VPAC2-R Mediates the Lipolytic Effects of Pituitary Adenylate Cyclase-Activating Polypeptide/Vasoactive Intestinal Polypeptide in Primary Ra
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内分泌学杂志 2005年第2期
Section for Molecular Signaling, Department of Cell and Molecular Biology (L.?., G.E., E.D.), and Department of Medicine (B.A.), Lund University, SE-221 84 Lund, Sweden
Address all correspondence and requests for reprints to: Dr. Lina ?kesson, Biomedical Center C11, Lund University, SE-221 84 Lund, Sweden. E-mail: lina@akesson@.nu.
Abstract
The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are structurally and functionally related. Their actions have been shown to be mediated by three different receptor subtypes: PAC1-R, which has exclusive affinity for PACAP, and VPAC1-R and VPAC2-R, which have equal affinity for PACAP and VIP. We recently showed that PACAP38 induces lipolysis in rat adipocytes, and in the present study we examined whether VIP has similar effects and which of the three receptors mediates this PACAP/VIP action. We showed by RT-PCR that all three receptor subtypes are present in rat adipocytes. We demonstrated that VIP (1–100 nM), like PACAP38, stimulates lipolysis in isolated adipocytes, as determined by glycerol release. By a pharmacological approach, using antagonists and agonists specific for the receptor subtypes, we elucidated the mechanisms by which PACAP38 and VIP mediate their lipolytic effects. We found that antagonists of PAC1-R [PACAP(6–38)] and VPAC1-R (PG97–269) did not affect lipolysis induced by 0.1–100 nM PACAP38 or VIP, and that a VPAC1-R agonist [K15, R16, L27VIP(1–7)GRF(8–27)] did not affect lipolysis at 1–1000 nM. However, two different VPAC2-R agonists [Hexa-VIP(1–28) and Ro25-1553] clearly mimicked the lipolytic effect of PACAP38 and VIP. In addition, the VPAC2-R antagonist PG99–465 (100 nM) caused right-shifted dose-response curves of PACAP38- and VIP-induced lipolysis. These results therefore provide evidence that all three PACAP/VIP receptor subtypes are expressed in primary rat adipocytes, but that the VPAC2-R subtype is responsible for mediating the lipolytic effects induced by PACAP38 and VIP.
Introduction
CYCLIC AMP IS a major modulator of lipid metabolism in adipocytes, and increased formation of cAMP is associated with enhanced hydrolysis of triglycerides to free fatty acids and glycerol (lipolysis). The cascade underlying this action is initiated by binding of catecholamines to ?-adrenergic receptors, which results in the formation of cAMP and the activation of protein kinase A (PKA), with subsequent activation of hormone-sensitive lipase (1). Lipolysis is counteracted by insulin, mainly via insulin-dependent activation of the cAMP-degrading enzyme phosphodiesterase 3B (2). The net effect of formation and degradation of cAMP, therefore, governs lipolysis; hence, compounds affecting cAMP in adipocytes are expected to affect lipolysis.
Two compounds that may regulate lipolysis are pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), because both are strong activators of cAMP formation, as demonstrated in several cell types. They belong to the glucagon superfamily of peptides, and they are extensively distributed in both the central and peripheral nervous system and seem to function as neuropeptides (3). PACAP exists in two isoforms, PACAP38 and PACAP27. PACAP27 consists of the 27 N-terminal amino acids of PACAP38 (4). PACAP shows 68% structural homology with VIP. Three receptor subtypes mediate the actions of PACAP and VIP; all are of the seven-transmembranous, G protein-coupled type. Two of the receptor subtypes, VPAC1-R and VPAC2-R, show equal affinity for PACAP and VIP, whereas the third receptor subtype, PAC1-R, has unique affinity for PACAP (5, 6). The PACAP/VIP receptors are distributed throughout the body, in the respiratory system, the gastrointestinal tract, and the central nervous system (6). The receptors have also been shown to be expressed in organs of importance for energy homeostasis, and there is an increasing amount of evidence that PACAP and VIP have effects on lipid and carbohydrate metabolism (7). For instance, PACAP/VIP have been shown to stimulate hepatic glucose output from the perfused rat liver (8, 9, 10) and to increase glucose-stimulated insulin secretion in mice (11, 12), calves (13), and humans (14). Furthermore, previous findings show that PACAP is able to enhance insulin-mediated glucose uptake in 3T3-L1 adipocytes (15). Our recent findings showed that PACAP38 has both catabolic and anabolic effects on primary rat adipocytes depending on the presence or absence of insulin. In the absence of insulin, lipolysis was increased, whereas in the presence of insulin, lipolysis was suppressed, and insulin-induced lipogenesis was increased (16). In this study we examined what receptor(s) is involved in the lipolytic effects of PACAP38 and VIP. We show that mRNAs for all three receptor subtypes are present in primary rat adipocytes. By the use of specific PACAP/VIP receptor agonists and antagonists, we present evidence that PACAP38/VIP-stimulated lipolysis is exclusively mediated by VPAC2-R, presumably by activating PKA.
Materials and Methods
Materials
VPAC1-R antagonist PG97–269 (Ac-His1, D-Phe2, K15, R16, L27)VIP(3, 4, 5, 6, 7)/GRF(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27), VPAC1-R agonist (K15, R16, L27)VIP(1, 2, 3, 4, 5, 6, 7)/GRF(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27), VPAC2-R antagonist PG99–465, and VPAC2-R agonists Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) and Ro25-1553 were gifts from Prof. Patrick Robberecht (University of Brussels, Brussels, Belgium). PACAP(6–38) was purchased from Bachem (Weil am Rhein, Germany). ATP, kemptide phosphate acceptor peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), PACAP38, VIP, antibodies against VPAC1-R and VPAC2-R, 3-isobutyl-1-isobutylxanthine, and protein kinase inhibitor were purchased from Sigma-Aldrich Corp. (Stockholm, Sweden). Glycerol-3-phosphate dehydrogenase, glycerokinase, and nicotinamide adenine dinucleotide were purchased from Roche (Stockholm, Sweden). Insulin was a gift from Novo Nordisk (Copenhagen, Denmark). OPC3911 was a gift from Otsuka Pharmaceutical Corp. (Tokyo, Japan). The primers used were synthesized by MWG Biotech (Ebersberg, Germany).
Animals
Male Sprague Dawley rats, 36 d of age, were obtained from B&K Universal (Stockholm, Sweden). The rats were maintained in a facility with controlled temperature and a 12-h light, 12-h dark cycle. The animals were given free access to food and water. All experiments were approved by the ethical committee of Lund University.
Preparation of adipocytes
Epididymal fat pads were isolated and digested in collagenase and washed as described previously (17). Packed cell volume was determined by centrifuging adipocyte suspensions in hematocrit tubes. The adipocytes were resuspended in wash buffer (KRH) containing 25 mM HEPES (pH 7.5), 120 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 1% BSA, 2 mM glucose, and 200 nM adenosine. In experiments in which antagonists were used, the cells were preincubated for 30 min with antagonists or water before proceeding with the stimulations with PACAP38/VIP.
