The agouti gene product inhibits lipolysis in human adipocytes via a Ca²⁺-dependent mechanism

^a Departments of Nutrition and Medicine, The University of Tennessee, Knoxville, Tennessee 37996, USA
^b Zen-Bio, Inc., Research Triangle Park, North Carolina 27709, USA

	ABSTRACT

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Overexpression of the murine agouti gene results in obesity. The human homologue of agouti is expressed primarily in human adipocytes, and we have shown recombinant agouti protein to increase adipocyte intracellular Ca²⁺([Ca²⁺]_i) and thereby stimulate lipogenesis. However, since recent data demonstrate that increasing adipocyte [Ca²⁺]_i may also inhibit lipolysis, we have investigated the role of agouti-induced [Ca²⁺]_i increases in regulating lipolysis in human adipocytes. Short-term (1 h) exposure to recombinant agouti (100 nM) protein had no effect on basal lipolysis, although longer term treatment (24 h) caused a 60% decrease in basal lipolysis (P<0.0001). Short-term agouti treatment totally inhibited ACTH-induced lipolysis (P<0.05). Since melanocortin receptors (MCR) are involved in some actions of agouti, we next determined whether agouti's antilipolytic effect is exerted through competitive antagonism of the ACTH receptor (MCR-2). Forskolin (1 µM), an adenylate cyclase activator, induced a 48% increase in lipolysis in human adipocytes (P<0.05); this effect was reversed by 100 nM agouti (P<005), demonstrating that the antilipolytic effect of agouti is distal to the ACTH receptor. To determine the role of [Ca²⁺]_i in the antilipolytic effect of agouti, human adipocytes were treated with KCl or arginine vasopressin to stimulate voltage- and receptor-stimulated Ca²⁺ influx, respectively. Both agents caused inhibition of forskolin-induced lipolysis (P<0.005). Furthermore, agouti's antilipolytic effect was also blocked by the Ca²⁺ channel blocker nitrendipine. These data demonstrate that agouti exerts a potent antilipolytic effect in human adipocytes via a Ca²⁺-dependent mechanism. This effect, combined with agouti-induced lipogenesis, represents a coordinate control of adipocyte lipid metabolism that may contribute to an agouti-induced obesity syndrome.—Xue, B., Moustaid-Moussa, N., Wilkison, W. O., Zemel, M. B. The agouti gene product inhibits lipolysis in human adipocytes via a Ca²⁺-dependent mechanism. FASEB J. 12, 1391–1396 (1998)

Key Words: calcium • agouti polypeptide • MSH • melanocortin receptor

	INTRODUCTION

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THE MOUSE agouti gene is normally involved in regulating coat color development (1). It is expressed only in the neonatal skin of wild-type mice during hair growth, such that the expression of agouti causes a transient switch from black (eumelanin) to yellow (pheomelanin) pigment. This gives rise to black hairs with a subapical yellow band (1, 2). However, dominant mutations in the promoter region of this gene, such as viable yellow (A^vy), cause overexpression of the normal protein throughout life, which results not only in an altered pattern of pigmentation (i.e., yellow coat color), but also in a syndrome of adult-onset obesity and insulin resistance (3, 4).

Agouti regulation of pigment development occurs via a paracrine action within the hair follicle (5, 6), where agouti competitively antagonizes the binding of melanocyte-stimulating hormone (-MSH)² to its receptor (melanocortin receptor 1, MCR-1). This results in an inhibition of cAMP production and a switch from eumelanin to pheomelanin production (7). Agouti has been demonstrated to similarly antagonize other classes of MCRs (8). Consequently, this competitive antagonism of melanocortin binding has been considered by some investigators to serve as a paradigm for agouti's action in obesity.

However, the actual mechanism of the action of agouti in the development of obesity is not clear. Agouti protein was recently demonstrated to inhibit melanogenesis independent of any melanocortin ligand (9, 10). Moreover, we have shown that agouti regulates several cellular functions, such as Ca²⁺ signaling, independent of melanocortin receptor antagonism (11). Furthermore, we have demonstrated that agouti-induced obesity is not antagonized by the melanocortin receptor agonist NDP-MSH (12).

