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Endocrinology, doi:10.1210/en.2003-1180
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Endocrinology Vol. 145, No. 5 2346-2356
Copyright © 2004 by The Endocrine Society

Identification of a Functional Peroxisome Proliferator-Activated Receptor Responsive Element within the Murine Perilipin Gene

So Nagai, Chikara Shimizu, Masaaki Umetsu, Satoshi Taniguchi, Mikiko Endo, Hideaki Miyoshi, Narihito Yoshioka, Mitsumasa Kubo and Takao Koike

Department of Medicine II (S.N., C.S., M.U., S.T., M.E., H.M., N.Y., T.K.), Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan; and Health Administration Center, Hokkaido University of Education (M.K.), Sapporo 002-8501, Japan

Address all correspondence and requests for reprints to: Chikara Shimizu, M.D., Ph.D, Department of Medicine II, Hokkaido University Graduate School of Medicine, N-15, W-7, Kita-ku, Sapporo 060-8638, Japan. E-mail: shimizch{at}med.hokudai.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Perilipin, a family of phosphoproteins located around lipid droplets in adipocytes, is essential for enlargement of lipid droplets and lipolytic reaction by hormone-sensitive lipase. Thiazolidinediones, peroxisome proliferator-activated receptor (PPAR) {gamma} agonists, have been shown to increase perilipin expression in fully differentiated adipocytes. However, the precise mechanism of transcriptional regulation of murine perilipin gene heretofore remains unclear. We determined the transcription start site of murine perilipin gene by RNA ligase-mediated rapid amplification of the cDNA ends method. We generated luciferase reporter gene constructs containing various lengths of the 5'-flanking region of the murine perilipin gene and assayed promoter/enhancer activities using differentiated 3T3-L1 adipocytes. We identified a functional PPAR-responsive element (PPRE) in the murine perilipin promoter, and this was confirmed by gel EMSAs using nuclear extracts from differentiated 3T3-L1 adipocytes. Furthermore, point mutations of the identified functional PPRE markedly reduced both the reporter gene activity in differentiated 3T3-L1 adipocytes and PPAR{gamma}/thiazolidinedione-induced transactivation in NIH-3T3 fibroblasts. Real-time RT-PCR revealed that thiazolidinedione up-regulates endogenous perilipin mRNA levels. We propose that PPAR{gamma} plays a significant role in the transcriptional regulation of murine perilipin gene via the PPRE in its promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MAMMALIAN ORGANISMS HAVE the capacity to store energy in adipose tissues. A typical adipocyte contains one or more large cytoplasmic triacylglycerol-rich droplets, surrounded by proteins such as perilipin, adipose differentiation-related protein, and tail-interacting protein of 47 kDa (TIP47) (1). Perilipins, identified by Londos and colleagues (2), are phosphoproteins, the expression of which was restricted to adipocytes and steroidogenic cells (3, 4, 5), whereas adipose differentiation-related protein and TIP47 are expressed ubiquitously (1). Murine perilipins consist of four isoforms of perilipin A, B, C, and D, encoded by a single copy gene with alternative and tissue-specific mRNA splicing (6). Perilipin A and B are expressed in both adipocytes and steroidogenic cells, whereas perilipin C and D are detected only in the latter cells. Each isoform of perilipins has a common amino-terminal region and has a unique carboxy-terminal end. The relationship between a molecular profile and tissue distribution of each isoform remains unclear (6). Perilipin A, the most abundant isoform in murine adipose tissue and differentiated cultured 3T3-L1 adipocytes (4), functions to increase triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis, and thus is fundamental to enlarging lipid droplets in adipocytes (7). Perilipin A has multiple phosphorylation sites within the molecule that are induced by protein kinase A (2). An important step of lipolysis in adipocytes is the translocation of hormone-sensitive lipase (HSL) from cytosol to the surface of the lipid droplet. Recent studies revealed that translocation of HSL required phosphorylation of both HSL and perilipin (8, 9). Conversely, perilipin A provides a protective barrier against lipolysis in the absence of protein kinase A stimulation.

Obesity, an excessive accumulation of adipose tissue, is a major risk factor for hypertension, impaired glucose tolerance, and hyperlipidemia (10). Aggregation of these disorders, referred to as metabolic syndrome, which is otherwise known as insulin resistance syndrome, leads to atherosclerosis and to cardiovascular disease (10). Morphologically, subjects with severe obesity have an increased number (hyperplasia) and size (hypertrophy) of fat cells concomitant with an increase in the size of lipid droplets. Hypertrophic fat cells increase insulin resistance-inducible factors, e.g. TNF-{alpha}, free fatty acid, and so on (10).

Troglitazone, one of the thiazolidinediones, improves insulin resistance and increases the number of small adipocytes containing small lipid droplets in white adipose tissues of obese Zucker rats, presumably via activating peroxisome proliferator-activated receptor (PPAR) {gamma} (11). Another study demonstrated that adipocytes in heterozygous PPAR{gamma}-deficient mice are smaller than those in wild-type mice (12). Thus, the size rather than the number of adipocytes plays an important role in the pathogenesis of insulin resistance, although it is not well known how PPAR{gamma} regulates the size of lipid droplets.

