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Role of the sulfonylurea receptor in regulating human adipocyte metabolism

HANG SHI^*, NAIMA MOUSTAID-MOUSSA^*, W. O. WILKISON and MICHAEL B. ZEMEL^*1

^* Department of Nutrition, The University of Tennessee, Knoxville, Tennessee 37996, USA; and
Zen-Bio, Inc., Research Triangle Park, North Carolina 27709, USA

¹Correspondence: University of Tennessee, 1215 West Cumberland Avenue, #229, Knoxville, TN 37996-1900, USA. E-mail: mzemel{at}utk.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A regulatory role for intracellular Ca²⁺ ([Ca²⁺]_i) in adipocyte lipogenesis, lipolysis and triglyceride accumulation has been demonstrated. Compounds acting on the pancreatic sulfonylurea receptor (SUR) to increase (e.g., glibenclamide) or decrease (e.g., diazoxide) [Ca²⁺]_i cause corresponding increases and decreases in weight gain. However, these weight gain and loss effects have been attributed to insulin release rather than to the primary effects of these compounds on the adipocyte SUR and its associated K_ATP channel. Accordingly, we have evaluated the direct role of the human adipocyte SUR in regulating adipocyte metabolism. We used RT-PCR with primers for a highly conserved region of SUR1 to demonstrate that human adipocytes express SUR1. The PCR product was confirmed by sequence analysis and used as a probe to demonstrate adipocyte SUR1 expression by Northern blot analysis. Adipocytes exhibited glibenclamide dose-responsive (0–20 µM) increases in [Ca²⁺]_i (P<0.05). Similarly, glibenclamide (10 µM) caused a 67% increase in adipocyte fatty acid synthase activity (P<0.001), a 48% increase in glycerol-3-phosphate dehydrogenase activity (P<0.01) and a 68% inhibition in lipolysis (P<0.01), whereas diazoxide (10 µM) completely prevented each of these effects. These data demonstrate that human adipocytes express a SUR that regulates [Ca²⁺]_i and, consequently, exerts coordinate control over lipogenesis and lipolysis. Accordingly, the adipocyte SUR1 may represent an important target for the development of therapeutic interventions in obesity.—Shi, H., Moustaid-Moussa, N., Wilkison, W. O., Zemel, M. B. Role of the sulfonylurea receptor in regulating human adipocyte metabolism.

Key Words: adipocytes • intracellular Ca²⁺ • fatty acid synthase • SUR

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
INTRACELLULAR CA²⁺ ([Ca²⁺]_i)² plays a key role in the metabolic disorders associated with obesity and insulin resistance (1 2 3) . Recombinant agouti protein, an obesity gene product, causes a dose-responsive increase in [Ca²⁺]_i in a variety of cells (4 , 5) , including both murine and human adipocyte. Moreover, agouti protein promotes the expression and activity of fatty acid synthase (FAS), a key enzyme in de novo lipogenesis, and thereby increases triglyceride storage in a Ca²⁺-dependent manner (6) . In addition, we have also shown that agouti inhibits basal and agonist-stimulated lipolysis in primary cultured human adipocytes via a Ca²⁺-dependent mechanism (7) . Therefore, increasing [Ca²⁺]_i appears to promote triglyceride accumulation in adipocytes by exerting a coordinated control over lipogenesis and lipolysis.

Sulfonylureas such as glibenclamide are insulin secretagogues widely used to stimulate insulin secretion in the treatment of non-insulin-dependent diabetes mellitus. Sulfonylureas depolarize pancreatic ß cells by blocking K_ATP channels, thereby resulting in depolarization and secondary Ca²⁺ influx via L-type Ca²⁺ channels, which in turn triggers insulin release (8 , 9) . The ß cell receptor for sulfonylureas, sulfonylurea receptor 1 (SUR1), has been cloned (10) .

Alemzadeh et al. (11 , 12) have reported that diazoxide, a drug that activates the ß cell K_ATP channel and subsequently reduces [Ca⁺]_i and inhibits insulin release, exerts an antiobesity effect in obese Zucker rats. Further, they recently reported that diazoxide exerted a significant antiobesity effect in hyperinsulinemic obese adults (13) . However, this action was attributed to actions of diazoxide on ß cells rather than to the direct effects of diazoxide on adipocyte metabolism.