We have found that recombinant agouti protein increases intracellular calcium ([Ca²⁺]_i) in several cell types (11), including murine and human adipocytes. Agouti protein also stimulated lipogenesis in both murine and human adipocytes via a Ca²⁺-dependent mechanism (13–15). Since the human homologue of agouti is expressed primarily in adipose tissue (16), and [Ca²⁺]_i plays an important role in the metabolic disorders of obesity and insulin resistance (17–19), agouti may act similarly via a paracrine mechanism on adipocytes Ca²⁺ signaling and thereby stimulate lipogenesis.

Both basal and agonist-stimulated adipocyte lipolysis is impaired in agouti mutant mice compared to their wild-type littermates (20–22). This impairment may either be a consequence of the obesity syndrome or it may result from a direct inhibitory effect of agouti on adipocyte lipolysis. Recent data have demonstrated that increasing [Ca²⁺]_i inhibits lipolysis in a dose-dependent manner (23). Accordingly, agouti may coordinately regulate both lipogenesis and lipolysis in adipocytes via Ca²⁺-mediated mechanisms. Alternatively, two melanocortin receptors, MCR-2 and MCR-5, are expressed in adipocytes (24). MCR-5 is a ubiquitously expressed receptor, whereas MCR-2 is the adrenocorticotropic hormone (ACTH) receptor, whose natural ligand is a potent lipolytic hormone (25). Thus, agouti may also exert an antilipolytic effect via antagonism of this melanocortin receptor.

The present study was conducted to determine the effect of recombinant agouti protein on human adipocyte lipolysis and address the role of both [Ca²⁺]_i and the ACTH receptor in this process. We report here that agouti inhibits basal and agonist-stimulated lipolysis in primary cultured human adipocytes. This action is Ca²⁺ dependent, as it can be mimicked by KCl, blocked by the Ca²⁺ channel blockade nitrendipine, and is not dependent on ACTH receptor antagonism.

	MATERIALS AND METHODS

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Isolation and culture of human adipocytes
Human subcutaneous abdominal adipose tissue was obtained from patients undergoing elective abdominal cosmetic surgery. These patients were normal, with no known history of metabolic disorders. This protocol was approved by the Institutional Review Board for Human Subjects and the Committee for Research Participation of the University of Tennessee. Adipocytes were isolated as previously described (26). Briefly, the tissue was washed several times with Hank's balanced salt solution (HBSS), minced into small fragments, and digested with type I collagenase (1 mg/ml) in a shaking water bath at 37°C for 30–40 min. Cells were then filtered through a sterile nylon filter (500 µm mesh) and cultured in Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum, 1% bovine serum albumin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamicin.

Production and purification of recombinant agouti polypeptide
A 614 bp Xbal/PstI fragment of the full-length mouse agouti cDNA (27) or human agouti cDNA (16) was subcloned into a baculovirus expression vector and expressed in Trichiphisia ni (T.ni) cells. Medium was collected 48 h after infection, filtered through a Whatman 3 filter, and purified as previously described (28).

Lipolysis experiments
Human adipocytes were incubated in 24-well plates with different treatments for either 1 h or 24 h, as indicated, and glycerol release into the culture medium was determined as an indicator for lipolysis using an one-step enzymatic fluorometric method (29). The media collected from 1 h treatments were used directly for glycerol measurements. For 24 h treatments, media were removed; cells were washed with HBSS and incubated in fresh medium for an additional 30 min. The media were then used for glycerol measurements for 24 h treatments. After medium was collected, HClO₄ was added to give a final concentration of 3% for deproteinization. The sample was centrifuged at 9000 x g for 20 min.. The supernatant was then collected and neutralized with 10N NaOH (23). Samples were stored at -80°C prior to glycerol assay.

Protein assay
Cells were collected, homogenized, and stored at -80°C for subsequent total protein correction by a modified Bradford method using Coomassie blue dye (PIERCE, Rockford, Ill.).

Statistics
All data are expressed as mean ±SE. Statistical analysis was performed by analysis of variance or Student's t test, as appropriate.

