Potential conflict of interest: Nothing to report.
Primary nonalcoholic fatty liver disease is one of the most common forms of chronic liver diseases and is associated with insulin-resistant states such as diabetes and obesity. Recent work has revealed potential implications of peroxisome proliferator-activated receptor-δ (PPARδ) in lipid homeostasis and insulin resistance. In this study, we examined the effect of PPARδ on sterol regulatory element-binding protein-1 (SREBP-1), a pivotal transcription factor controlling lipogenesis in hepatocytes. Treatment with GW0742, the PPARδ agonist, or overexpression of PPARδ markedly reduced intracellular lipid accumulation. GW0742 and PPARδ overexpression in hepatocytes induced the expression of insulin-induced gene-1 (Insig-1), an endoplasmic reticulum protein braking SREBP activation, at both the mRNA and the protein levels. PPARδ inhibited the proteolytic processing of SREBP-1 into the mature active form, thereby suppressing the expression of the lipogenic genes fatty acid synthase, stearyl CoA desaturase-1, and acetyl coenzyme A carboxylase. Our results revealed a direct binding of PPARδ to a noncanonical peroxisome proliferator responsive element motif upstream of the transcription initiation site of human Insig-1. The disruption of this site diminished the induction of Insig-1, which suggested that Insig-1 is a direct PPARδ target gene in hepatocytes. Knockdown of endogenous Insig-1 attenuated the suppressive effect of GW0742 on SREBP-1 and its target genes, indicating PPARδ inhibited SREBP-1 activation via induction of Insig-1. Furthermore, overexpression of PPARδ by intravenous infection with the PPARδ adenovirus induced the expression of Insig-1, suppressed SREBP-1 activation, and, consequently, ameliorated hepatic steatosis in obese db/db mice. Conclusion: Our study reveals a novel mechanism by which PPARδ regulates lipogenesis, suggesting potential therapeutic applications of PPARδ modulators in obesity and type 2 diabetes, as well as related steatotic liver diseases. (HEPATOLOGY 2008.)
Obesity, a type of aberrant lipogenesis, is a major health issue in Western countries as well as in Asia and is closely associated with several metabolic disorders such as diabetes, coronary heart disease, and fatty liver disease. Nonalcoholic fatty liver disease is the most common form of chronic liver disease and is considered the hepatic manifestation of the insulin resistance syndrome. Treatment strategies specific for nonalcoholic fatty liver disease aim to improve insulin sensitivity and modify underlying metabolic risk factors.1, 2 Peroxisome proliferator-activated receptors (PPARs), with three isoforms, α, β/δ, and γ, are lipid-activated transcription factors that play important roles in controlling lipid and glucose homeostasis. PPARα and PPARγ have been extensively studied as therapeutic targets for dyslipidemia and type 2 diabetes.3 In contrast, PPARδ remains less studied despite its ubiquitous expression. Recently, genetic approaches and the use of newly developed selective agonists have revealed the potential implications of PPARδ in lipid homeostasis and insulin resistance, thus suggesting the potential therapeutic application of PPARδ in the metabolic syndrome.4, 5 PPARδ enhances fatty acid catabolism and energy uncoupling in adipose tissue and muscle and exerts antidiabetic effects in animals.5–8
Sterol regulatory element-binding proteins (SREBPs) are transcription factors controlling the synthesis of cholesterol and fatty acids. Mammalian cells contain three isoforms, 1a, 1c, and 2, encoded by two distinct genes. SREBPs are bound to the SREBP cleavage-activating protein (SCAP) in the endoplasmic reticulum (ER).9 Under ample level of sterols, the retention of the SCAP–SREBP complex in the ER is mediated by sterol-dependent binding of the complex to one of two ER retention proteins—namely, insulin-induced gene (Insig)-1 and Insig-2.10, 11 Under low levels of lipids or sterols in the ER, the SCAP–SREBP complex dissociates from Insig and