There are reports describing the regulation of perilipin expression. TNF-{alpha} decreases perilipin A mRNA by counteracting insulin. On the other hand, BRL 49653, another thiazolidinedione, increases perilipin A mRNA in fully differentiated 3T3-L1 adipocytes (13, 14, 15), which suggests that activation of PPAR{gamma} may up-regulate perilipin A mRNA in adipocytes, although it is unclear whether signals via PPAR{gamma} are directly related to the regulation of perilipin A expression. Because data on the transcriptional regulation of the murine perilipin gene have not been documented, we analyzed the promoter region of this gene and identified a cis regulatory element involved in gene expression during differentiation of 3T3-L1 adipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dexamethasone and 3-isobutyl-2-methylxanthine were purchased from WAKO Pure Chemicals (Richmond, VA). Insulin and GW9662 were purchased from Sigma Chemical Co. (St. Louis, MO). Pioglitazone was kindly provided by Takeda Chemical Industries (Osaka, Japan). 3T3-L1 preadipocytes and NIH-3T3 fibroblasts were purchased from the American Tissue Culture Collection (Manassas, VA). The anti-PPAR{gamma} antibody, PPAR{gamma} N-20X, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Isolation of the 5' untranslated region of the murine perilipin gene
Poly(A)+ RNA was prepared using the Fastrack mRNA isolation kit according to the manufacturer’s protocol (Invitrogen Corp., Carlsbad, CA). The 5'-rapid amplification of cDNA ends (RACE) procedure was done using the MARATHON cDNA Amplification kit according to the manufacturer’s protocol (Clontech Laboratories Inc., Palo Alto, CA). Two micrograms of poly(A)+ RNA derived from mouse adipose tissues were reverse transcribed and used as a template. Gene-specific antisense primers for 5'-RACE were synthesized based on the sequence obtained from the mouse expressed sequence tag database (dEST). PCR was done using AmpliTaq GOLD DNA polymerase (PerkinElmer Corp., Foster City, CA) and primers (all specific oligonucleotide primers used in these studies are listed in Table 1Go) mperiAS1 and the adaptor primer (CAP-1) (5'-CCATCCTAATACGACTCACTATAGGGC) provided with the kit. PCR conditions were as follows: denaturing at 94 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 3 min; cycle number was 30. The PCR products were gel-purified, subcloned into the TA cloning vector (Invitrogen Corp.), and sequenced using a Big Dye Terminator cycle sequence kit (Applied Biosystems, Foster City, CA).


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  		TABLE 1. Sequences of oligonucleotides

 
Isolation of the 5'-flanking region of the murine perilipin gene
Phage clones (1.2 x 106) from 129SV/J mouse genomic library (Stratagene, La Jolla, CA) were screened using a [32P]-labeled 175-bp 5'-RACE product according to standard method (16). After purification and mapping of the positive clones, a 4.3-kb XbaI fragment containing the first exon was subcloned into pBluescript II SK-vector (pBS) at an XbaI site (designated pX4.3). A 2.9-kb XbaI/PstI fragment was subcloned into pBS at an XbaI and PstI site (designed pX/P2.9), and both strands were sequenced.

RNA ligase-mediated rapid amplification of cDNA ends
Total RNA was extracted using an RNeasy mini kit according to the manufacturer’s protocol (Qiagen, Bothell, WA). To obtain full-length 5'- untranslated region of the perilipin cDNA, RNA ligase-mediated (RLM)-RACE method was used according to the manufacturer’s instructions with the FirstChoice RLM-RACE kit (Ambion Inc., Austin, TX), which is designed to amplify cDNA only from full-length, capped mRNA. Briefly, 10 µg of total RNA derived from 3T3-L1 adipocytes 4 d after differentiation was treated with calf intestinal alkaline phosphatase (CIP) for 1 h at 37 C then subjected to phenol/chloroform (1:1, vol/vol) extraction and ethanol precipitation. CIP-treated RNA was incubated with tobacco acid pyrophosphatase (TAP) for 1 h at 37 C to remove the 7-methylguanosine cap at structures from mRNA, leaving the 5'-monophosphates to be ligated to an RNA adaptor with T4 RNA ligase. After reverse transcription with random decamers, PCR was done using primer mperiAS2 and an outer RNA adaptor primer provided with the kit. PCR conditions were as follows: preheat denaturing at 95 C for 9 min, followed by 35 cycles of denaturing at 95 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 30 sec. Nested PCR was done using primer mperiAS1 and an inner RNA adaptor primer provided with the kit. PCR conditions were the same as described above. PCR products were directly subcloned using a TOPO TA cloning kit (Invitrogen Corp.). The nucleotide sequences of PCR products were determined by sequencing.