	RESULTS

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To investigate the role of agouti on lipolysis, we first studied the effect of agouti on both basal and ACTH-induced lipolysis in human adipocytes. Short-term (1 h) exposure of human adipocytes to recombinant agouti protein (100 nM) had no effect on basal lipolysis. However, recombinant agouti protein completely blocked 50 nM ACTH-induced lipolysis (P<0.05, Fig. 1). Similar results were obtained in 3T3-L1 murine adipocytes (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 1. The effect of 1 h agouti treatment on ACTH-induced lipolysis in human adipocytes. Primary cultured human adipocytes were incubated with 100 nM recombinant agouti protein, 50 nM ACTH, or both for 1 h. Lipolysis was determined as described in Materials and Methods. Data are expressed as mean ±SD. Data are normalized to control value of 0.29 ± 0.009 µmol glycerol/mg protein. *P < 0.05.

Since agouti is a potent MCR antagonist (7), we next determined whether agouti's antilipolytic effect is exerted through competitive antagonism of the ACTH receptor (MCR-2). Forskolin (1 µM), an adenylate cyclase activator, induced a 48% increase in lipolysis in human adipocytes (P<0.05); this effect was also reversed by 100 nM recombinant agouti protein ( Fig. 2), demonstrating that the antilipolytic effect of agouti includes an action distal to the ACTH receptor.

View larger version (12K): [in this window] [in a new window]   Figure 2. The effect of 1 h agouti treatment on forskolin-induced lipolysis in human adipocytes. Human adipocytes were incubated with 1 µM forskolin or 1 µM forskolin plus 100 nM recombinant agouti protein for 1 h. Lipolysis was determined as described in Materials and Methods. Data are presented as mean ±SD. Data are normalized to control value of 0.35 ± 0.006 µmol glycerol/mg protein. *P < 0.005.

We have previously shown recombinant agouti protein to increase intracellular Ca²⁺ ([Ca²⁺]_i) in several cell types, including human adipocytes (11). To determine the role of [Ca²⁺]_i in the antilipolytic effect of agouti, human adipocytes were treated with KCl or arginine vasopressin (AVP) to stimulate voltage- and receptor-operated Ca²⁺ influx, respectively. As shown in Fig. 3, depolarizing human adipocytes with 25 mM and 40 mM KCl for 1 h inhibited 1 µM forskolin-induced lipolysis by 42 and 50%, respectively (P<0.05). Similarly, short-term treatment with 1 nM AVP caused a 56% inhibition in forskolin-induced lipolysis in human adipocytes (P<0.01, Fig. 4). This suggests that increasing [Ca²⁺]_i by either KCl or AVP mimicked the antilipolytic effect of agouti. Furthermore, the inhibitory effect of agouti on forskolin-induced lipolysis was reversed by coincubation of human adipocytes with the Ca²⁺ channel blocker nitrendipine (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 3. The effect of 1 h KCl treatment on forskolin-induced lipolysis in human adipocytes. Human adipocytes were treated with 1 µM forskolin alone or 1 µM forskolin plus either 25 mM KCl or 40 mM KCl for 1 h. Lipolysis was determined as described in Materials and Methods. Data are presented as mean ±SD. Data are normalized to control value of 0.35 ± 0.006 µmol glycerol/mg protein. *P < 0.05 vs. control; **P < 0.05 vs. forskolin or control.

View larger version (11K): [in this window] [in a new window]   Figure 4. The effect of 1 h AVP treatment on basal and forskolin-induced lipolysis in human adipocytes. Human adipocytes were incubated with 1 µM forskolin, 1 nM AVP or both for 1 h. Lipolysis was determined as described in Materials and Methods. Data are presented as mean ±SD. Data are normalized to control value of 0.35 ± 0.006 µmol glycerol/mg protein. *P < 0.05 vs. control; **P < 0.01 vs. forskolin or control.

Although short-term exposure of human adipocytes to 100 nM recombinant agouti did not affect basal lipolysis, acute treatment of adipocytes with AVP or KCl did inhibit basal lipolysis, as shown in Fig. 4and Fig. 5. To further investigate whether agouti affects basal lipolysis in the absence of lipolytic agents, we incubated human adipocytes with 100 nM agouti for up to 24 h. As shown in Fig. 6, 24 h treatment of agouti caused a 60% decrease in lipolysis compared to control (P<0.0001). This effect was blocked by 30 µM nitrendipine ( Fig. 6) and mimicked by 10 mM KCl ( Fig. 7).