RT-PCR of PACAP/VIP receptors
Total RNA was extracted from adipocytes, using RNAzol (Tel-Test, Inc., Friendswood, TX), and cDNA was produced using the RT-for-PCR-kit (BD Clontech, Stockholm, Sweden) from 1 μg RNA, using oligo(deoxythymidine)18 priming. For amplification of PACAP and VIP receptors, PAC1-R, VPAC1-R, and VPAC2-R, the following receptor-specific primer combinations were used: PAC1-R, 5'-CTGACTGCATCTAGAAGG-3' (exon 1) and 5'-TGGATGAAGTTGCGAGTGCA-3' (exon 5; expected product, 498 bp); VPAC1-R, 5'-CAGCTGGAGAATGAAACCAC-3' (exon unknown) and 5'-CATGAAGAGATGCATGTGGA-3' (exon unknown; expected product, 389 bp); and VPAC2-R, 5'-GTGAGCAGCATCCACCCAGAAT-3' (exon 2) and 5'-GCTGGAGTAGAGCACACTGT-3' (exon 5; expected product, 506 bp). cDNA (5 μl) was subjected to one cycle of 94 C for 10 min before 40 cycles of amplification (94 C for 1 min, 57 C for 1 min, and 72 C for 1.5 min) using 1 U AmpliTaq Gold (PerkinElmer, Stockholm, Sweden), 1.2 mM MgCl2, and 0.3 mM deoxy-NTP.
Western blot analysis of VPAC1-R and VPAC2-R
Cells were prepared and washed as described above and suspended in 50 mM N-Tris[hydroxymethyl]metyl-2-aminoethanesulfonic acid (pH 7.4), 250 mM sucrose, 1 mM EDTA, and 0.1 mM EGTA (10% cell suspension). Homogenization was carried out in the same buffer supplemented with 10 μg /ml antipain, 1 μg/ml pepstatin A, and 10 μg/ml leupeptin. Homogenates were briefly centrifuged for 10 min at 13,000 rpm at 4 C. The fat cake was discarded as well as cell debris. The fat-free supernatant was withdrawn and centrifuged again at 50,000 x g for 60 min at 4 C. The resulting membrane fractions were rehomogenized and solubilized on ice for 60 min in homogenization buffer containing 1% C13E12 (nonionic alkyl polyoxyethylene glycol detergent, Berol Kemi AB, Stenungsund, Sweden) and 150 mM NaCl. A final centrifugation was carried out to remove unsolubilized membranes at 10,000 rpm for 10 min at 4 C. Homogenates were subjected to 7% SDS-PAGE, followed by electrotransfer to polyvinylidene difluoride membranes (Millipore Corp., Sundbyberg, Sweden). The membrane was blocked for 60 min using 0.5% gelatin in wash buffer consisting of 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% (vol/wt) Tween 20. Membranes were incubated overnight with a monoclonal VPAC1-R or VPAC2-R antibody, followed by incubation with a secondary mouse antibody conjugated with horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK) for 45 min. Proteins were detected using chemiluminescent Super Signal West PicoLuminol/Enhancer solution (Boule Nordic AB, Huddinge, Sweden) and were visualized using the Fuji LAS 1000 Plus system (Fuji Photo Film Co. Ltd., Tokyo, Japan).
PKA assay
Adipocytes were suspended in KRH and diluted to 6% cell suspension. Stimulations were carried out as indicated in the figure legends and were stopped by adding 250 μl ice-cold homogenization buffer containing 50 mM Tris (pH 7.4), 50 mM EDTA, 2 mM 3-isobutyl-1-isobutylxanthine, 50 μM OPC3911 (phosphodiesterase inhibitor), 10 μg/ml antipain, 10 μg/ml leupeptin, and 1 μg/ml pepstatin A. Samples were immediately put on ice and subsequently centrifuged at 13,000 rpm for 10 min. The fat cake and pellet were discarded, and 10 μl infranatants were incubated for 20 min at 30 C with 5 μl phosphorylation mix containing 20 mM Tris-EDTA-sucrose (pH 7.4), 50 mM MgSO4, 0.2 mM ATP, 5 mM dithiothreitol, 4 mg/ml substrate peptide (kemptide), and 5 μCi [-32P]ATP with or without 10 μM protein kinase inhibitor (to correct for non-PKA activity). The reactions were stopped by the addition of 10 μl 1% BSA and 1 mM ATP (pH 3.0) and were precipitated with 10 μl 31% trichloroacetic acid. Samples were left to precipitate for 15 min, then centrifuged at 10,000 rpm for 3 min at 4 C. Ten-microliter aliquots were transferred to p81 membranes and after drying were washed three times with 75 mM H3PO4 and once with acetone. The amount of 32P incorporated was determined by scintillation counting.
Lipolysis measurements
As a measurement of lipolysis, glycerol release was determined as previously described (18). Briefly, 5% cell suspension (in KRH) was stimulated with PACAP/VIP or agonists as indicated in figure legends. When studies using antagonists were performed, cells were preincubated with water or antagonists before proceeding with PACAP/VIP stimulations. Incubations were stopped after 30 min of shaking (150 rpm, 37 C) and were put on ice for 20 min. One milliliter of hydrazine buffer containing 50 mM glycine (pH 9.8), 0.05% hydrazine hydrate, 1 mM MgCl2 supplemented with 0.75 mg/ml ATP, 0.375 mg/ml nicotinamide adenine dinucleotide, 25 μg/ml glycerol-3-phposphate dehydrogenase, and 0.5 μg/ml glycerokinase was added to 200 μl collected medium. After incubation for 40 min at room temperature, OD340 was measured, and glycerol release was calculated.
Statistical analysis
All data are presented as the mean ± SEM. Statistical analysis was performed using the unpaired t test. Statistical significance was defined as P < 0.05.
Results
Effects of VIP on lipolysis in primary rat adipocytes
We have previously shown that PACAP38 induces lipolysis in rat adipocytes (16). The structural homology and the fact that PACAP and VIP have equal affinity for two of the three PACAP receptors indicate that VIP could stimulate lipolysis in these cells (6). In accordance with previous results reported by Hauner et al. (19), we achieved dose-dependent glycerol release by stimulating adipocytes for 30 min with 0.1–100 nM VIP (Fig. 1). VIP had no effect on lipolysis at 0.1 or 1 nM VIP. At 10 nM VIP, glycerol release was 5-fold compared with that of nonstimulated cells, whereas 100 nM VIP induced a 7-fold increase. In addition, we studied the effect of 1.7 nM insulin on VIP-induced lipolysis. Isolated cells were incubated with 0.1–100 nM VIP in the absence or presence of 1.7 nM insulin. Insulin almost completely abolished VIP-induced lipolysis (Fig. 1). Lipolysis was inhibited 70% by insulin when induced by 100 nM PACAP or 10 or 100 nM VIP. This is in agreement with our previous finding that insulin is able to suppress PACAP-induced lipolysis.