Accordingly, the present study was conducted to assess the direct role of the human adipocyte SUR in adipocyte metabolism. We report here that human adipocytes express SUR and exhibit a glibenclamide dose-responsive increase in [Ca²⁺]_i. Moreover, glibenclamide exerts lipogenic and antilipolytic effects in human adipocytes, whereas diazoxide completely blunts each of these effects. These data demonstrate that SUR in human adipocyte regulates [Ca²⁺]_i and thereby exerts coordinate control over lipogenesis and lipolysis.

	MATERIALS AND METHODS

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Isolation and culture of human adipocytes
Human subcutaneous adipose tissue was obtained from patients undergoing abdominal plastic surgery 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 described previously (14) . Briefly, adipose tissue was first washed several times with Hanks' balanced salt solution (HBSS), minced into small pieces, and digested with 0.8 mg/ml type 1 collagenase in a shaking water bath at 37°C for 30 min. Adipocytes were then filtered through a sterile 500 µM nylon mesh and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% fetal bovine serum, 100 Ul/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamicin. Cells were cultured in suspension and maintained in a thin layer at the top of culture media, which were changed every day. Cells were maintained viable and metabolically responsive under this culture condition for 7 days.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA from human adipocytes was extracted using CsCl₂ density centrifugation. mRNA from human adipocytes was isolated according to the manufacturer's instructions (micro poly(A) pure kit, Ambion Inc., Austin, Tex.). RT-PCR was performed essentially as described before (7) . Briefly, 400 ng of human adipocyte mRNA was reverse-transcribed to first-strand cDNA using random hexamer and reverse transcriptase (Perkin Elmer, Norwalk, Conn.) and amplified by PCR (Perkin Elmer). The PCR conditions were as follows: initial denaturation at 94°C for 5 min, followed by 34 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 1 min, and extension at 72°C for 2 min, with a final extension step at 72°C for 8 min with 0.5 µM 5' primer (5'-CATCATTGATGGCATTGACATCCGC) and 3' primer (5'-CTCTGGCTTATCGAACTCAAGGATGG), which correspond to nucleotide positions 4206–4230 and 4664–4689 in HSU63421, respectively. The amplified PCR products were then visualized by 1.2% agarose gel electrophoresis, purified (Geneclean kit, bio 101, Inc.), and subjected to sequence analysis using ABI PRISM system (model version 2. 1. 1).

Northern blot analysis
Northern blot analysis was conducted as described (15) . Human adipocyte mRNA (4 µg/well) was run in 1% agarose gel and transferred to nylon membrane, which was hybridized with human SUR1 cDNA probes eluted from PCR products, and radiolabeled using a random primer method. Unbound probe was removed by rinsing the membrane with 2x SSC for 30 min at room temperature and 0.1x SSC/0.1% SDS for 45 min at 60°C. Finally, the membrane was exposed to X-ray film at -80°C.

Intracellular Ca²⁺ ([Ca²⁺]_i) measurement
[Ca²⁺]_i in isolated human adipocytes was determined fluorometrically as described previously (2 , 16) . Briefly, the human adipocytes isolated as described above were incubated in DMEM medium overnight for cell recovery. Prior to [Ca²⁺]_i measurement, adipocytes were preincubated in serum-free medium for 2 h and rinsed with HBSS solution containing the following components (in mM): NaCl 138, CaCl₂ 1.8, MgSO₄ 0.8, NaH₂PO₄ 0.9, NaHCO₃ 4, glucose 20, glutamine 6, HEPES 20, and bovine serum albumin 1%. Cells were then loaded with Fura-2 acetoxymethyl ester (10 µM) in the same buffer for 45 min at 37°C in the dark with continuous shaking. To remove extracellular dye, cells were rinsed with HBSS three times and resuspended in this solution at a concentration of 2 x 10⁵ cells/ml. [Ca²⁺]_i was measured using dual excitation (340 and 380 nm) and single emission (510 nm) fluorometry. After the establishment of stable baseline, the response to glibenclamide (10 and 20 µM) was determined. Digitonin (25 µM) and Tris/EGTA (100 mM) were used to measure maximal and minimal fluorescence to calibrate the signals, and final [Ca²⁺]_i was calculated by the equation of Grynkiewicz et al. (17) .