View larger version (14K): [in this window] [in a new window]   Figure 5. The effect of 1 h KCl treatment on basal lipolysis in human adipocytes. Human adipocytes were treated with 25 mM KCl or 40 mM KCl for 1 h. Lipolysis was determined as described in Materials and Methods. Data are presented as mean ±SD. Data are normalized to control value of 0.35 ± 0.006 µmol glycerol/mg protein. *P < 0.05.

View larger version (10K): [in this window] [in a new window]   Figure 6. The effect of 24 h agouti treatment on basal lipolysis in human adipocytes. Human adipocytes were incubated with either 100 nM agouti or 100 nM agouti plus 30 (M nitrendipine for 24 h. Lipolysis was determined as described in Materials and Methods. Data are presented as mean ±SD. Data are normalized to control value of 0.13±0.006 µmol glycerol/mg protein. *P < 0.0001.

View larger version (9K): [in this window] [in a new window]   Figure 7. The effect of 24 h KCl treatment on basal lipolysis in human adipocytes. Human adipocytes were incubated with or without 10 mM KCl for 24 h. Lipolysis was determined as described in Materials and Methods. Data are presented as mean ±SD. Data are normalized to control value of 0.10 ± 0.007 µmol glycerol/mg protein. *P < 0.005.

	DISCUSSION

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Previous studies of isolated adipose tissue in vitro demonstrated that the basal lipolytic rate of adipose tissue from yellow agouti mice was about 50% that of wild-type littermates (20–22). The response of this obese adipose tissue to epinephrine (21), theophylline (21), and the ß agonists LY79771 (22) and LY104119 (20) was also lower than normal. It was proposed that this may not result from the direct action of agouti protein, as decreased lipolysis was also observed in other obese models, including ob/ob and db/db mice, compared to controls (3).

However, our data demonstrated that recombinant agouti protein directly inhibited basal and agonist-stimulated lipolysis in primary cultured human adipocytes. We propose that this may represent a coordinated regulation of lipid metabolism in adipose tissue by agouti, as agouti inhibits lipolysis at the same dosage at which it stimulates lipogenesis (13). This coordinated process may be responsible for the accumulation of triglyceride in adipose tissue and may thereby contribute to the development of obesity. As the human homologue of agouti is expressed in adipose tissue (16), agouti may exert its action on adipocytes via a paracrine mechanism, similar to its action in pigment regulation within the hair bulb.

Agouti regulation of coat color is exerted through competitive antagonism of -MSH binding to the melanocortin receptor (MCR-1; 7). Agouti is also a potent antagonist of other melanocortin receptors (8). Since MCR-2 (the ACTH receptor) and MCR-5 are expressed in adipocytes (24) and ACTH is a potent lipolytic hormone (25), it is possible that agouti's antilipolytic effect may similarly involve competitive antagonism of this receptor. However, our data show that, although agouti blocked ACTH-induced lipolysis in primary cultured human adipocytes ( Fig. 1), it also blocked forskolin-induced lipolysis ( Fig. 2). This demonstrates that although agouti may competitively antagonize MCR-2, the antilipolytic action of agouti also includes a significant effect distal to the ACTH receptor. This concept is further supported by agouti inhibition of basal lipolysis in the 24 h treatment, even in the absence of ACTH ( Fig. 6).

Agouti may contribute to the obese phenotype via both MCR-dependent and independent mechanisms. It has recently been demonstrated that homologous recombinant knockout mice of the MCR-4 gene became obese and insulin resistant with a time course similar to that found in yellow (A^vy) mice (30). Moreover, intracerebroventricular administration of selective MCR-4 agonists has been shown to inhibit feeding behavior (31). These data clearly support the hypothesis that agouti-induced hyperphagia is mediated via MCRs, particularly MCR-4. Recent studies also demonstrate that blockade of MCR-4 prevented the anorexic effects of leptin, providing direct evidence for a role of MCR-4 signaling in leptin action (32). Although obese yellow mice do express high levels of leptin, this `leptin resistance' is not due to an intrinsic defect blocking leptin action, as removal of leptin from these mice restores complete leptin sensitivity (33). Accordingly, it is reasonable to propose that increased leptin secretion in obese yellow mice may serve peripherally to limit agouti-induced obesity, whereas central effects of leptin are attenuated by agouti antagonism of MCR-4.