FIG. 1. Effect of insulin on VIP-induced lipolysis. Adipocytes were stimulated with 0.1–100 nM VIP with () or without () 1.7 nM insulin for 30 min. PACAP38 (100 nM) was used as a positive control. Glycerol release was calculated and expressed as the fold increase compared with the unstimulated control (Ctrl). Statistical significance was assessed by t test (**, P < 0.01). Data are presented as the mean ± SEM. Each condition was performed in triplicate in three independent experiments.
Expression of PAC1-R, VPAC1-R, and VPAC2-R in rat adipocytes
It has previously been shown by ribonuclease protection assay that PAC1-R, VPAC1-R, and VPAC2-R mRNAs are expressed in human adipose tissue (20). To confirm the occurrence of these receptors in rat adipocytes, RT-PCR was performed using the primers indicated in Materials and Methods. mRNA from rat whole brain tissue was used as a positive control for all three receptors (data not shown). As shown in Fig. 2A, mRNAs of PAC1-R (PCR product, 498 bp), VPAC1-R (PCR product, 389 bp), and VPAC2-R (PCR product, 506 bp) were all present in rat primary adipocytes. In addition, Western blot analysis showed bands corresponding to the expected molecular masses of VPAC1-R (45–50 kDa) and VPAC2-R (49–52 kDa) proteins (Fig. 2B).
FIG. 2. Expression and effects on PKA activity of PACAP/VIP receptors in primary rat adipocytes. A, PCR analysis was performed on rat cDNA from adipocytes using the primers listed in Materials and Methods (lane 1, PAC1-R; lane 2, VPAC1-R; lane 3, VPAC2-R). PCR products were separated on 1% agarose and visualized with ethidium bromide. B, SDS-PAGE and Western blot analysis were performed on membrane fractions from primary rat adipocytes (Adip.) and whole rat brain (Brain) using a VPAC1-R antibody (upper panel) or a VPAC2-R antibody (lower panel). C, Adipocytes were stimulated with 30 nM isoproterenol (Iso), 100 nM VPAC1-R agonist (VPAC1-R Ago), or VPAC2-R agonists (Hexa-VIP and Ro25-1553). PKA activity was calculated and expressed as the fold increase compared with unstimulated cells (Ctrl). Statistical significance was assessed by t test (***, P < 0.001). Data are presented as the mean ± SEM. Each condition was performed in duplicate in four independent experiments.
Effects of VPAC1-R and VPAC2-R agonists on PKA activity
The VPAC1-R-specific agonist (21) and the VPAC2-R-selective agonistic peptides, Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) and Ro25-1553, have been previously described (22, 23). To demonstrate that VPAC1-R and VPAC2-R are present and functional, adipocytes were incubated with 100 nM of the respective agonist for 30 min, and PKA activity was measured. Figure 2C shows that all three of these agonists were able to stimulate PKA activity to the same extent as 30 nM isoproterenol, i.e. approximately 1.5-fold compared with unstimulated control.
Effects of PAC1-R antagonist PACAP(6–38) on lipolysis
To evaluate which receptor(s) is involved in adipocyte lipolysis, we studied the effects of receptor-specific antagonists and agonists on lipolysis. PACAP(6–38) is a truncated form of PACAP38 that inhibits the binding of PACAP to PAC1-R (24). To evaluate the ability of PACAP(6–38) to inhibit lipolysis induced by PACAP38 and VIP, rat primary adipocytes were incubated with water or 10 or 100 nM PAC1-R antagonist for 30 min before stimulation with the indicated concentration (0–100 nM) of PACAP38 (Fig. 3A) or VIP (Fig. 3B), and glycerol release was measured. As shown in Fig. 3A, PACAP38-induced lipolysis could not be inhibited by PACAP(6–38). In addition, PACAP(6–38) did not have any effect on basal lipolysis, i.e. cells not treated with PACAP or VIP (Fig. 3A). VIP has little or no affinity for PAC1-R and should therefore not be affected by PACAP(6–38). As expected, PACAP(6–38) was not able to inhibit VIP-induced lipolysis (Fig. 3B).
FIG. 3. Effect of the PAC1-R antagonist PACAP(6–38) on PACAP38-induced (A) and VIP-induced (B) lipolysis. Adipocytes were pretreated with water () or 10 nM () or 100 nM () PACAP(6–38) for 30 min, followed by treatment with 0–100 nM PACAP38 (A) or VIP (B). Glycerol release was calculated and is expressed as the fold increase compared with unstimulated control cells (Ctrl). Data are presented as the mean ± SEM. Each condition was performed in triplicate in three independent experiments.
Effects of VPAC1-R antagonist and agonist on lipolysis
The peptide PG97–269 is an antagonist of VPAC1-R receptor and has been previously described by Gourlet et al. (25). Rat adipocytes were incubated with water or 10 or 100 nM of the antagonist for 30 min before stimulating the cells with PACAP38 (Fig. 4A) or VIP (Fig. 4B). Neither PACAP38- nor VIP-induced lipolysis was affected by treatment of the cells with this antagonist, and no statistically significant differences were detected when comparing PACAP/VIP-induced lipolysis to isoproterenol-induced lipolysis. In addition, adipocytes were incubated with 1–1000 nM of the VPAC1-R-selective agonist (21). As shown in Fig. 4C, the VPAC1-R agonist did not induce glycerol release.
FIG. 4. Effect of VPAC1-R antagonist and agonist on PACAP38- and VIP-induced lipolysis. Adipocytes were pretreated with water () or 10 nM () or 100 nM () VPAC1-R antagonist for 30 min, followed by treatment with 0.1–100 nM PACAP38 (A) or VIP (B). C, Adipocytes were treated with 0–1000 nM VPAC1-R agonist for 30 min. Glycerol release was calculated and is expressed as the fold increase compared with unstimulated control cells (Ctrl). Data are presented as the mean ± SEM. Each condition was performed in triplicate in three independent experiments.