FAS and glycerol-3-phosphate dehydrogenase (GPDH) activity assay
Human adipocytes were incubated in 24-well plates with the treatments indicated for 48 h. FAS and GPDH activities were determined spectrophotometrically in crude cytosolic extracts of human adipocytes by measuring the oxidation rate of NADPH or NADH, respectively, as described previously (18) . The protein correction was measured by a modified Bradford method using Coomassie blue dye (Pierce, Rockford, Ill.).

Lipolysis assay
Human adipocytes were treated as described above; glycerol released into the culture medium was determined as an indicator for lipolysis, using a one-step enzymatic fluorometric method (19) . After treatment medium was obtained, HClO₄ was added for deproteinization. The sample was then centrifuged to precipitate protein and the supernatant was neutralized with NaOH before glycerol assay.

Statistical analysis
All data are expressed as mean ± SE. Data were evaluated for statistical significance by analysis of variance or t test.

	RESULTS

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Human SUR1 is expressed in adipocytes. Using RT-PCR, we detected a 490 bp SUR1 fragment, which represents a highly conserved region in SUR gene (Fig. 1 ). Sequence analysis confirmed the identity of this PCR product, which was 100% identical to human SUR1. No SUR1 transcript was detectable in mRNA isolated from human preadipocytes (Fig. 1) . The PCR product was purified and used as a probe to detect adipocyte SUR1 expression by Northern blot analysis, which showed an ~5 Kb transcript of SUR1 mRNA in adipose tissue (Fig. 2 ).

View larger version (60K): [in this window] [in a new window]   Figure 1. Detection of the expression of SUR1 gene in human adipocyte by RT-PCR. The PCR product, a 490 bp SUR1 fragment (designated by arrow), was visualized by 1.2% agarose gel electrophoresis. Lane 1: human pancreatic ß cells as positive control; lane 2: human preadipocytes without reverse transcriptase; lane 3: human preadipocyte with reverse transcriptase; lane 4: human adipocytes without reverse transcriptase; lane 5: human adipocytes with reverse transcriptase; lane 6: 18s fragment amplified as RT-PCR system positive control; lane 7: DNA marker. RT-PCR was performed as described in Materials and Methods.

View larger version (30K): [in this window] [in a new window]   Figure 2. Detection of the expression of SUR1 mRNA in human adipocyte by Northern blot analysis. A 5 Kb transcript of SUR1 mRNA was detected in human adipocyte. Lane 1: human pancreas mRNA as positive control; lane 2: human adipocyte mRNA. Northern blot analysis was conducted as described in Materials and Methods.

To determine the functional significance of this adipocyte SUR, the [Ca²⁺]_i response to glibenclamide was evaluated. Glibenclamide (10 and 20 µM) induced sustained increases of [Ca²⁺]_i in human adipocytes in a dose-dependent manner, with 34.5 ± 2.6 nM and 69.7 ± 4.0 nM increases over baseline (P<0.05), respectively (Fig. 3 and Fig. 4). However, human preadipocytes did not exhibit any [Ca²⁺]_i response to glibenclamide. This is consistent with the absence of SUR1 in human preadipocytes noted above.

View larger version (10K): [in this window] [in a new window]   Figure 3. Effects of glibenclamide (20 µM) on stimulation of [Ca2+]i in isolated human adipocyte. Glibenclamide was added at time designated by the arrow. [Ca2+]i measurement as described in Materials and Methods.

To study the role of adipocyte SUR1 in regulating lipogenesis, we treated human adipocyte with the SUR1 agonist and antagonist, using FAS and GPDH activities as lipogenic markers. Glibenclamide (10 µM) caused a 67% increase in adipocyte FAS activity (0.692±0.052 NADPH nM·min^-1·mg protein^-1 vs. 1.154±0.010 NADPH nM·min^-1·mg protein^-1; P<0.001, Fig. 5 ), which was completely blocked by 10 µM diazoxide, a K_ATP channel activator, and partially inhibited by nitrendipine, an L-type Ca²⁺ channel antagonist. Similarly, 10 µM glibenclamide stimulated a 48% increase in GPDH activity (554.0±33.0 NADPH nM·min^-1·mg protein^-1 vs. 821.0±73.4 NADPH nM·min^-1·mg protein^-1; P<0.01, Fig. 6 ), which was totally blocked by 10 µM diazoxide.