Confirmation of transcription start site (TSS) by RT-PCR
To confirm the TSS, RT-PCR was done using gene-specific sense primers; mperiRT1S and mperiRT2S, upstream and downstream of TSS determined using the RLM-RACE method. The antisense primer used was mperiAS1, located on exon 3. Total RNA (0.5 µg) of 3T3-L1 adipocytes 4 d after differentiation was used in the RT-PCR. RT was done according to the manufacturer’s protocol (Superscript III RNase H– Reverse Transcriptase, Invitrogen). PCR conditions were as follows: preheat denaturing at 95 C for 9 min, denaturing at 95 C for 30 sec, annealing at 58 C for 30 sec, and extension at 72 C for 30 sec; cycle number was 30. PCR products were analyzed electrophoretically using 2.5% agarose gels.

Plasmid construction
After the digestion of pX/P2.9 with EcoRV and PstI, a 2.5-kb DNA fragment was subcloned into EcoRV/PstI-digested pBS (designated pE/P 2.5), which was digested with KpnI and SmaI, and the DNA fragment was subcloned into KpnI/SmaI-digested luciferase expression vector, pGL3 basic vector (Promega, Madison, WI), designated pGL3(–2422/+103). pGL3(–932/+103), pGL3(–504/+103), and pGL3(+64/+103) were generated by digestion with HincII, SacI, or PvuII as well as KpnI, blunting and self-ligating. pGL3(–219/+103), pGL3(–2008/+103), pGL3(–1827/+103), pGL3(–1678/+103), and pGL3(–1442/+103) were generated by PCR using pGL3(–2422/+103) as template and the specific primers shown in Table 1Go. PCR products were digested with KpnI and PstI and subcloned into the KpnI/PstI-digested pGL3 basic vector. To further delineate enhancer region, various DNA fragments (–2008 to –1802 bp) were similarly ligated into the minimal simian virus 40 (SV40) containing the pGL3 promoter luciferase expression vector (Promega). The largest promoter construct, designated pGL3pro(–2008/–1802), and subsequent truncated fragments (–1957/–1802, –1933/–1802, and –1858/–1802 bp) were PCR-amplified using pGL3(–2422/+103) as template. These promoter fragments were subcloned into the SacI-digested pGL3 promoter vector. In addition to these forward direction constructs, we generated another series of luciferase constructs in which reverse directional DNA fragments were ligated. The largest promoter construct was named pGL3pro(–1802/–2008), and subsequent truncated constructs were designated as well. Sequences of all primers used are given in Table 1Go. Purification of plasmids was done using a modified alkaline lysis method (Plasmid Maxi kit, Qiagen), and the vector-insert junctions were confirmed by sequencing.

Mutational modifications of the murine perilipin gene
Mutations of a putative PPAR-responsive element (PPRE) were generated using a QuikChange XL Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Briefly, 10 ng of pGL3pro(–2008/–1802) was used as a template and the mutated nucleotides as described (see Fig. 4BGo). PCR conditions were as follows: denaturing at 95 C for 1 min, followed by 18 cycles of denaturing at 95 C for 50 sec, annealing at 60 C for 50 sec, and extension at 68 C for 12 min. After digestion with DpnI, 2 µl of PCR products were used to transform XL10-Gold competent cells provided with the kit. Appropriate clones were verified by sequencing.



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  		FIG. 4. Identification of PPRE in the 5'-flanking region of the mouse perilipin gene. A, PPRE in the 5'-flanking region of the mouse perilipin gene is compared with the PPRE sequence in various murine PPAR{gamma}-responsive genes: mouse FATP (fatty acid transport protein) PPRE (18 ); mouse UCP1 (uncoupling protein 1) PPRE (31 ); aP2 (adipocyte fatty acid binding protein 2) PPRE, ARE6 and ARE7 (19 ); and consensus PPRE sequence (18 ). DR1 motif is boxed. B, A scheme of luciferase constructs using transfection experiments. The sequences of PPRE wild-type, pGL3pro(–2008/–1802), and two kinds of mutated constructs (mut), pGL3pro-PPREmut and pGL3pro-PPRE3mut, are described. Small letters indicate mutated nucleotides. C, pGL3pro(–2008/–1802) vectors were transfected into NIH-3T3 fibroblasts with a total 2 µg of mPPAR{gamma}2 expression vector and pSV-CMV mock vector with or without 1 µM pioglitazone. At least three experiments were done in duplicate. Normalized luciferase activities are shown as the means ± SEM.

 
Cell culture and transfection
3T3-L1 preadipocytes were grown in DMEM containing 10% fetal bovine serum (FBS) and seeded on 12-well plates. Forty-eight hours after reaching confluence, cells began to differentiate under the treatment with 174 nM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1 µM pioglitazone in DMEM containing 10% FBS. After 48 h of incubation, medium was replaced with DMEM only containing 10% FBS, and transfection was done using a LipofectAMINE2000 reagent (Invitrogen Corp.) according to the manufacturer’s protocol. Briefly, 2 µl of LipofectAMINE2000 reagent was gently mixed into 100 µl of OPTI-MEM (Life Technologies, Inc./BRL, Rockville, MD) and the preparation incubated for 5 min at room temperature. One microgram of luciferase reporter construct and 0.3 µg of pCMV-SPORT ß-gal expression vector (Invitrogen) in 100 µl OPTI-MEM were mixed with 100 µl of reagent containing OPTI-MEM and the mixture incubated for an additional 15 min. After the incubation, transfection mixtures were added to the cells, the preparation was incubated for 48 h, then the cells harvested.