Effects of VPAC2-R antagonist and agonists on lipolysis
The peptide PG99–465 has previously been shown to antagonize VPAC2-R receptors exclusively (26). As described above, adipocytes were pretreated with water or 10 or 100 nM PG99–465 for 30 min before stimulation with 0–100 nM PACAP38 (Fig. 5A) or VIP (Fig. 5B). Pretreatment with 10 nM VPAC2-R antagonist did not result in any inhibition of PACAP38- or VIP-induced lipolysis. However, 100 nM VPAC2-R antagonist significantly decreased the ability of both PACAP38 and VIP to induce lipolysis at submaximal concentrations, thus resulting in a right-shifted lipolysis curve. Furthermore, VPAC2-R antagonist (10–100 nM) did not affect isoproterenol-induced lipolysis, showing that the inhibiting effect of the antagonist was specific for PACAP/VIP-induced lipolysis. We did not find any statistically significant difference between PACAP-induced lipolysis and the lipolysis induced by VIP at maximal concentrations (100 nM) in the absence or presence of VPAC2-R antagonist (data not shown). These results were also confirmed by the use of two highly potent VPAC2-R agonists, Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) and Ro25-1553 (22, 23). As shown in Fig. 5C, both agonists were able to increase glycerol release to the same extent as PACAP38, showing that VPAC2-R mediates the lipolytic responses induced by PACAP and VIP. The agonist-induced lipolysis [4.3 ± 0.7 for Ro 25–1553 and 4.3 ± 0.64 for Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)] was comparable to the lipolysis induced by VIP (4.8 ± 0.83) and isoproterenol (4.2 ± 0.16); no statistically significant differences were found when comparing agonists to isoproterenol (data not shown).
FIG. 5. Effect of VPAC2-R antagonist and agonists on PACAP38- and VIP-induced lipolysis. Adipocytes were pretreated with water () or 10 nM () or 100 nM () VPAC2-R antagonist for 30 min, followed by treatment with 0–100 nM PACAP38 (A) or VIP (B). C, Adipocytes were treated with 0–100 nM PACAP () or the VPAC2-R agonists Hexa-VIP () and Ro 25–1553 () for 30 min. Glycerol release was calculated and is expressed as the fold increase compared with the unstimulated control cells (Ctrl). Statistical significance was assessed by t test (**, P < 0.01). Data are presented as the mean ± SEM. Each condition was performed in triplicate in four independent experiments.
Discussion
This study showed that VIP, as previously reported for PACAP38 (16), stimulates lipolysis in primary rat adipocytes. The main aim of this study was to elucidate the receptor subtype that mediates the actions of PACAP38 and VIP to increase lipolysis in rat adipocytes. We pursued this question by evaluating the effect of PACAP/VIP-receptor specific antagonists and agonists on adipocyte lipolysis. We show here that VPAC2-R is the sole receptor responsible for this metabolic event. It has previously been debated whether VPAC2-R is present in adipocytes. Wei et al. (20) detected VPAC2-R mRNA by ribonuclease protection assay in human adipose tissue, whereas Harmar et al. (27) could not detect VPAC2-R in mouse adipocytes, as shown by binding assays. In this study we report that mRNA from all three PACAP/VIP receptor subtypes are detected by RT-PCR in rat adipocytes. Furthermore, the presence of VPAC1-R and VPAC2-R proteins was confirmed by Western blot analysis, whereas the presence of PAC1-R protein has been shown by others (15).
Our data show that the VPAC2-R is the main receptor mediating PACAP38- and VIP-induced lipolysis in primary rat adipocytes. This conclusion is based on a pharmacological approach, using previously described receptor subtype-specific agonists and antagonists. We found that the lipolytic actions of PACAP38 and VIP were mimicked by VPAC2-R agonism and were prevented by VPAC2-R antagonism, whereas no such actions were observed for agonists or antagonists of VPAC1-R or PAC1-R. The validity of the conclusion is based on the validity of the agonists and antagonists used, and there are some arguments regarding the specificities of the antagonists and agonists used in this work. Thus, although the VPAC2-R agonist Ro25-1553 has been described as highly selective for VPAC2-R (28), its specificity has been questioned, because the compound also has partial agonistic effects on rat PAC1-R (29) and human VPAC1-R (26). Therefore, we used another VPAC2-R agonist, Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), in addition to Ro25-1553. We found that Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), like Ro25-1553, efficiently increased lipolysis in these cells. To support these data, we used a VPAC2-R antagonist (PG99–465), which caused inhibition of PACAP38- and VIP-induced lipolysis. The VPAC2-R antagonist has previously been shown to have weak agonistic effects on VPAC1-R (26). However, the highly selective VPAC1-R antagonist used in this work did not counteract PACAP38- or VIP-induced lipolysis, suggesting that the inhibitory action of PG99–465 on PACAP38- and VIP-induced lipolysis is indeed due to its VPAC2-R antagonistic property. The PAC1-R antagonist PACAP(6–38) has previously been shown to act antagonistically on VPAC2-R in addition to PAC1-R (29). However, VIP-induced lipolysis was not affected by PACAP(6–38), suggesting that PACAP(6–38) did not bind VIP receptors (including VPAC2-R). Thus, both PAC1-R and VPAC1-R can be excluded from a role in mediating the lipolytic effects induced by PACAP38 and VIP, whereas a role for VPAC2-R is strongly supported.
An interesting observation in this study was that the two different VPAC2-R agonists and the VPAC1-R agonist were able to induce PKA activity to the same extent as isoproterenol, yet only the VPAC2-R receptor agonists induced lipolysis in these cells. This suggests that distinct, subcellular compartments of cAMP/PKA exist to provide the substrate specificity of this signaling pathway, as previously reviewed (30). The presence of PKA-anchoring proteins in adipocytes has not yet been studied in great detail, although it has been suggested that PKA-anchoring proteins may be crucial for full lipolysis to occur in adipocytes (31). The occurrence of such scaffolding proteins would raise the possibility that only the PKA activated by VPAC2-R, not by VPAC1-R, is able to access the lipid droplet and subsequently induce lipolysis via hormone-sensitive lipase.
The physiological role for VPAC2-R-mediated lipolysis remains to be established. It is conceivable that VPAC2-R contributes to maintain energy homeostasis by increasing the release of free fatty acids in times of energy deprivation. It is also possible that the lipolytic action of PACAP/VIP potentiates insulin secretion early after food intake, because it is known that increased free fatty acid levels have a positive effect on glucose-stimulated insulin secretion (32). However, as a consequence of increased insulin release, the lipolytic action of PACAP and VIP is abolished, suggesting a short-term effect of these peptides. In studies using a VPAC2-R agonist (BAY 55–9837), increased plasma free fatty acid levels could not be detected. However, these measurements were conducted after 3 d of continuous infusion of the agonist (33). At this time point, VPAC2-R-mediated lipolysis may be antagonized by insulin. Furthermore, VPAC2-R-deficient mice have been generated, but there are no reports of lipid metabolism in these mice. The mice demonstrate no deterioration of glucose elimination, although insulin levels are decreased (34).
We have shown that VPAC2-R mediates the lipolytic effects of PACAP38 and VIP. The physiological role of this effect remains to be established, but it is possible that PACAP and VIP are important for energy homeostasis. Evaluation of the effects exerted by the different receptors is important, because the specific receptors could be important targets of drug design for treatment of type 2 diabetes and other syndromes of metabolic disorders (33).