translocates from the ER to the Golgi apparatus, where the SREBPs encounter proteases S1P and S2P and undergo proteolytic processing to generate the active mature transcription factors, which results in the activation of lipid synthesis. SREBP-2 is mainly involved in cholesterol biosynthesis and uptake. SREBP-1c is the major isoform in the liver and predominantly activates the biosynthesis of fatty acid and metabolism of triglycerides.12 SREBP-1c activation up-regulates its target genes involved in lipogenic pathways, such as fatty acid synthase (FAS), stearyl coenzyme A desaturase-1 (SCD-1), glycerol-3-phosphate acyltransferase (GPAT), and acetyl coenzyme A carboxylase (ACC). Aberrant expression and activity of SREBP-1c is associated with obesity and fatty liver.13, 14 Upregulation of Insig-1 inhibits the SREBP proteolytic processing and brakes the lipid synthesis and lipogenesis.15–17
Because PPARδ exerts profound effects on lipid metabolism, in which SREBP-1 plays pivotal regulatory roles, we aimed to examine whether PPARδ regulates the activity of SREBP-1. In this study, we demonstrate in hepatocytes that PPARδ reduced proteolytic processing of SREBP-1 by inducing Insig-1 gene expression and thus ameliorated hepatic steatosis in obese diabetic db/db mice.
HepG2 cells, a human hepatoma cell line, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). GW0742 was a generous gift from Dr. Timothy Wilson (GlaxoSmithKline). GW501516, GW9662, and BRL49653 were obtained from Cayman Chemical (Ann Arbor, MI). The antibodies against Insig-1, SREBP-1, PPARδ, β-actin, or HA tag were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Adenoviral Vectors and Infection.
The adenovirus expressing PPARδ (AdPPARδ) was constructed by subcloning the full-length complementary DNA coding region of human PPARδ with an HA tag, a generous gift from Dr. Bart Staels (Pasteur Institute, France), into the shuttle vector pAdlox. The expression cassette is driven by the minimal cytomegalovirus promoter controlled by a 7× tet operon. The recombinant adenovirus was produced by homologous recombination in CRE8 (a 293 cell expressing Cre recombinase), plaque purified, propagated, and cesium chloride–purified.18, 19 Adenoviruses expressing the tetracycline-responsive transactivator (AdtTA) and green fluorescence protein (AdGFP) were described previously.20 Adenovirus-mediated gene transfer was performed by exposing confluent HepG2 cells to the adenoviral vectors at a multiplicity of infection of 50–100 for 2 hours (AdtTA was coinfected to induce the transgene expression in all adenoviral infections).
Protein was extracted from the cells or liver tissue with lysis buffer supplemented with the protease inhibitor cocktail (Roche Diagnostics, Rotkreuz, Switzerland). The samples were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted to nitrocellulose membranes. Immunoblots were reacted with primary antibodies to Insig-1, SREBP-1, β-actin, or HA, detected with horseradish peroxidase–conjugated secondary antibodies, and visualized with use of the ECL chemiluminescence system (Cell Signaling Technology Inc., Beverly, MA).21
Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction and Northern Blotting.
Total RNA was isolated with TRIzol reagent (Invitrogen). For quantitative real-time polymerase chain reaction (PCR) analysis, the complementary DNA was synthesized with the use of SuperscriptIII reverse transcriptase (Invitrogen) and oligo-(dT) primer (Promega, Madison, MI). Quantitative PCR was performed with the iQ SYBR Green PCR Supermix in the DNA Engine Opticon real-time system (Bio-Rad Laboratories Inc., Hercules, CA), with β-actin used as an internal control. The sequences of primers are shown in Table 1. For northern blotting, total RNA samples were separated on formaldehyde-agarose gels, blotted onto nylon membranes and hybridized with [32P]dCTP labeled probes for Insig-1 and β-actin.22
Table 1. Primers for Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction
Plasmids and Transient Transfection Assays.
