic expression of PPAR-
in nonadipogenic fibroblast promotes
lipid accumulation and characteristics of mature adipocytes (41). The elevated levels of PPAR-
in the aP2-agouti
mice are consistent with the increased adipocyte hypertrophy and
increased insulin sensitivity. The elevated expression of STAT1 and
STAT3 by agouti is interesting. Agouti does not appear to have any
direct effect on STAT phosphorylation in adipocytes, but the expression of these two STATs has been shown to be highly regulated in fat cells
(1). The expression of STAT1 is highly induced during adipocyte differentiation and can be controlled by thiazoldidnedione treatment (38, 39). STAT3 may also be important in
adipogenesis because STAT3 antisense oligonucletodies have been shown
to inhibit fat cell differentiation (11). Although the
functions of STATs in fat cells have not been identified, various
studies suggest that these transcription factors are important
regulators of adipocyte gene expression.
In summary, these data provide direct in vivo and in vitro evidence that agouti and/or melanocortin signaling may govern adipogenesis. These mice have significantly increased fat mass, accompanied by a substantial increase in three key adipocyte transcription factors that are also upregulated in agouti-treated 3T3-L1 adipocytes. The modest weight gain in these mice suggests that the normal hypothalamic pathways regulating food intake are intact and that the observed adiposity is within the ranges that can be achieved by this restricted physiological mechanism at the adipocyte level.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by American Heart Association Scientist Development Grant 9630120N (to R. L. Mynatt) and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-52968-02 to (J. M. Stephens).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: R. L Mynatt, Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge, LA 70808 (E-mail: mynattrl{at}pbrc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 November 2000; accepted in final form 10 November 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Balhoff, JP,
and
Stephens JM.
Highly specific and quantitative activation of STATs in 3T3-L1 adipocytes.
Biochem Biophys Res Commun
247:
894-900,
1998[ISI][Medline].
2.
Boston, BA.
The role of melanocortins in adipocyte function.
Ann NY Acad Sci
885:
75-84,
1999
3.
Boston, BA,
and
Cone RD.
Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line.
Endocrinology
137:
2043-2050,
1996[Abstract].
4.
Bousquet-Melou, A,
Galitzky J,
Lafontan M,
and
Berlan M.
Control of lipolysis in intra-abdominal fat cells of nonhuman primates: comparison with humans.
J Lipid Res
36:
451-461,
1995[Abstract].
5.
Bultman, SJ,
Michaud EJ,
and
Woychik RP.
Molecular characterization of the mouse agouti locus.
Cell
71:
1195-1204,
1992[ISI][Medline].
6.
Butler, AA,
Kesterson RA,
Khong K,
Cullen MJ,
Pelleymounter MA,
Dekoning J,
Baetscher M,
and
Cone RD.
A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse.
Endocrinology
141:
3518-3521,
2000
7.
Chagnon, YC,
Chen WJ,
Perusse L,
Chagnon M,
Nadeau A,
Wilkison WO,
and
Bouchard C.
Linkage and association studies between the melanocortin receptors 4 and 5 genes and obesity-related phenotypes in the Quebec family study.
Mol Med
3:
663-673,
1997[ISI][Medline].
8.
Chen, AS,
Marsh DJ,
Trumbauer ME,
Frazier EG,
Guan XM,
Yu H,
Rosenblum CI,
Vongs A,
Feng Y,
Cao L,
Metzger JM,
Strack AM,
Camacho RE,
Mellin TN,
Nunes CN,
Min W,
Fisher J,
Gopal-Truter S,
MacIntyre DE,
Chen HY,
and
Van Der Ploeg LH.
Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.
Nat Genet
26:
97-102,
2000[ISI][Medline].
9.
Cone, RD,
Mountjoy KG,
Robbins LS,
Nadeau JH,
Johnson KR,
Roselli-Rehfuss L,
and
Mortrud MT.
Cloning and functional characterization of a family of receptors for the melanotropic peptides.
Ann NY Acad Sci
680:
342-363,
1993[ISI][Medline].
10.
Darnell, JEJ
STATs and gene regulation.
Science
277:
1630-1635,
1997
11.
Deng, J,
Hua K,
Lesser SS,
and
Harp JB.
Activation of signal transducer and activator of transcription-3 during proliferative phases of 3T3-L1 adipogenesis.
Endocrinology
141:
2370-2376,
2000
12.
Duhl, DM,
Stevens ME,
Vrieling H,
Saxon PJ,
Miller MW,
Epstein CJ,
and
Barsh GS.