In other experiments, 3T3-L1 preadipocytes and NIH-3T3 fibroblasts were grown to 70–80% confluence in DMEM and 10% FBS, and luciferase reporter constructs were transfected using LipofectAMINE2000 as described above. In NIH-3T3 fibroblasts, a total of 2 µg of various amounts of mPPAR{gamma}2 expression vector and mock vector [pSV-CMV vector (17)] was transfected, with or without pioglitazone, in addition to 1 µg of luciferase reporter plasmid. Murine (m) PPAR{gamma}2 expression vector was constructed by inserting a PCR-amplified mPPAR{gamma}2 cDNA fragment into pSV-SPORT vector (Life Technologies/BRL). The dose of pioglitazone used was 1 µM. Cells were harvested 48 h after transfection. Cells were washed twice with PBS, lysed, and luciferase activities were measured using a TD20/20 luminometer (Turner BioSystems Inc., Sunnyvale, CA) and a Luciferase assay system (Promega). Transfection efficiency was adjusted depending on ß-galactosidase activities. Data are presented as fold-increase of luciferase activity over control vector (pGL3 basic or pGL3 promoter vector).

Gel EMSA (GEMSA)
Nuclear extracts were prepared from 3T3-L1 adipocytes 4 d after differentiation using a commercially available kit (NE-PER nuclear and cytoplasmic extraction reagent, PIERCE Chemical Corp., Rockford, IL). GEMSA was done using reagents in the Gel Shift Assay kit (Promega). To analyze the binding of nuclear hormone receptors to the putative PPRE, sense oligonucleotides of probes given in Fig. 6AGo were end-labeled using [{gamma}32P]ATP (3000 Ci/mmol, 10 mCi/ml Amersham Biosciences, Piscataway, NJ) and T4 polynucleotide kinase using a standard method (16) and were column-purified (Microspin G-25 Columns, Amersham Biosciences) (see Fig. 6AGo). After annealing with sequence-matched complementary oligonucleotides, these probes were used for GEMSA. The binding reaction was done according to the manufacturer’s instructions using 5 µg of nuclear extract and 0.2 pmol of each labeled probe. Competition analysis was made using a 100-fold amount of unlabeled double-stranded oligonucleotides corresponding to the labeled probe. Samples were electrophoresed on 4% polyacrylamide gels in 0.5x TBE buffer (9 mM Tris/HCl, 90 mM boric acid, and 20 mM EDTA, pH 8.0) at 4 C, dried, and analyzed by autoradiography.



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  		FIG. 6. Mutational analysis of putative PPRE. A, pGL3pro(–2008/–1802) vector and two kinds of mutated pGL3pro(–2008/–1802) vectors were transfected into NIH-3T3 fibroblasts under different conditions of mPPAR{gamma}2 expression vector and pioglitazone. B, pGL3pro(–2008/–1802) vector and two kinds of mutated pGL3pro(–2008/–1802) were transfected into 3T3-L1 cells under undifferentiated and differentiated conditions. At least three experiments were done in duplicate. Normalized luciferase activities are shown as the means ± SEM.

 
Effects of PPAR{gamma} agonist and antagonist
The regulation of perilipin mRNA levels by PPAR{gamma} agonist and antagonist were examined in differentiated 3T3-L1 adipocytes. 3T3-L1 preadipocytes were cultured and differentiated as described above. Pioglitazone, a PPAR{gamma} agonist, was added to the media 48 h after differentiation. On the other hand, GW9662, a PPAR{gamma} antagonist, was added to the media at the beginning of differentiation. Both reagents were maintained until the cells were harvested.

Expression analysis of perilipin mRNA by real-time RT-PCR
Two micrograms of total RNA of 3T3-L1 adipocytes 4 d after differentiation-treated with PPAR{gamma} agonist and antagonist were reverse transcribed in a 20-µl reaction volume. Real-time PCR was performed using ABI PRISM 7700 Sequence Detection System and SYBR Green I as a double-stranded DNA-specific binding dye, according to the manufacturer’s instructions (PE Applied Biosystems, Foster City, CA). Amplifications were carried out using 15 µl SYBR Green PCR mastermix (Applied Biosystems), 11.2 µl sterile deionized water, 2 µl reverse transcript sample, and 1.8 µl primer pair (30 nM) in a 30-µl reaction volume. The real-time PCR conditions were as follows: preheat denaturing at 95 C for 10 min, followed by 40 cycles of heat denaturing at 95 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 1 min. The melting temperature profile for perilipin and ß-actin demonstrated single peak at 87 and 88 C, respectively. The interassay and intraassay coefficients of variation were calculated to be 5.9 and 2.2%, respectively, using primers for ß-actin and reverse transcript sample.