The genomic fragment harboring −1259 to +192 bp in relation to transcription start site was cloned by PCR from the genomic DNA with the sense primer 5′-GTC GAG GAG ACA CAG CAC AG and the antisense primer 5′-GTC ACT GAT ATC TTA TCC C according to the published sequence (U96876).23 The amplified product was subcloned into the pGL3-luciferase reporter vector to generate Insig-1-luc. The Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to generate the mInsig-1-luc as described.22 The putative peroxisome proliferator responsive element (PPRE) sites (at −769 and −1234) were disrupted with the mutagenic primers 5′-GGA AGA CTT CTT TTT Tta gaa gTC TaG CTT AAA GCC ACA ACC-3′ and 5′-CAC AGC ACA GGC TTC TGA Aaa taa CCC TGT CCA TTA GGG AGC AC-3′, respectively (sense sequence with the mutated bases shown in lower case). The 4× SRE-luc and pRSV-β-gal plasmids have been described previously.22 Transient transfection was performed in HepG2 cells grown in 12-well plates with lipofectamine 2000 (Invitrogen). The pRSV-β-gal was cotransfected to normalize the transfection efficiency. Luciferase and β-galactosidase activities were measured with the luciferase activity assay kit and the β-galactosidase enzyme assay kit (Promega), respectively.
Cells were cross-linked with 1% formaldehyde and quenched prior to harvest and sonication. The sheared chromatin was immunoprecipitated with anti-PPARδ antibody (or control immunoglobulin G [IgG]) in conjugation with protein A/G Sepharose beads. The eluted immunoprecipitates were digested with proteinase K, and DNA was extracted and underwent PCR with different primer sets (Table 2) flanking the putative PPRE elements within Insig-1. The supernatant of the control group was used as an input control.24
Table 2. Probes for Chromatin Immunoprecipitation Assay and Electrophoretic Mobility Shift Assay
ChIP 1 (−799
ChIP 2 (−1249
Consensus PPRE (AOX)
Electrophoretic Mobility Shift Assay.
HepG2 cells were infected with AdPPARδ or AdGFP for 24 hours, and nuclear proteins were extracted. The oligonucleotides containing the putative PPRE motif (−769 to −757) in the Insig-1 promoter or the consensus PPRE (AOX-PPRE) were annealed, end-labeled with [γ-32P]ATP, and column-purified. Cold competition was involved adding an excess amount of the unlabeled Insig-1-PPRE, consensus PPRE or irrelevant oligonucleotides into the binding reactions. Supershift experiments were involved by the use of anti-PPARδ antibody or control IgG.22
Adenoviral Infection In Vivo.
Adult male C57BL Lepr(db)/Lepr(db) mice (The Jackson Laboratory) were used for the in vivo studies. Investigations were conducted conforming to the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institute of Health (NIH Publication No. 85-23, revised 1996). AdPPAR or AdGFP, together with AdtTA, was intravenously injected at a combined dose of 1 × 109 plaque-forming units. The mice were killed 7 days after the adenoviral infection, and liver tissues were dissected for further processing.
To stain the lipid droplets, liver sections or cultured cells were stained with filtered Oil-red-O solution using standard procedures.25 Immunofluorescence images were obtainec with fluorescence microscopy (Leica, Heidelberg, Germany).
Small Interfering RNA and Transfection.
For small interfering RNA (siRNA)-mediated gene knockdown, the siRNA targeting human Insig-1 messenger RNA (mRNA) was synthesized with the sense sequence 5′-UGGUGUCUAUCAGUAUACATT. The double-strand RNAs were transfected into HepG2 cells using Lipofectamine 2000 (Invitrogen). A scrambled siRNA was used as a control.
Quantitative data are expressed as the mean ± standard error of the mean. Differences were analyzed via one-way analysis of variance followed by Student-Newman-Keuls test. A P value of <0.05 was considered significant. Nonquantitative results were representative of at least three independent experiments.
PPARδ Inhibits Lipogenesis and SREBP-1 Activation in Hepatocytes.