However, data in this report as well as in our previous reports of agouti modulation of Ca²⁺ signaling and lipogenesis (11, 13–15) argue for peripheral effects of agouti that are independent of MCR antagonism. Moreover, we have recently reported that daily administration of NDP-MSH, a potent ligand and agonist of all of the MCRs, into transgenic mice ubiquitously overexpressing the wild-type agouti protein, failed to exert any effect on the obese diabetic phenotype (12). This lack of effect was not due to lack of systemic availability, as marked effects on coat pigmentation and core temperature were noted. Thus, although agouti modulation of feeding behavior appears to be mediated via MCR-4 antagonism, peripheral effects that contribute to the obese/diabetic phenotype appear to be, in part, independent of MCR antagonism.

We have found agouti to regulate Ca²⁺ signaling in several cell types (11), including adipocytes. Recent data demonstrated that increasing [Ca²⁺]_i by AVP and epidermal growth factor in rat adipocytes inhibits agonist-induced lipolysis in a dose-dependent manner (24). Our data show that increasing Ca²⁺-influx into adipocytes through either voltage- or receptor-operated Ca²⁺ channels, using KCl or AVP, respectively, also inhibited forskolin-induced lipolysis ( Figs. 3 and 5). KCl (10 mM) also inhibited basal lipolysis in the long-term (24 h) treatment ( Fig. 7). Further, the inhibitory effect of agouti on basal and forskolin-induced lipolysis was blocked by the Ca²⁺ channel antagonist nitrendipine. These data demonstrate that agouti's antilipolytic effect is mediated by Ca²⁺ signaling.

[Ca²⁺]_i may play an important role in the metabolic disorder of obesity and insulin resistance (17–19). Obese patients exhibit elevated basal [Ca²⁺]_i in adipocytes (19). An optimal range of [Ca²⁺]_i has been demonstrated for maximizing insulin-stimulated glucose transport, with elevation beyond this optimal range causing diminished cellular responsiveness to insulin (18). Similarly, although Ca²⁺ is known to be necessary for ACTH-induced lipolysis in adipocytes (34, 35), our data demonstrated that elevating [Ca²⁺]_i results in reduced lipolysis. This suggests that an optimal range of [Ca²⁺]_i may also exist for maximizing agonist-stimulated lipolysis.

Although we have demonstrated that recombinant agouti protein exerted antilipolytic effect in human adipocytes, the target of agouti inhibition of lipolysis is not clear. Since agouti inhibited both ACTH- and forskolin-induced lipolysis, it is possible that agouti may cause a defect in cAMP signaling beyond adenylate cyclase. Yen et al. (22) demonstrated that whereas adipose tissue from yellow agouti mice exhibited a lower lipolytic rate in response to epinephrine and theophylline, the response to dibutyryl cAMP, a permeable cAMP analog, was normal. This indicates that agouti may inhibit the generation of cAMP. The sustained inhibition of lipolysis seen in long-term treated human adipocytes may be due to prolonged defect in cAMP production. Alternatively, we have demonstrated that there is an agouti response element in the fatty acid synthase promoter (36). It is possible that hormone-sensitive lipase expression may be similarly modulated by agouti, although this possibility has not yet been investigated.

This study shows that recombinant agouti protein exhibits potent antilipolytic effect via a Ca²⁺-dependent mechanism in primary cultured human adipocytes. This appears to be a direct effect that is not mediated by MCR antagonism. This inhibition of lipolysis, combined with agouti-induced lipogenesis, represents a coordinate control of adipocyte lipid metabolism, which may contribute to agouti-induced obesity.

	FOOTNOTES

 
¹ Correspondence: University of Tennessee, 1215 W. Cumberland Ave., Rm. 229,Knoxville, TN 37996–1900, USA. E-mail: mzemel{at}utk.edu

² Abbreviations: MSH, melanocyte-stimulating hormone; MCR, melanocortin receptor(s); ACTH, adrenocorticotropic hormone; HBSS, Hank's balanced salt solution; AVP, arginine vasopressin.

Received for publication February 27, 1998.

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