Acknowledgments
Special thanks to Prof. Patrick Robberecht for providing us with VPAC1-R and VPAC2-R antagonists and agonists. and to Eva Ohlson for excellent technical assistance.
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Address all correspondence and requests for reprints to: Dr. Lina ?kesson, Biomedical Center C11, Lund University, SE-221 84 Lund, Sweden. E-mail: lina@akesson@.nu.
Abstract
The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are structurally and functionally related. Their actions have been shown to be mediated by three different receptor subtypes: PAC1-R, which has exclusive affinity for PACAP, and VPAC1-R and VPAC2-R, which have equal affinity for PACAP and VIP. We recently showed that PACAP38 induces lipolysis in rat adipocytes, and in the present study we examined whether VIP has similar effects and which of the three receptors mediates this PACAP/VIP action. We showed by RT-PCR that all three receptor subtypes are present in rat adipocytes. We demonstrated that VIP (1–100 nM), like PACAP38, stimulates lipolysis in isolated adipocytes, as determined by glycerol release. By a pharmacological approach, using antagonists and agonists specific for the receptor subtypes, we elucidated the mechanisms by which PACAP38 and VIP mediate their lipolytic effects. We found that antagonists of PAC1-R [PACAP(6–38)] and VPAC1-R (PG97–269) did not affect lipolysis induced by 0.1–100 nM PACAP38 or VIP, and that a VPAC1-R agonist [K15, R16, L27VIP(1–7)GRF(8–27)] did not affect lipolysis at 1–1000 nM. However, two different VPAC2-R agonists [Hexa-VIP(1–28) and Ro25-1553] clearly mimicked the lipolytic effect of PACAP38 and VIP. In addition, the VPAC2-R antagonist PG99–465 (100 nM) caused right-shifted dose-response curves of PACAP38- and VIP-induced lipolysis. These results therefore provide evidence that all three PACAP/VIP receptor subtypes are expressed in primary rat adipocytes, but that the VPAC2-R subtype is responsible for mediating the lipolytic effects induced by PACAP38 and VIP.
Introduction
CYCLIC AMP IS a major modulator of lipid metabolism in adipocytes, and increased formation of cAMP is associated with enhanced hydrolysis of triglycerides to free fatty acids and glycerol (lipolysis). The cascade underlying this action is initiated by binding of catecholamines to ?-adrenergic receptors, which results in the formation of cAMP and the activation of protein kinase A (PKA), with subsequent activation of hormone-sensitive lipase (1). Lipolysis is counteracted by insulin, mainly via insulin-dependent activation of the cAMP-degrading enzyme phosphodiesterase 3B (2). The net effect of formation and degradation of cAMP, therefore, governs lipolysis; hence, compounds affecting cAMP in adipocytes are expected to affect lipolysis.
Two compounds that may regulate lipolysis are pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), because both are strong activators of cAMP formation, as demonstrated in several cell types. They belong to the glucagon superfamily of peptides, and they are extensively distributed in both the central and peripheral nervous system and seem to function as neuropeptides (3). PACAP exists in two isoforms, PACAP38 and PACAP27. PACAP27 consists of the 27 N-terminal amino acids of PACAP38 (4). PACAP shows 68% structural homology with VIP. Three receptor subtypes mediate the actions of PACAP and VIP; all are of the seven-transmembranous, G protein-coupled type. Two of the receptor subtypes, VPAC1-R and VPAC2-R, show equal affinity for PACAP and VIP, whereas the third receptor subtype, PAC1-R, has unique affinity for PACAP (5, 6). The PACAP/VIP receptors are distributed throughout the body, in the respiratory system, the gastrointestinal tract, and the central nervous system (6). The receptors have also been shown to be expressed in organs of importance for energy homeostasis, and there is an increasing amount of evidence that PACAP and VIP have effects on lipid and carbohydrate metabolism (7). For instance, PACAP/VIP have been shown to stimulate hepatic glucose output from the perfused rat liver (8, 9, 10) and to increase glucose-stimulated insulin secretion in mice (11, 12), calves (13), and humans (14). Furthermore, previous findings show that PACAP is able to enhance insulin-mediated glucose uptake in 3T3-L1 adipocytes (15). Our recent findings showed that PACAP38 has both catabolic and anabolic effects on primary rat adipocytes depending on the presence or absence of insulin. In the absence of insulin, lipolysis was increased, whereas in the presence of insulin, lipolysis was suppressed, and insulin-induced lipogenesis was increased (16). In this study we examined what receptor(s) is involved in the lipolytic effects of PACAP38 and VIP. We show that mRNAs for all three receptor subtypes are present in primary rat adipocytes. By the use of specific PACAP/VIP receptor agonists and antagonists, we present evidence that PACAP38/VIP-stimulated lipolysis is exclusively mediated by VPAC2-R, presumably by activating PKA.
Materials and Methods
Materials
VPAC1-R antagonist PG97–269 (Ac-His1, D-Phe2, K15, R16, L27)VIP(3, 4, 5, 6, 7)/GRF(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27), VPAC1-R agonist (K15, R16, L27)VIP(1, 2, 3, 4, 5, 6, 7)/GRF(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27), VPAC2-R antagonist PG99–465, and VPAC2-R agonists Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) and Ro25-1553 were gifts from Prof. Patrick Robberecht (University of Brussels, Brussels, Belgium). PACAP(6–38) was purchased from Bachem (Weil am Rhein, Germany). ATP, kemptide phosphate acceptor peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), PACAP38, VIP, antibodies against VPAC1-R and VPAC2-R, 3-isobutyl-1-isobutylxanthine, and protein kinase inhibitor were purchased from Sigma-Aldrich Corp. (Stockholm, Sweden). Glycerol-3-phosphate dehydrogenase, glycerokinase, and nicotinamide adenine dinucleotide were purchased from Roche (Stockholm, Sweden). Insulin was a gift from Novo Nordisk (Copenhagen, Denmark). OPC3911 was a gift from Otsuka Pharmaceutical Corp. (Tokyo, Japan). The primers used were synthesized by MWG Biotech (Ebersberg, Germany).
Animals
Male Sprague Dawley rats, 36 d of age, were obtained from B&K Universal (Stockholm, Sweden). The rats were maintained in a facility with controlled temperature and a 12-h light, 12-h dark cycle. The animals were given free access to food and water. All experiments were approved by the ethical committee of Lund University.
Preparation of adipocytes
Epididymal fat pads were isolated and digested in collagenase and washed as described previously (17). Packed cell volume was determined by centrifuging adipocyte suspensions in hematocrit tubes. The adipocytes were resuspended in wash buffer (KRH) containing 25 mM HEPES (pH 7.5), 120 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 1% BSA, 2 mM glucose, and 200 nM adenosine. In experiments in which antagonists were used, the cells were preincubated for 30 min with antagonists or water before proceeding with the stimulations with PACAP38/VIP.