To study the role of PPARδ in lipogenesis, HepG2 cells maintained in DMEM containing 10% FBS were treated with the specific PPARδ agonist GW0742 (1 μM) or infected with the adenovirus expressing PPARδ for 24 hours. Dimethyl sulfoxide (DMSO) or AdGFP was used as a control for GW0742 or AdPPARδ, respectively. As revealed by Oil-red-O staining (Fig. 1A), either GW0742 or overexpression of PPARδ markedly reduced the intracellular lipid accumulation. SREBP-1 is the major isoform of SREBPs that controls lipogenesis in hepatocytes. Western blotting analysis revealed that serum depletion triggered the proteolytic processing of the 120-kDa SREBP-1 precursor into the 68-kDa mature nuclear form (nSREBP-1) (Fig. 1B). The level of the cleaved mature form was diminished in GW0742-treated or AdPPARδ-infected cells, suggesting that PPARδ inhibited SREBP-1 activation by inhibiting its proteolytic processing.
To further explore the role of PPARδ in SREBP-1 activation, HepG2 cells were transfected with the SRE-responsive luciferase reporter gene 4× SRE-luc. Serum depletion resulted in a transcriptional activation of the reporter, which was significantly suppressed by pretreatment with GW0742 or infection with AdPPARδ (Fig. 2A). More importantly, the induction of the endogenous target genes of SREBP-1, including FAS, SCD-1 and GPAT, was significantly inhibited by PPARδ activation (Fig. 2B), which indicates that PPARδ inhibited the SREBP-1-mediated transcriptional activity.
PPARδ Induces Expression of Insig-1 in Hepatocytes.
The SCAP–SREBP complex may be trapped in the ER by Insig-1 or Insig-2, thereby preventing SREBPs from entering the Golgi apparatus for proteolytic maturation of the transcriptionally active nuclear forms. To investigate whether the activation of PPARδ induces Insig-1 gene expression, HepG2 cells were treated with GW0742 or infected with PPARδ adenovirus for 24 hours. The expression of Insig-1 was examined at both the mRNA and protein levels with the use of northern blotting, quantitative real-time reverse-transcription PCR (RT-PCR), and western blotting. As shown in Fig. 3A,C,D, the mRNA expression of Insig-1 was significantly increased by GW0742 treatment or PPARδ overexpression in a time-dependent and dose-dependent manner. GW0742 treatment or PPARδ overexpression also increased the protein level of Insig-1 (Fig. 3B). GW0742 did not significantly induce expression of Insig-2 (data not shown), suggesting that induction of Insig-1 may contribute to the inhibitory effect of PPARδ on SREBP-1 processing.
Knockdown of Insig-1 Attenuates the Suppression of GW0742 on the Processing of SREBP-1.
To examine whether the suppressive effect of PPARδ agonist on the processing of SREBP-1 depended on an adequate level of Insig-1 expression, HepG2 cells were transfected with the Insig-1 siRNA or scramble siRNA as a control. As shown in Fig. 4A, Insig-1 siRNA decreased the protein level of endogenous Insig-1 in HepG2 cells. As a result, in hepatocytes transfected with siRNA for Insig-1, the suppressive effects of GW0742 on SREBP-1 processing and on the induction of lipogenic genes were greatly reduced (Fig. 4B,C). These results indicate that PPARδ inhibited SREBP-1 activation via induction of Insig-1 in hepatocytes.
PPARδ Induces Insig-1 via a Functional PPRE Site.
Sequence analysis of the 5′ flanking regions of human Insig-1 revealed putative binding sites for PPAR. To examine whether PPARδ binds to these regions, chromatin immunoprecipitation (ChIP) assay was performed with the anti-PPARδ antibody, then PCR amplification with the specific primers flanking these putative PPRE-like motifs within the regulatory region of human Insig-1, as depicted in relation to the transcription start site. The positive binding site was located within the −799/−566 region that harbors the proximal putative PPRE (−769/−757) (Fig. 5A). Gel shift assay (Fig. 5B) revealed that PPARδ bound to this proximal PPRE, and the binding specificity was confirmed on cold competition and super-shift experiments.