Primer sequences used are described in Table 1Go. Primers were designed to recognize a different exon in each gene, and the size of PCR products was verified by agarose gel electrophoresis. Exogenous cDNA standards for perilipin and ß-actin were produced by inserting PCR products, which were generated using sample primers noted above and 3T3-L1 adipocytes cDNA as templates, into the pCR2.1 vector using the TOPO TA cloning kit. Inserts of control vectors for perilipin and ß-actin were verified by sequencing. The concentration of each standard was determined by measuring the OD260, and the copy number was calculated. The ß-actin was quantified to normalize perilipin mRNA levels, and the final results were expressed as the ratio of the copy number of perilipin to the copy number of ß-actin.

Expression of PPAR{gamma} mRNA of each sample was validated by RT-PCR using specific primers. PCR conditions were as follows: preheat denaturing at 95 C for 9 min, denaturing at 95 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 30 sec; cycle number was 30. PCR products were analyzed electrophoretically using 2.5% agarose gels.

DNA sequence analysis
We used the web-based search programs Transcription Element Search Software (TESS) (http://www.cbil.upenn.edu/tess/) and TRANSFAC (http://transfac.gbf.de/TRANSFAC/) to analyze the 5'-flanking region of the perilipin gene. In addition to these programs, we visually inspected minimal suspicious promoter regions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Determination of TSS
Because the nucleotide sequence of perilipin cDNA had not been reported when our experiment was started, we searched an expressed sequence tag database (dEST) using a 200-bp sequence of the rat and human perilipin cDNA located around the translation initiation site. Two antisense primers (mperiAS1 and mperiAS2) were generated based on the sequence hit (GenBank accession nos. AA458173, AA510052, AA711707, AA982036, AI158797, and AI180560). Primer extension analysis, 5'-RACE, and RLM-RACE were done using these antisense primers.

Initially, the standard 5'-RACE and primer extension analysis were done to determine TSS (data not shown). While doing these experiments, the nucleotide sequence of mouse perilipin cDNA was reported (6). Because TSS derived from our experiments was incompatible with that described in the report, RLM-RACE was done. With this method, cDNA is generated only from full-length, capped mRNA, by treatment of RNA with CIP and TAP before reverse transcription. The PCR products obtained by RLM-RACE showed a broad band from approximately 120–210 bp (lane 1 in Fig. 1AGo). Sequencing of the PCR products showed that they were composed of several kinds of clones (11 different clones of the 29 sequenced) (Fig. 1CGo). Three alternative splicings were evident with a little difference compared with the previous report (6) (Fig. 1CGo). Presumably, the RLM-RACE experiment revealed that the 5' end of the longest cDNA was extended 7 bp upstream compared with that in previous report (6) and was designated +1 as TSS (Fig. 1CGo; bold right-angled arrow). Analysis of sequences in the promoter region revealed no canonical TATA, CAAT, or GC motif in the murine perilipin gene, as there was no typical initiator sequence.



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  		FIG. 1. Determination of the TSS of murine perilipin gene. A, Gel electrophoresis of the PCR product, deduced from RLM-RACE experiments with (lane 1) and without TAP treatment (lane 2); 100-bp DNA ladder (lane M). B, Gel electrophoresis of PCR product, obtained using RT-PCR and a gene-specific sense primer located upstream (mperiRT1S, lane 1) and downstream (mperiRT2S, lane 2) of TSS determined by RLM-RACE. The antisense primer was located in exon 3 (mperiAS2); 100-bp DNA ladder (lane M). C, Genomic DNA sequence in the vicinity of TSS of mouse perilipin gene. Bold, right-angled arrow and +1 indicate the most upstream TSS deduced from RLM-RACE experiment. Sense primers used in RT-PCR experiment are indicated by arrows. Figure with asterisks indicated above the sequence shows number of the clones in which the 5' end of cDNA was determined by sequencing (a total of 29 clones were sequenced). The clone with a single asterisk indicated above the sequence lacks a DNA segment shown by a single underline. However, the clone with double asterisks has a single underline sequence. The clone with triple asterisks has another alternative splicing; additional DNA segment indicated by double underlines. The sequence shown by italic letters corresponds to the intron of major mRNA. Bold letters indicate a translation initiation codon, ATG.

 
To confirm the TSS, a RT-PCR experiment was done using a gene-specific sense primer upstream and downstream of the TSS determined by RLM-RACE (mperiRT1S and mperiRT2S, respectively, in Fig. 1CGo). When the upstream sense primer was used, no PCR product could be detected (lane 1 in Fig. 1BGo). Two bands yielded by alternative splicing were found when the downstream sense primer was used (lane 2 in Fig. 1BGo). These results indicate that mRNA encoding mouse perilipin protein did not extend to the position of upstream sense primer.

Identification of cis regulatory element(s) in the 5'-flanking region of the perilipin gene
To identify upstream cis element(s), a series of 5' deletion reporter constructs were generated and transfected into 3T3-L1 cells before and 2 d after differentiation to measure luciferase activity. No significant change of luciferase activity was detected when 3T3-L1 preadipocytes were used (Fig. 2AGo). However, when transfected into differentiated 3T3-L1 adipocytes, the constructs showed a significant reporter gene activity. Especially, the construct with the largest DNA fragment [pGL3(–2422/+103)] revealed approximately 2000-fold luciferase activity compared with that of pGL3 basic vector (Fig. 2AGo). When the 5' end of DNA fragment was shortened to –1442 bp, luciferase activity prominently diminished. Then, to further analyze the region between –2422 and –1442 bp, additional reporter gene constructs [pGL3(–2219/+103), pGL3(–2008/+103), pGL3(–1827/+103), and pGL3(–1678/+103)] were generated, and luciferase activity was measured (Fig. 2BGo). When the 5' end of reporter gene constructs was abbreviated from –2008 to –1827 bp, the reporter activity was prominently diminished.