RT-PCR of PACAP/VIP receptors
Total RNA was extracted from adipocytes, using RNAzol (Tel-Test, Inc., Friendswood, TX), and cDNA was produced using the RT-for-PCR-kit (BD Clontech, Stockholm, Sweden) from 1 μg RNA, using oligo(deoxythymidine)18 priming. For amplification of PACAP and VIP receptors, PAC1-R, VPAC1-R, and VPAC2-R, the following receptor-specific primer combinations were used: PAC1-R, 5'-CTGACTGCATCTAGAAGG-3' (exon 1) and 5'-TGGATGAAGTTGCGAGTGCA-3' (exon 5; expected product, 498 bp); VPAC1-R, 5'-CAGCTGGAGAATGAAACCAC-3' (exon unknown) and 5'-CATGAAGAGATGCATGTGGA-3' (exon unknown; expected product, 389 bp); and VPAC2-R, 5'-GTGAGCAGCATCCACCCAGAAT-3' (exon 2) and 5'-GCTGGAGTAGAGCACACTGT-3' (exon 5; expected product, 506 bp). cDNA (5 μl) was subjected to one cycle of 94 C for 10 min before 40 cycles of amplification (94 C for 1 min, 57 C for 1 min, and 72 C for 1.5 min) using 1 U AmpliTaq Gold (PerkinElmer, Stockholm, Sweden), 1.2 mM MgCl2, and 0.3 mM deoxy-NTP.
Western blot analysis of VPAC1-R and VPAC2-R
Cells were prepared and washed as described above and suspended in 50 mM N-Tris[hydroxymethyl]metyl-2-aminoethanesulfonic acid (pH 7.4), 250 mM sucrose, 1 mM EDTA, and 0.1 mM EGTA (10% cell suspension). Homogenization was carried out in the same buffer supplemented with 10 μg /ml antipain, 1 μg/ml pepstatin A, and 10 μg/ml leupeptin. Homogenates were briefly centrifuged for 10 min at 13,000 rpm at 4 C. The fat cake was discarded as well as cell debris. The fat-free supernatant was withdrawn and centrifuged again at 50,000 x g for 60 min at 4 C. The resulting membrane fractions were rehomogenized and solubilized on ice for 60 min in homogenization buffer containing 1% C13E12 (nonionic alkyl polyoxyethylene glycol detergent, Berol Kemi AB, Stenungsund, Sweden) and 150 mM NaCl. A final centrifugation was carried out to remove unsolubilized membranes at 10,000 rpm for 10 min at 4 C. Homogenates were subjected to 7% SDS-PAGE, followed by electrotransfer to polyvinylidene difluoride membranes (Millipore Corp., Sundbyberg, Sweden). The membrane was blocked for 60 min using 0.5% gelatin in wash buffer consisting of 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% (vol/wt) Tween 20. Membranes were incubated overnight with a monoclonal VPAC1-R or VPAC2-R antibody, followed by incubation with a secondary mouse antibody conjugated with horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK) for 45 min. Proteins were detected using chemiluminescent Super Signal West PicoLuminol/Enhancer solution (Boule Nordic AB, Huddinge, Sweden) and were visualized using the Fuji LAS 1000 Plus system (Fuji Photo Film Co. Ltd., Tokyo, Japan).
PKA assay
Adipocytes were suspended in KRH and diluted to 6% cell suspension. Stimulations were carried out as indicated in the figure legends and were stopped by adding 250 μl ice-cold homogenization buffer containing 50 mM Tris (pH 7.4), 50 mM EDTA, 2 mM 3-isobutyl-1-isobutylxanthine, 50 μM OPC3911 (phosphodiesterase inhibitor), 10 μg/ml antipain, 10 μg/ml leupeptin, and 1 μg/ml pepstatin A. Samples were immediately put on ice and subsequently centrifuged at 13,000 rpm for 10 min. The fat cake and pellet were discarded, and 10 μl infranatants were incubated for 20 min at 30 C with 5 μl phosphorylation mix containing 20 mM Tris-EDTA-sucrose (pH 7.4), 50 mM MgSO4, 0.2 mM ATP, 5 mM dithiothreitol, 4 mg/ml substrate peptide (kemptide), and 5 μCi [-32P]ATP with or without 10 μM protein kinase inhibitor (to correct for non-PKA activity). The reactions were stopped by the addition of 10 μl 1% BSA and 1 mM ATP (pH 3.0) and were precipitated with 10 μl 31% trichloroacetic acid. Samples were left to precipitate for 15 min, then centrifuged at 10,000 rpm for 3 min at 4 C. Ten-microliter aliquots were transferred to p81 membranes and after drying were washed three times with 75 mM H3PO4 and once with acetone. The amount of 32P incorporated was determined by scintillation counting.
Lipolysis measurements
As a measurement of lipolysis, glycerol release was determined as previously described (18). Briefly, 5% cell suspension (in KRH) was stimulated with PACAP/VIP or agonists as indicated in figure legends. When studies using antagonists were performed, cells were preincubated with water or antagonists before proceeding with PACAP/VIP stimulations. Incubations were stopped after 30 min of shaking (150 rpm, 37 C) and were put on ice for 20 min. One milliliter of hydrazine buffer containing 50 mM glycine (pH 9.8), 0.05% hydrazine hydrate, 1 mM MgCl2 supplemented with 0.75 mg/ml ATP, 0.375 mg/ml nicotinamide adenine dinucleotide, 25 μg/ml glycerol-3-phposphate dehydrogenase, and 0.5 μg/ml glycerokinase was added to 200 μl collected medium. After incubation for 40 min at room temperature, OD340 was measured, and glycerol release was calculated.
Statistical analysis
All data are presented as the mean ± SEM. Statistical analysis was performed using the unpaired t test. Statistical significance was defined as P < 0.05.
Results
Effects of VIP on lipolysis in primary rat adipocytes
We have previously shown that PACAP38 induces lipolysis in rat adipocytes (16). The structural homology and the fact that PACAP and VIP have equal affinity for two of the three PACAP receptors indicate that VIP could stimulate lipolysis in these cells (6). In accordance with previous results reported by Hauner et al. (19), we achieved dose-dependent glycerol release by stimulating adipocytes for 30 min with 0.1–100 nM VIP (Fig. 1). VIP had no effect on lipolysis at 0.1 or 1 nM VIP. At 10 nM VIP, glycerol release was 5-fold compared with that of nonstimulated cells, whereas 100 nM VIP induced a 7-fold increase. In addition, we studied the effect of 1.7 nM insulin on VIP-induced lipolysis. Isolated cells were incubated with 0.1–100 nM VIP in the absence or presence of 1.7 nM insulin. Insulin almost completely abolished VIP-induced lipolysis (Fig. 1). Lipolysis was inhibited 70% by insulin when induced by 100 nM PACAP or 10 or 100 nM VIP. This is in agreement with our previous finding that insulin is able to suppress PACAP-induced lipolysis.
