Agouti regulates adipocyte transcription factors

Randall L. Mynatt and Jacqueline M. Stephens

Pennington Biomedical Research Center, Baton Rouge 70808; and Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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)-gamma 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-gamma .

adipose tissue; signal transducers and activators of transcription; peroxisome proliferator-activated receptor; melanocortins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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, alpha -melanocyte-stimulating hormone (alpha -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 alpha -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, alpha -MSH, and beta -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 alpha -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 alpha -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)-gamma , 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-beta and CEBP-delta , a condition that results in adipogenesis (45). In these studies, the expression of STAT1, STAT5A, and STAT5B was induced in a PPAR-gamma 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-gamma 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-gamma expression results in the regulation of various genes associated with the adipocyte phenotype.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-gamma 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 1.   Comparison of body weights between mice at Oak Ridge National Laboratory (ORNL) and Pennington Biomedical Research Center (PBRC). Body weights are from mice housed at ORNL and mice that were rederived from the ORNL mice and established at PBRC. All mice were maintained on the FVB/N background, fed a diet containing 11% fat by weight, and weaned at 21-25 days of age. Data are from mice that are hemizygous for the transgene or their nontransgenic littermates. There were between 12 and 28 mice per data point. Data are presented as means ± SE. *Significantly different (P >=  0.01) from ORNL mice; **significantly different (P >=  0.01) from nontransgenic and ORNL mice



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Fig. 2.   Body weight and individual fat pad weights. Body weight (A) and individual fat depot weights (B-D) were measured at 4, 6, 8, and 10 wk of age. Data are from mice that are hemizygous for the transgene or their nontransgenic littermates. There were between 5 and 8 mice per data point. Data are presented as means ± SE.

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.


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Fig. 3.   Agouti regulation of leptin mRNA. A: total RNA (20 µg) from individual fat depots from 10-wk-old aP212 transgenic (Tg/+) mice and nontransgenic (+/+) mice was hybridized with a radiolabeled cDNA probe for agouti and, subsequently, with a cDNA probe for 18S ribosomal RNA as a loading control. B: total RNA (20 µg) from combined fat depots of 10-wk-old aP212 (aP2a) transgenic mice (n = 10), obese beta -actin promoter-agouti (BAP20; BAPa) transgenic mice (21), and nontransgenic littermates (n = 4 aP2-agouti littermates, n = 1 BAPa littermate) was hybridized with a radiolabeled cDNA probe for leptin and, subsequently, with a cDNA probe for 18S ribosomal RNA as a loading control.

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-gamma 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-gamma 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-gamma 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-gamma 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-gamma expression was substantially elevated in transgenic mice compared with controls.


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Fig. 4.   Agouti regulation of signal transducers and activators of transcription (STATs) and peroxisome proliferator-activated receptor (PPAR)-gamma . A: retroperitoneal fat pad extracts were prepared as described in EXPERIMENTAL PROCEDURES from transgenic (Tg/+) and nontransgenic (+/+) 10-wk-old mice. Each extract (100 µg) was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. The molecular mass of each protein is indicated to the left of the blot in kilodaltons. The detection system was horseradish peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence (Pierce). B: a digital photograph of the Western blots was taken with a white backlight using the Nucleovision system (Nucleotech). The bands were quantitated using the Nucleovision software. Data are reported as relative optical density (OD) units after background substraction. STAT5A is shown for each group to demonstrate even loading of the samples.

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-gamma 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-alpha -MSH binding to cells stably expressing mouse melanocortin receptors (20). The regulation of STAT1, STAT3, and PPAR-gamma by the addition of recombinant agouti to cultured adipocytes unequivocally demonstrates that agouti directly influences adipocyte metabolism.


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Fig. 5.   In vitro effects of agouti on adipocytes. A: whole cell extracts were prepared from fully differentiated 3T3-L1 adipocytes after a treatment with 50 nM agouti for 0, 4, 8, 12, 18, and 24 h as described in EXPERIMENTAL PROCEDURES. Each extract (100 µg) was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis. The molecular mass of each protein is indicated to the left of the blot in kilodaltons. The detection system was horseradish peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence (Pierce). B: a digital photograph of the Western blots was taken with a white backlight using the Nucleovision system (Nucleotech). The bands were quantitated using the Nucleovision software. Data are reported as the degree of increase in OD above zero time point. STAT5A is shown for each group to demonstrate even loading of the samples.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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-gamma . Several studies (14, 27, 28, 36, 39, 42, 46) have shown that PPAR-gamma is an essential transcription factor for differentiation and maturation of adipocytes. Additionally, ectopic expression of PPAR-gamma in nonadipogenic fibroblast promotes lipid accumulation and characteristics of mature adipocytes (41). The elevated levels of PPAR-gamma 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

33.   Rosen, ED, Walkey CJ, Puigserver P, and Spiegelman BM. Transcriptional regulation of adipogenesis. Genes Dev 14: 1293-1307, 2000[Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].


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