View larger version (14K): [in this window] [in a new window]   Figure 5. The effect of glibenclamide on FAS activity in human adipocytes. Human adipocytes were treated with 10 µM glibenclamide alone, 10 µM glibenclamide plus 10 µM diazoxide, or 10 µM glibenclamide plus 30 nM nitrendipine for 48 h. FAS activity was measured as described in Materials and Methods. *P < 0.001 vs. control.

View larger version (14K): [in this window] [in a new window]   Figure 6. The effect of glibenclamide on GPDH activity in human adipocytes. Human adipocytes were treated with 10 µM glibenclamide or 10 µM glibenclamide plus 10 µM diazoxide for 48 h. GPDH activity was measured as described in Materials and Methods. *P < 0.01 vs. control.

We next investigated the role of adipocyte SUR1 on lipolysis. A 48 h treatment with 10 µM glibenclamide caused a 68% inhibition in lipolysis (0.193±0.050 NADH µM/mg protein vs. 0.061 ± 0.009 NADH µM/mg protein, P<0.05; Fig. 7 ), which was recovered to 75% and 50% of control by diazoxide and nitrendipine, respectively.

View larger version (13K): [in this window] [in a new window]   Figure 7. The effect of glibenclamide on lipolysis in human adipocytes. Human adipocytes were treated with 10 µM glibenclamide alone, 10 µM glibenclamide plus 10 µM diazoxide, or 10 µM glibenclamide plus 30 µM nitrendipine for 48 h. Glycerol release was determined as described in Materials and Methods. *P < 0.01 vs. control.

	DISCUSSION

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We have demonstrated that the sulfonylurea receptor is expressed in human adipose tissue and found that this receptor responds to glibenclamide, a typical agonist for the pancreatic form of the receptor. This receptor mediates physiological responses such as lipogenesis and lipolysis in the adipocytes. These results implicate the SUR in mediating energy homeostasis in an extrapancreatic tissue and suggest that the adipocyte SUR may be a suitable target for the identification of novel antiobesity agents.

Intracellular Ca²⁺ ([Ca²⁺]_i) appears to play a key role in the metabolic disorders associated with obesity and insulin resistance, and sustained high levels of [Ca²⁺]_i may contribute to these derangements (1 2 3) . Several reports from our laboratory demonstrated that [Ca²⁺]_i modulates de novo lipogenesis and lipolysis in both rodent and human adipocytes (4 5 6 7) . Sulfonylureas are a family of oral drugs used to promote insulin release in the treatment of type II diabetes. These insulin secretagogues bind to the sulfonylurea receptor (SUR) of pancreatic ß cells and then block the conductance of an ATP-dependent potassium channel (K_ATP channel) (8) . The attenuation of potassium current by blocking this channel depolarizes the ß cells and thereby induces Ca²⁺ entry via L-type calcium channels (20) , leading to increased insulin secretion (21) . The SUR is a member of the ATP binding cassette proteins, with multiple membrane-spanning domains and two nucleotide binding folds (10) . The SUR itself does not form the ion-conducting part of the K_ATP channel. However, SUR combines and interacts with an inward rectifier K⁺ channel (Kir6.2) to generate K_ATP channel (22) . Thomas et al. (23) reported that familial persistent hyperinsulinemic hypoglycemia of infancy, an autosomal recessive disorder characterized by unregulated insulin secretion, was associated with two separate SUR gene splice site mutations.

Patients treated with glibenclamide frequently experience weight gain as a side effect. Conversely, reports by Alemzadeh et al. (11 12 13) showed that diazoxide, which inhibits SUR by activating K_ATP channels, exerts an antiobesity effect in obese Zucker rats and hyperinsulinemic obese humans. These effects of glibenclamide and diazoxide on body weight have been attributed to their effect on circulating insulin rather than to any direct effect on adipocytes (11 12 13) . However, our data indicate that these effects may also be attributable in part to direct effects on the adipocyte SUR and K_ATP channel.