FIG. 1. Effect of insulin on VIP-induced lipolysis. Adipocytes were stimulated with 0.1–100 nM VIP with () or without () 1.7 nM insulin for 30 min. PACAP38 (100 nM) was used as a positive control. Glycerol release was calculated and expressed as the fold increase compared with the unstimulated control (Ctrl). Statistical significance was assessed by t test (**, P < 0.01). Data are presented as the mean ± SEM. Each condition was performed in triplicate in three independent experiments.
Expression of PAC1-R, VPAC1-R, and VPAC2-R in rat adipocytes
It has previously been shown by ribonuclease protection assay that PAC1-R, VPAC1-R, and VPAC2-R mRNAs are expressed in human adipose tissue (20). To confirm the occurrence of these receptors in rat adipocytes, RT-PCR was performed using the primers indicated in Materials and Methods. mRNA from rat whole brain tissue was used as a positive control for all three receptors (data not shown). As shown in Fig. 2A, mRNAs of PAC1-R (PCR product, 498 bp), VPAC1-R (PCR product, 389 bp), and VPAC2-R (PCR product, 506 bp) were all present in rat primary adipocytes. In addition, Western blot analysis showed bands corresponding to the expected molecular masses of VPAC1-R (45–50 kDa) and VPAC2-R (49–52 kDa) proteins (Fig. 2B).
FIG. 2. Expression and effects on PKA activity of PACAP/VIP receptors in primary rat adipocytes. A, PCR analysis was performed on rat cDNA from adipocytes using the primers listed in Materials and Methods (lane 1, PAC1-R; lane 2, VPAC1-R; lane 3, VPAC2-R). PCR products were separated on 1% agarose and visualized with ethidium bromide. B, SDS-PAGE and Western blot analysis were performed on membrane fractions from primary rat adipocytes (Adip.) and whole rat brain (Brain) using a VPAC1-R antibody (upper panel) or a VPAC2-R antibody (lower panel). C, Adipocytes were stimulated with 30 nM isoproterenol (Iso), 100 nM VPAC1-R agonist (VPAC1-R Ago), or VPAC2-R agonists (Hexa-VIP and Ro25-1553). PKA activity was calculated and expressed as the fold increase compared with unstimulated cells (Ctrl). Statistical significance was assessed by t test (***, P < 0.001). Data are presented as the mean ± SEM. Each condition was performed in duplicate in four independent experiments.
Effects of VPAC1-R and VPAC2-R agonists on PKA activity
The VPAC1-R-specific agonist (21) and the VPAC2-R-selective agonistic peptides, Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) and Ro25-1553, have been previously described (22, 23). To demonstrate that VPAC1-R and VPAC2-R are present and functional, adipocytes were incubated with 100 nM of the respective agonist for 30 min, and PKA activity was measured. Figure 2C shows that all three of these agonists were able to stimulate PKA activity to the same extent as 30 nM isoproterenol, i.e. approximately 1.5-fold compared with unstimulated control.
Effects of PAC1-R antagonist PACAP(6–38) on lipolysis
To evaluate which receptor(s) is involved in adipocyte lipolysis, we studied the effects of receptor-specific antagonists and agonists on lipolysis. PACAP(6–38) is a truncated form of PACAP38 that inhibits the binding of PACAP to PAC1-R (24). To evaluate the ability of PACAP(6–38) to inhibit lipolysis induced by PACAP38 and VIP, rat primary adipocytes were incubated with water or 10 or 100 nM PAC1-R antagonist for 30 min before stimulation with the indicated concentration (0–100 nM) of PACAP38 (Fig. 3A) or VIP (Fig. 3B), and glycerol release was measured. As shown in Fig. 3A, PACAP38-induced lipolysis could not be inhibited by PACAP(6–38). In addition, PACAP(6–38) did not have any effect on basal lipolysis, i.e. cells not treated with PACAP or VIP (Fig. 3A). VIP has little or no affinity for PAC1-R and should therefore not be affected by PACAP(6–38). As expected, PACAP(6–38) was not able to inhibit VIP-induced lipolysis (Fig. 3B).
FIG. 3. Effect of the PAC1-R antagonist PACAP(6–38) on PACAP38-induced (A) and VIP-induced (B) lipolysis. Adipocytes were pretreated with water () or 10 nM () or 100 nM () PACAP(6–38) for 30 min, followed by treatment with 0–100 nM PACAP38 (A) or VIP (B). Glycerol release was calculated and is expressed as the fold increase compared with unstimulated control cells (Ctrl). Data are presented as the mean ± SEM. Each condition was performed in triplicate in three independent experiments.
Effects of VPAC1-R antagonist and agonist on lipolysis
The peptide PG97–269 is an antagonist of VPAC1-R receptor and has been previously described by Gourlet et al. (25). Rat adipocytes were incubated with water or 10 or 100 nM of the antagonist for 30 min before stimulating the cells with PACAP38 (Fig. 4A) or VIP (Fig. 4B). Neither PACAP38- nor VIP-induced lipolysis was affected by treatment of the cells with this antagonist, and no statistically significant differences were detected when comparing PACAP/VIP-induced lipolysis to isoproterenol-induced lipolysis. In addition, adipocytes were incubated with 1–1000 nM of the VPAC1-R-selective agonist (21). As shown in Fig. 4C, the VPAC1-R agonist did not induce glycerol release.
FIG. 4. Effect of VPAC1-R antagonist and agonist on PACAP38- and VIP-induced lipolysis. Adipocytes were pretreated with water () or 10 nM () or 100 nM () VPAC1-R antagonist for 30 min, followed by treatment with 0.1–100 nM PACAP38 (A) or VIP (B). C, Adipocytes were treated with 0–1000 nM VPAC1-R agonist for 30 min. Glycerol release was calculated and is expressed as the fold increase compared with unstimulated control cells (Ctrl). Data are presented as the mean ± SEM. Each condition was performed in triplicate in three independent experiments.