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  		FIG. 2. Luciferase activities of the murine perilipin promoter in 3T3-L1 cells. A, Luciferase assay using fused promoter constructs. B, Luciferase assay using constructs abbreviated at approximately 200-bp length from –2242 to –1442 bp. The locations of putative cis regulatory elements are indicated by open (PPRE), closed (C/EBP{alpha}), and checkered (C/EBPß) vertical ovals on the top solid line and directionally localized by the first base pair of each motif. At least three experiments were carried out in duplicate. Normalized luciferase activities are shown as the means ± SEM. Luc, Luciferase.

 
To further delineate location of the cis element(s), another series of 5' truncated constructs were generated. Sequential DNA segments ranging from –2008/–1802 to –1858/–1802 bp were ligated to the upstream of the minimal SV40 promoter in pGL3-promoter vector (Fig. 3AGo). The longest construct, containing the –2008 to –1802-bp DNA segment, consistently showed more than a 10-fold higher luciferase activity than did the insertless pGL3 promoter vector when transfected into differentiated 3T3-L1 adipocytes. In contrast, no different luciferase activity could be detected in 3T3-L1 preadipocytes. The experiments with reverse direction vectors gave the same results (Fig. 3BGo). Taking these data together, a 51-bp region located within –2008 bp to –1957 bp seems to be critical to up-regulate the perilipin gene transcription during adipocyte differentiation.



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  		FIG. 3. Luciferase activities of murine perilipin promoter ligated pGL3-promoter vector in 3T3-L1 cells. A, Sequential DNA segments ranging from –2008/–802 bp to –1858/–1802 bp were ligated to upstream of the minimal SV40 promoter in pGL3-promoter vector as described in Materials and Methods. B, DNA fragments ligated reverse directionally in panel A to upstream of the minimal SV40 promoter in pGL3-promoter vector. The locations of putative cis regulatory elements are indicated by open (PPRE) and checkered (C/EBPß) vertical ovals on the top solid line. At least three experiments were done in duplicate. Normalized luciferase activities are shown as the means ± SEM. Luc, Luciferase.

 
Identification of a functional PPRE in the 5'-flanking region of the mouse perilipin gene
In comparison with functional PPRE identified in other genes, a minimal 51-bp region between –2008 bp to –1957 bp seemed to contain a putative PPRE at –1980 bp to –1968 bp (Fig. 4BGo), which is similar to direct repeat 1 (DR1) motifs (Fig. 4AGo). The pGL3pro(–2008/–1802) vector was then transfected into NIH-3T3 fibroblasts with 0–2 µg of the mPPAR{gamma}2 expression vector (Fig. 4CGo). In this experiment, PPAR{gamma}2 with PPAR{gamma} ligand, pioglitazone, augmented reporter gene activity in a dose-dependent manner, whereas PPAR{gamma}2 alone weakly increased the reporter activity. These results indicated that PPAR{gamma}2 functioned via this PPRE, either directly or indirectly.

GEMSA
Nuclear extracts from differentiated 3T3-L1 adipocytes were incubated with radiolabeled wild-type (PPRE wild-type) and two mutant PPRE probes with different positions (PPREmut and PPRE3mut) plus or minus unlabeled competitors (Fig. 5AGo). When the radiolabeled wild-type probe was used, an up-shifted band was detected (lane 1 in Fig. 5BGo). The signal of the shifted bands disappeared by competition with an excessive amount of the unlabeled wild-type probe (lane 2 in Fig. 5BGo), but not with the mutant-type probes (lanes 4 and 5 in Fig. 5BGo). The radiolabeled wild-type probe with antibody against PPAR{gamma} produced a supershifted band (lane 3 in Fig. 5BGo). When radiolabeled mutant type probes were used, no shifted bands were produced (lanes 6 and 7 in Fig. 5BGo).



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  		FIG. 5. GEMSA using nuclear extracts of differentiated 3T3-L1 adipocytes. A, Nucleotide sequences of wild-type (PPREwt) and two mutant PRE probes with different positions (PPREmut and PPRE3mut); PPRE is shown by bold lettering, and small letters indicate mutated nucleotides. B, Result of GEMSA. Ratio of competitor (comp.) to labeled probe was 100:1. Arrowhead indicates a probe-nuclear extract complex. A supershifted band with PPAR{gamma} antibody is indicated by an asterisk.