SUR agonists have previously been demonstrated to exert direct effects on adipocytes. Draznin et al. (2 , 16) reported that glibenclamide increased [Ca²⁺]_i in isolated rat adipocytes in a dose-dependent manner by promoting Ca²⁺ influx through voltage-dependent Ca²⁺ channels, while this effect was blocked by nitrendipine. Moreover, glibenclamide has been reported to potentiate peripheral insulin effect in isolated adipocytes (24 , 25) . These data support our observation of a direct effect of glibenclamide on adipocyte metabolism. In contrast, Rajan et al. (26) were unable to identify high-affinity SUR in either isolated rat adipocytes or 3T3-L1 adipocytes. Moreover, they were unable to inhibit ⁸⁶Rb⁺ efflux (a surrogate for K_ATP channel activity) or increase [Ca²⁺]_i with glibenclamide. The reason for this discrepancy is not clear. However, several other investigators have reported both specific binding of sulfonylurea and specific postreceptor effects in murine and rat adipocytes. For example, rat adipocytes exhibit specific, saturable glibenclamide binding (K_D of 1–3 µM), which is displaced by other sulfonylureas, and sulfonylurea treatment of isolated rat adipocytes potentiates insulin receptor of glucose transport (24 , 25 , 27) . Further, Muller et al. (28 , 29) reported that glimepiride exhibits specific binding to 3T3-L1 and rat adipocytes, resulting in an insulin-mediated stimulation of glucose transport and nonoxidative glucose disposal. These effects were attributed to sulfonylurea-induced inhibition of cAMP level and protein kinase A activity. Thus, rodent adipocytes appear to exhibit SUR binding and functional response to this binding.

Similarly, data presented here demonstrate that SUR appears to play an important role in adipocyte metabolism. We have demonstrated that human adipocytes express SUR1 by both RT-PCR and Northern blot analysis, whereas human preadipocytes do not. This provides a mechanism for SUR agonists and antagonists to modulate [Ca²⁺]_i levels. Indeed, glibenclamide elicited sustained increases in human adipocyte [Ca²⁺]_i, whereas human preadipocytes were not responsive to glibenclamide. Similarly, glibenclamide (10 µM) caused a 67% increase in FAS activity, a 48% increase in GPDH activity, and a 68% inhibition in lipolysis, whereas diazoxide (10 µM) completely prevented each of these effects. These observations are consistent with our previous reports that modulation of [Ca²⁺]_i in adipocytes may exert coordinated regulation of lipogenesis and lipolysis, and demonstrate effects of SUR agonists that are similar to the physiological effects of agouti protein in both human and murine adipocytes (6 , 7) . Thus, these data suggest that previous observations of diazoxide-induced weight loss may be attributed to the effects of this compound on the adipocyte SUR rather than primarily to effects on insulin release.

The human chromosome region encoding both the SUR and the associated inward rectifying K⁺ channel, 11p15.1 (30) , also contains the human homologue of Tubby, a locus responsible for severe obesity in mice (31) . Moreover, a significant association has been reported between an exon 22 allelic variant of the SUR gene and obesity in French Caucasians (32) . In addition, linkage between the SUR region and a subgroup of morbidly obese families was also noted (32) . Thus, the SUR locus may contribute to genetic susceptibility to obesity (32) . Whether this contribution is based on alteration in insulin secretion or an extrapancreatic role of the SUR is unknown. However, our data suggest a potential role for the human adipocyte SUR in modulating energy storage and thereby potentially contributing to obesity.

We conclude that modulation of the human adipocyte SUR results in corresponding significant modulation of adipocyte energy storage. This provides a potential new mechanism for previous observations of sulfonylurea-induced weight gain and diazoxide-induced weight loss in addition to the insulin-mediated effects of these compounds. Our data suggest that diazoxide-induced antagonism of adipocyte lipogenesis and promotion of lipolysis, possibly coupled with suppression of insulin release, provide a likely mechanism for the antiobesity effect of diazoxide. Accordingly, the human adipocyte SUR1 appears to represent an important target for further development of therapeutic intervention in obesity.

View larger version (14K): [in this window] [in a new window]   Figure 4. The dose-responsive effect of glibenclamide (10 µM and 20 µM) on [Ca2+]i in human adipocytes. *P < 0.05 vs. control; **P < 0.05 vs. 10 µM glibenclamide.

	FOOTNOTES

 
² Abbreviations: [Ca²⁺]_i, intracellular Ca²⁺; DMEM, Dulbecco's modified Eagle's medium; FAS, fatty acid synthase; GPDH, glycerol-3-phosphate dehydrogenase; HBSS, Hanks' buffered salt solution; SUR, sulfonylurea receptor.

Received for publication February 23, 1999. Revised for publication March 23, 1999.

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