Effects of VPAC2-R antagonist and agonists on lipolysis
The peptide PG99–465 has previously been shown to antagonize VPAC2-R receptors exclusively (26). As described above, adipocytes were pretreated with water or 10 or 100 nM PG99–465 for 30 min before stimulation with 0–100 nM PACAP38 (Fig. 5A) or VIP (Fig. 5B). Pretreatment with 10 nM VPAC2-R antagonist did not result in any inhibition of PACAP38- or VIP-induced lipolysis. However, 100 nM VPAC2-R antagonist significantly decreased the ability of both PACAP38 and VIP to induce lipolysis at submaximal concentrations, thus resulting in a right-shifted lipolysis curve. Furthermore, VPAC2-R antagonist (10–100 nM) did not affect isoproterenol-induced lipolysis, showing that the inhibiting effect of the antagonist was specific for PACAP/VIP-induced lipolysis. We did not find any statistically significant difference between PACAP-induced lipolysis and the lipolysis induced by VIP at maximal concentrations (100 nM) in the absence or presence of VPAC2-R antagonist (data not shown). These results were also confirmed by the use of two highly potent VPAC2-R agonists, Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) and Ro25-1553 (22, 23). As shown in Fig. 5C, both agonists were able to increase glycerol release to the same extent as PACAP38, showing that VPAC2-R mediates the lipolytic responses induced by PACAP and VIP. The agonist-induced lipolysis [4.3 ± 0.7 for Ro 25–1553 and 4.3 ± 0.64 for Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)] was comparable to the lipolysis induced by VIP (4.8 ± 0.83) and isoproterenol (4.2 ± 0.16); no statistically significant differences were found when comparing agonists to isoproterenol (data not shown).
FIG. 5. Effect of VPAC2-R antagonist and agonists on PACAP38- and VIP-induced lipolysis. Adipocytes were pretreated with water () or 10 nM () or 100 nM () VPAC2-R antagonist for 30 min, followed by treatment with 0–100 nM PACAP38 (A) or VIP (B). C, Adipocytes were treated with 0–100 nM PACAP () or the VPAC2-R agonists Hexa-VIP () and Ro 25–1553 () for 30 min. Glycerol release was calculated and is expressed as the fold increase compared with the unstimulated control cells (Ctrl). Statistical significance was assessed by t test (**, P < 0.01). Data are presented as the mean ± SEM. Each condition was performed in triplicate in four independent experiments.
Discussion
This study showed that VIP, as previously reported for PACAP38 (16), stimulates lipolysis in primary rat adipocytes. The main aim of this study was to elucidate the receptor subtype that mediates the actions of PACAP38 and VIP to increase lipolysis in rat adipocytes. We pursued this question by evaluating the effect of PACAP/VIP-receptor specific antagonists and agonists on adipocyte lipolysis. We show here that VPAC2-R is the sole receptor responsible for this metabolic event. It has previously been debated whether VPAC2-R is present in adipocytes. Wei et al. (20) detected VPAC2-R mRNA by ribonuclease protection assay in human adipose tissue, whereas Harmar et al. (27) could not detect VPAC2-R in mouse adipocytes, as shown by binding assays. In this study we report that mRNA from all three PACAP/VIP receptor subtypes are detected by RT-PCR in rat adipocytes. Furthermore, the presence of VPAC1-R and VPAC2-R proteins was confirmed by Western blot analysis, whereas the presence of PAC1-R protein has been shown by others (15).
Our data show that the VPAC2-R is the main receptor mediating PACAP38- and VIP-induced lipolysis in primary rat adipocytes. This conclusion is based on a pharmacological approach, using previously described receptor subtype-specific agonists and antagonists. We found that the lipolytic actions of PACAP38 and VIP were mimicked by VPAC2-R agonism and were prevented by VPAC2-R antagonism, whereas no such actions were observed for agonists or antagonists of VPAC1-R or PAC1-R. The validity of the conclusion is based on the validity of the agonists and antagonists used, and there are some arguments regarding the specificities of the antagonists and agonists used in this work. Thus, although the VPAC2-R agonist Ro25-1553 has been described as highly selective for VPAC2-R (28), its specificity has been questioned, because the compound also has partial agonistic effects on rat PAC1-R (29) and human VPAC1-R (26). Therefore, we used another VPAC2-R agonist, Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), in addition to Ro25-1553. We found that Hexa-VIP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), like Ro25-1553, efficiently increased lipolysis in these cells. To support these data, we used a VPAC2-R antagonist (PG99–465), which caused inhibition of PACAP38- and VIP-induced lipolysis. The VPAC2-R antagonist has previously been shown to have weak agonistic effects on VPAC1-R (26). However, the highly selective VPAC1-R antagonist used in this work did not counteract PACAP38- or VIP-induced lipolysis, suggesting that the inhibitory action of PG99–465 on PACAP38- and VIP-induced lipolysis is indeed due to its VPAC2-R antagonistic property. The PAC1-R antagonist PACAP(6–38) has previously been shown to act antagonistically on VPAC2-R in addition to PAC1-R (29). However, VIP-induced lipolysis was not affected by PACAP(6–38), suggesting that PACAP(6–38) did not bind VIP receptors (including VPAC2-R). Thus, both PAC1-R and VPAC1-R can be excluded from a role in mediating the lipolytic effects induced by PACAP38 and VIP, whereas a role for VPAC2-R is strongly supported.
An interesting observation in this study was that the two different VPAC2-R agonists and the VPAC1-R agonist were able to induce PKA activity to the same extent as isoproterenol, yet only the VPAC2-R receptor agonists induced lipolysis in these cells. This suggests that distinct, subcellular compartments of cAMP/PKA exist to provide the substrate specificity of this signaling pathway, as previously reviewed (30). The presence of PKA-anchoring proteins in adipocytes has not yet been studied in great detail, although it has been suggested that PKA-anchoring proteins may be crucial for full lipolysis to occur in adipocytes (31). The occurrence of such scaffolding proteins would raise the possibility that only the PKA activated by VPAC2-R, not by VPAC1-R, is able to access the lipid droplet and subsequently induce lipolysis via hormone-sensitive lipase.
The physiological role for VPAC2-R-mediated lipolysis remains to be established. It is conceivable that VPAC2-R contributes to maintain energy homeostasis by increasing the release of free fatty acids in times of energy deprivation. It is also possible that the lipolytic action of PACAP/VIP potentiates insulin secretion early after food intake, because it is known that increased free fatty acid levels have a positive effect on glucose-stimulated insulin secretion (32). However, as a consequence of increased insulin release, the lipolytic action of PACAP and VIP is abolished, suggesting a short-term effect of these peptides. In studies using a VPAC2-R agonist (BAY 55–9837), increased plasma free fatty acid levels could not be detected. However, these measurements were conducted after 3 d of continuous infusion of the agonist (33). At this time point, VPAC2-R-mediated lipolysis may be antagonized by insulin. Furthermore, VPAC2-R-deficient mice have been generated, but there are no reports of lipid metabolism in these mice. The mice demonstrate no deterioration of glucose elimination, although insulin levels are decreased (34).
We have shown that VPAC2-R mediates the lipolytic effects of PACAP38 and VIP. The physiological role of this effect remains to be established, but it is possible that PACAP and VIP are important for energy homeostasis. Evaluation of the effects exerted by the different receptors is important, because the specific receptors could be important targets of drug design for treatment of type 2 diabetes and other syndromes of metabolic disorders (33).
Acknowledgments
Special thanks to Prof. Patrick Robberecht for providing us with VPAC1-R and VPAC2-R antagonists and agonists. and to Eva Ohlson for excellent technical assistance.
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