Pleiotropic effects of the mouse lethal yellow (Ay) mutation explained by deletion of a maternally expressed gene and the simultaneous production of agouti fusion RNAs.
Development
120:
1695-1708,
1994
13.
Fan, W,
Boston BA,
Kesterson RA,
Hruby VJ,
and
Cone RD.
Role of melanocortinergic neurons in feeding and the agouti obesity syndrome.
Nature
385:
165-168,
1997[ISI][Medline].
14.
Hamm, JK,
el Jack AK,
Pilch PF,
and
Farmer SR.
Role of PPAR gamma in regulating adipocyte differentiation and insulin-responsive glucose uptake.
Ann NY Acad Sci
892:
134-145,
1999
15.
Huszar, D,
Lynch CA,
Fairchild-Huntress V,
Dunmore JH,
Fang Q,
Berkemeier LR,
Gu W,
Kesterson RA,
Boston BA,
Cone RD,
Smith FJ,
Campfield LA,
Burn P,
and
Lee F.
Targeted disruption of the melanocortin-4 receptor results in obesity in mice.
Cell
88:
131-141,
1997[ISI][Medline].
16.
Iozumi, K,
Hoganson GE,
Pennella R,
Everett MA,
and
Fuller BB.
Role of tyrosinase as the determinant of pigmentation in cultured human melanocytes.
J Invest Dermatol
100:
806-811,
1993[Abstract].
17.
Ito, S,
and
Fujita K.
Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography.
Anal Biochem
144:
527-536,
1985[ISI][Medline].
18.
Ito, S,
Fujita K,
Takahashi H,
and
Jimbow K.
Characterization of melanogenesis in mouse and guinea pig hair by chemical analysis of melanins and of free and bound dopa and 5-S-cysteinyldopa.
J Invest Dermatol
83:
12-14,
1984[Abstract].
19.
Jones, BH,
Kim JH,
Zemel MB,
Woychik RP,
Michaud EJ,
Wilkison WO,
and
Moustaid N.
Upregulation of adipocyte metabolism by agouti protein: possible paracrine actions in yellow mouse obesity.
Am J Physiol Endocrinol Metab
270:
E192-E196,
1996
20.
Kiefer, LL,
Veal JM,
Mountjoy KG,
and
Wilkison WO.
Melanocortin receptor binding determinants in the agouti protein.
Biochemistry
37:
991-997,
1998[ISI][Medline].
21.
Klebig, ML,
Wilkinson JE,
Geisler JG,
and
Woychik RP.
Ectopic expression of the agouti gene in transgenic mice causes obesity, features of type II diabetes, and yellow fur.
Proc Natl Acad Sci USA
92:
4728-4732,
1995[Abstract].
22.
Kwon, HY,
Bultman SJ,
Loffler C,
Chen WJ,
Furdon PJ,
Powell JG,
Usala AL,
Wilkison W,
Hansmann I,
and
Woychik RP.
Molecular structure and chromosomal mapping of the human homolog of the agouti gene.
Proc Natl Acad Sci USA
91:
9760-9764,
1994
23.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
24.
Michaud, EJ,
Bultman SJ,
Stubbs LJ,
and
Woychik RP.
The embryonic lethality of homozygous lethal yellow mice (Ay/Ay) is associated with the disruption of a novel RNA-binding protein.
Genes Dev
7:
1203-1213,
1993[Abstract].
25.
Michaud, EJ,
van Vugt MJ,
Bultman SJ,
Sweet HO,
Davisson MT,
and
Woychik RP.
Differential expression of a new dominant agouti allele (Aiapy) is correlated with methylation state and is influenced by parental lineage.
Genes Dev
8:
1463-1472,
1994[Abstract].
26.
Miller, MW,
Duhl DM,
Vrieling H,
Cordes SP,
Ollmann MM,
Winkes BM,
and
Barsh GS.
Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation.
Genes Dev
7:
454-467,
1993[Abstract].
27.
Morrison, RF,
and
Farmer SR.
Insights into the transcriptional control of adipocyte differentiation.
J Cell Biochem Suppl
32-33:
59-67,
1999.
28.
Morrison, RF,
and
Farmer SR.
Role of PPARgamma in regulating a cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) and p21(Waf1/Cip1), during adipogenesis.
J Biol Chem
274:
17088-17097,
1999
29.
Mountjoy, KG,
and
Wong J.
Obesity, diabetes and functions for proopiomelanocortin-derived peptides.
Mol Cell Endocrinol
128:
171-177,
1997[ISI][Medline].
30.
Mynatt, RL,
Miltenberger RJ,
Klebig ML,
Zemel MB,
Wilkinson JE,
Wilkinson WO,
and
Woychik RP.