 
Mutational analysis of putative PPRE
To determine whether putative PPRE was functional as the element bound to PPAR{gamma}, transfection experiments were done using two kinds of mutated pGL3pro(–2008/–1802) vector and two cell lines, NIH-3T3 fibroblasts and 3T3-L1 adipocytes (Fig. 6Go). In the experiment using NIH-3T3 fibroblasts, luciferase activities were diminished in the presence of PPAR{gamma}2 and pioglitazone when each mutated pGL3pro(–2008/–1802) vector was transfected (Fig. 6AGo). In addition, luciferase activities of both mutated constructs were significantly reduced compared with that of wild-type pGL3pro(–2008/–1802) in experiments using differentiated 3T3-L1 adipocytes (right columns in Fig. 6BGo). These results indicated that the ability to respond to PPAR{gamma} lies within the DR1-type PPRE.

Regulation of perilipin mRNA levels by PPAR{gamma} agonist and antagonist in differentiated 3T3-L1 adipocytes
Finally, we examined whether endogenous perilipin gene was regulated by PPAR{gamma}. For this purpose, real-time RT-PCR was used to quantify perilipin mRNA levels in differentiated 3T3-L1 adipocytes treated with different concentrations of PPAR{gamma} agonist and antagonist. As shown in Fig. 7AGo, increasing concentrations of pioglitazone resulted in a dose-dependent increase in perilipin mRNA. On the other hand, GW9662 was able to decrease perilipin mRNA and reduce the effect of pioglitazone in a dose-dependent manner. Meanwhile, PPAR{gamma} mRNA of each condition was expressed in a similar manner. The effect of PPAR{gamma} agonist and antagonist for perilipin gene was also revealed in the reporter activity of pGL3(–2422/+103), containing the full murine perilipin promoter, transfected into differentiated 3T3-L1 adipocytes (Fig. 7BGo). These findings indicated that identified PPRE functions to regulate transcriptional levels on the murine perilipin gene in differentiated 3T3-L1 adipocytes.



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  		FIG. 7. Regulation of endogenous perilipin mRNA by PPAR{gamma} agonist and antagonist in differentiated 3T3-L1 adipocytes. A, Expression of murine perilipin mRNA in differentiated 3T3-L1 adipocytes under different conditions of pioglitazone and GW9662, as determined by real-time RT-PCR. Column heights represent the mean ratio of the mRNA copy number of perilipin to the mRNA copy number of ß-actin. At least three experiments were done in duplicate. Error bar indicates SEM. Expression of mPPAR{gamma} mRNA detected by RT-PCR was shown. B, pGL3(–2422/+103) containing the full murine perilipin promoter transfected into differentiated 3T3-L1 adipocytes treated with pioglitazone and GW9662. At least three experiments were done in duplicate. Data are calculated as fold-increase of luciferase activity over control condition [pioglitazone (–) and GW9662 (–)] and are shown as the means ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The first purpose of the current study was to confirm the TSS of murine perilipin mRNA. Our data of TSS determined by primer extension analysis and a standard 5'-RACE procedure (data not shown) did not correspond to findings in a previous report (6). The RLM-RACE procedure was done and resulted in identification of the existence of multiple transcription start sites. The promoter region in the murine perilipin gene has no canonical TATA, CAAT, and GC motif. In addition, there are no typical initiator sequences around TSS. These appear to be responsible for the diversity of start sites.

The second purpose was to identify regulatory factor(s) during adipocyte differentiation by analyzing the 5'-flanking region of perilipin gene. Consequently, a functional PPRE was identified and proved to be a key element in transcriptional activation of the murine perilipin gene during adipogenesis. Perilipin, one of the genes related to lipid metabolism as are CD36, adipocyte fatty acid binding protein, fatty acid transport protein, and acyl-coenzyme A synthetase, which also have functional PPRE in the promoter region (18, 19, 20, 21), is a possible candidate gene regulated by this group of nuclear transcription factor, PPARs. Previous studies have shown that treatment of fully differentiated 3T3-L1 adipocytes with thiazolidinediones, which activate PPAR{gamma}, leads to an increase in murine perilipin mRNA or protein (13, 14, 15). A sequence in the 5'-flanking region of the murine perilipin gene identified in our study is similar to the consensus sequence of previously identified PPREs (see Fig. 4AGo).

Real-time RT-PCR experiments revealed that transcription of perilipin mRNA can be activated in a dose-dependent fashion by pioglitazone and inhibited in a dose-dependent fashion by GW9662. Additionally, transfection experiments using pGL3(–2422/+103) into differentiated 3T3-L1 adipocytes showed that full murine perilipin promoter containing identified PPRE is regulated by pioglitazone and GW9662. These results demonstrated that endogenous perilipin mRNA levels are regulated by PPAR{gamma} and its ligands via identified PPRE. These findings support the notion that the expression of murine perilipin mRNA is limited in tissues expressing PPAR{gamma} mRNA (19, 22).

PPAR{gamma} is a key transcriptional nuclear factor that regulates not only adipogenesis but also glucose and lipid metabolism. Previous studies using PPAR{gamma} null embryonic fibroblasts (12) revealed that PPAR{gamma} is essential for adipogenesis. Furthermore, CCAAT/enhancer-binding protein (C/EBP) {alpha}, adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element binding protein 1c, and other transcriptional factors expressed in adipocytes, are regulated downstream of PPAR{gamma}. Our present study revealed that perilipin is another gene regulated downstream of PPAR{gamma} during adipogenesis. Kubota et al. (12) demonstrated that the size of adipocytes is smaller in heterozygous PPAR{gamma}-deficient mice [PPAR{gamma}(+/–)] than in the wild-type under conditions of a high-fat diet. Taking our results into account, morphological change of adipocytes in PPAR{gamma}(+/–) mice may be due to suppression of perilipin expression by loss of one PPAR{gamma} allele.