Combined effects of insulin treatment and adipose tissue-specific agouti expression on the development of obesity.
Proc Natl Acad Sci USA
94:
919-922,
1997
31.
Oelofsen, W,
and
Ramachandran J.
Studies of corticotropin receptors on rat adipocytes.
Arch Biochem Biophys
225:
414-421,
1983[ISI][Medline].
32.
Ollmann, MM,
Wilson BD,
Yang YK,
Kerns JA,
Chen Y,
Gantz I,
and
Barsh GS.
Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein.
Science
278:
135-138,
1997
33.
Rosen, ED,
Walkey CJ,
Puigserver P,
and
Spiegelman BM.
Transcriptional regulation of adipogenesis.
Genes Dev
14:
1293-1307,
2000
34.
Ross, SR,
Graves RA,
Greenstein A,
Platt KA,
Shyu HL,
Mellovitz B,
and
Spiegelman BM.
A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo.
Proc Natl Acad Sci USA
87:
9590-9594,
1990[Abstract].
35.
Shutter, JR,
Graham M,
Kinsey AC,
Scully S,
Luthy R,
and
Stark KL.
Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice.
Genes Dev
11:
593-602,
1997[Abstract].
36.
Spiegelman, BM.
Peroxisome proliferator-activated receptor gamma: a key regulator of adipogenesis and systemic insulin sensitivity.
Eur J Med Res
2:
457-464,
1997[Medline].
37.
Stephens, JM,
Lumpkin SJ,
and
Fishman JB.
Activation of signal transducers and activators of transcription 1 and 3 by leukemia inhibitory factor, oncostatin-M, and interferon-gamma in adipocytes.
J Biol Chem
273:
31408-31416,
1998
38.
Stephens, JM,
Morrison RF,
and
Pilch PF.
The expression and regulation of STATs during 3T3-L1 adipocyte differentiation.
J Biol Chem
271:
10441-10444,
1996
39.
Stephens, JM,
Morrison RF,
Wu Z,
and
Farmer SR.
PPARgamma ligand-dependent induction of STAT1, STAT5A, and STAT5B during adipogenesis.
Biochem Biophys Res Commun
262:
216-222,
1999[ISI][Medline].
40.
Stewart, WC,
Morrison RF,
Young SL,
and
Stephens JM.
Regulation of signal transducers and activators of transcription (STATs) by effectors of adipogenesis: coordinate regulation of STATs 1, 5A, and 5B with peroxisome proliferator-activated receptor-gamma and C/AAAT enhancer binding protein-alpha.
Biochim Biophys Acta
1452:
188-196,
1999[ISI][Medline].
41.
Tontonoz, P,
Hu E,
and
Spiegelman BM.
Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor.
Cell
79:
1147-1156,
1994[ISI][Medline]. [Erratum. Cell 80: Mar 24, 1995, following p. 957].
42.
Tontonoz, P,
Hu E,
and
Spiegelman BM.
Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor gamma.
Curr Opin Genet Dev
5:
571-576,
1995[ISI][Medline].
43.
Wilson, BD,
Ollmann MM,
Kang L,
Stoffel M,
Bell GI,
and
Barsh GS.
Structure and function of ASP, the human homolog of the mouse agouti gene.
Hum Mol Genet
4:
223-230,
1995[Abstract].
44.
Wolff, GL.
Growth of inbred yellow and non-yellow mice in parabiosis.
Genetics
48:
1041-1058,
1963
45.
Wu, Z,
Bucher NL,
and
Farmer SR.
Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta, and glucocorticoids.
Mol Cell Biol
16:
4128-4136,
1996[Abstract].
46.
Wu, Z,
Rosen ED,
Brun R,
Hauser S,
Adelmant G,
Troy AE,
McKeon C,
Darlington GJ,
and
Spiegelman BM.
Cross-regulation of C/EBP alpha and PPAR gamma controls the transcriptional pathway of adipogenesis and insulin sensitivity.
Mol Cell
3:
151-158,
1999[ISI][Medline].
47.
Xue, B,
Moustaid N,
Wilkison WO,
and
Zemel MB.
The agouti gene product inhibits lipolysis in human adipocytes via a Ca2+-dependent mechanism.
FASEB J
12:
1391-1396,
1998
48.
Yen, TT,
Gill AM,
Frigeri LG,
Barsh GS,
and
Wolff GL.
Obesity, diabetes, and neoplasia in yellow A(vy)/ mice: ectopic expression of the agouti gene.
FASEB J
8:
479-488,
1994