Overexpression of perilipin in 3T3-L1 preadipocytes led to an accumulation of numerous small lipid droplets (7), and mutant perilipin transfections into NIH-3T3 fibroblasts or Chinese hamster ovary cells resulted in inhibitory effects on hormone-mediated lipolysis (8, 9). A recent study demonstrated that adipocytes in perilipin-null mice [Peri(–/–)] were smaller than in wild-type mice and prevented body weight gain under conditions of a high-fat diet (23). Serum leptin, which is one of insulin-sensitizing adipocytokines, is increased in Peri(–/–), whereas PPAR{gamma} mRNA expression in adipocytes is unchanged (23). Another study on perilipin knockout mice showed that breeding the Peri(–/–) alleles into Lprdb/db mice reversed the obesity (24). These results suggest that perilipin may participate in signaling mechanisms related to energy homeostasis, independent of PPAR{gamma} activation.

Assessing a role of PPAR{gamma} in the pathogenesis of obesity is difficult. It was reported that PPAR{gamma} antagonist reduced body weight and insulin resistance in KKAy mice on high-fat diet (25). This indicates that PPAR{gamma} antagonist has potential as an antiobesity drug. However, a more recent study revealed that PPAR{gamma} may induce synthesis of adiponectin, known as the antidiabetic (26) and antiatherogenic (27) adipocytokine. Therefore, control of PPAR{gamma} expression may aggravate metabolic conditions. Although direct suppression of perilipin gene expression may be a therapeutic target for obesity, further supportive study is needed. Otherwise, enhanced energy expenditure, which is accompanied by lipolysis in adipocytes, is another strategy of therapy for obesity. It is reasonably postulated that constitutional activating phosphorylation of perilipin may prevent maturing of lipid droplets. Recently, Wang et al. (28) showed that PPAR{delta}, which binds PPRE, inhibited obesity by activating fat burning. It was not mentioned whether PPAR{delta} affects expression of perilipin. Our GEMSA experiments demonstrated two extra bands in addition to a main shifted band. These extra bands remain, even when using specific PPAR{gamma} antibody, indicating that identified PPRE may bind to other ligands such as PPAR{delta}. PPAR{delta} may repress PPAR{gamma}-mediated transcriptional activity (29). Thus, it will be of interest to determine whether expression of the perilipin gene is controlled by PPAR{delta} through the identified PPRE. Furthermore, it was not clarified whether increase of phosphorylated perilipin modulates energy expenditure. The relationship between PPAR{delta} activation and phosphorylated perilipin in fat burning requires attention.

Finally, Nishiu et al. (30) isolated human perilipin cDNA and identified the gene on human chromosome 15. At this time, the human perilipin promoter has not been analyzed. We compared the sequences between human chromosome 15 upstream of the perilipin gene (GenBank accession nos. AC079075 and NM_002666) and the 5'-flanking region of the murine perilipin gene and identified the PPRE-like sequence at –3277 to –3265 in the human genome (adenosine of translation initiation codon, ATG, is designated +1), which is the DR1 type of PPRE and is similar to the PPRE of the murine perilipin gene identified in the present study. Whereas there is a 65% homology within a 500-bp region upstream of TSS between the murine and human perilipin gene, the nucleotide identity within a region of more than 500 bp upstream of TSS was generally less than 50%. However, nucleotide sequences in the vicinity of this PPRE-like motif are highly conserved between human and murine perilipin gene (71.8% in 201 bp) (Fig. 8Go). It will be of interest to determine whether these elements in human perilipin function as well.



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  		FIG. 8. Alignment of the nucleotide sequences in the vicinity of PPRE in the murine perilipin (mPeri) gene and its human counterpart (hPeri). In human perilipin gene, sequence number is designated adenosine of translation initiation codon, ATG, as +1. PPRE of murine perilipin gene and PPRE like sequence of its human gene are boxed. Identities are indicated by screen background. There is a 71.8% homology within this region.

 


   Acknowledgments
 
We thank M. Ohara (Fukuoka, Japan) for critical reading of the manuscript and M. Ishida (Hokkaido University) for expert technical assistance.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research (C: 15590965) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to C.S.).

Abbreviations: C/EBP, CCAAT/enhancer-binding protein; CIP, calf intestinal alkaline phosphatase; DR1, direct repeat 1; FBS, fetal bovine serum; GEMSA, gel EMSA; HSL, hormone-sensitive lipase; mPPAR, murine PPAR; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PPRE, PPAR-responsive element; RACE, 5'-rapid amplification of cDNA ends; RLM, RNA ligase-mediated; SV40, simian virus 40; TAP, tobacco acid pyrophosphatase; TSS, transcription start site.

Received September 8, 2003.

Accepted for publication January 8, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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