As shown in Fig. 5C, the Insig-1 promoter was activated in hepatocytes by GW0742. Similarly, another specific ligand for PPARδ, GW501516, significantly induced the Insig-1 promoter activity. However, the PPARγ agonist BRL49653 only slightly increased the Insig-1 promoter activity. In addition, the PPARγ antagonist GW9662 did not inhibit the GW0742 induction of Insig-1, which indicates that the GW0742 effect was PPARδ-specific but not due to activation of PPARγ. Furthermore, site-directed mutagenesis showed that the proximal PPRE site was required for the induction of Insig-1 by PPARδ, whereas mutation of the distal site did not affect the luciferase activity of the Insig-1 promoter (Fig. 5D). Taken together, these results indicate that Insig-1 was a direct target gene of PPARδ in human hepatocytes.
PPARδ Induces Insig-1 Expression and Inhibits SREBP-1 Activation in Obese Diabetic Mice.
To determine the in vivo effect of PPARδ on Insig-1 expression and hepatic steatosis, obese diabetic db/db mice were intravenously infected with AdPPARδ or AdGFP, a control adenovirus, for 7 days. Transgene expression was confirmed via western blotting with an antibody against HA-tag fused to PPARδ (Fig. 6A) and by visualizing GFP in the liver on immunofluorescence microscopy (Fig. 6C). Western blots showed that adenovirus-mediated overexpression of PPARδ induced Insig-1 and reduced the proteolytic processing of SREBP-1 in vivo: the mature form of SREBP-1 was decreased, whereas the precursor of SREBP-1 was markedly increased (Fig. 6A). Real-time RT-PCR results showed that PPARδ overexpression resulted in induction of Insig-1 and suppression of SREBP-1 target genes, including FAS, ACC, and SCD-1, in the livers of db/db mice (Fig. 6B). As revealed with Oil-red-O staining (Fig. 6C), hepatic steatosis in db/db mice was clearly ameliorated by PPARδ overexpression, suggesting that PPARδ-induced Insig-1 expression attenuated the SREBP-1 activation and diminished the hepatic steatosis in obese diabetic mice.
In this study, we uncovered a novel mechanism by which PPARδ regulates lipogenesis in hepatocytes both in vitro and in vivo. Our findings are as follows: (1) PPARδ induces the expression of Insig-1, thus diminishing the proteolytic processing and, hence, suppressing the activity of SREBP-1, a crucial transcription factor controlling lipogenesis; (2) Insig-1 is a direct target of PPARδ; (3) PPARδ inhibits the gene expression of the SREBP-1 target genes FAS, ACC and SCD-1, which encode key enzymes in the biosyntheses of fatty acids; and (4) PPARδ decreases the intracellular fat accumulation in cultured hepatocytes and ameliorated hepatic steatosis in diabetic db/db mice.
Studies involving pharmacological and genetic approaches have unveiled important roles of PPARδ in lipid metabolism in peripheral tissues and in diabetes and obesity. The mechanisms involved in the lipid modulating effects of PPARδ include (1) promotion of cholesterol efflux by inducing ABCA1, the reverse cholesterol transporter, in macrophages and other types of cells;26 (2) inhibition of intestinal cholesterol absorption by down-regulating the Niemann-Pick C1-like 1 gene;27 and (3) stimulation of fatty acid oxidization and use by increasing the expression of the target genes controlling these metabolic pathways.5–8 Although SREBPs are known to be important transcription factors and play a central role in lipid homeostasis and obesity-related disorders, no evidence as yet links SREBP to the lipid-lowering and antilipogenic effect of PPARδ. Our results suggest that in hepatocytes, PPARδ decreases the proteolytic processing of SREBP-1, thus leading to a potent suppression of SREBP-1 target genes and the lipogenic process. PPARδ acts on SREBP-1 via a direct induction of Insig-1 in the liver and the PPARδ action on SREBP-1 is predominantly at the posttranslational level.
PPARδ induces Insig-1 in hepatocytes in vitro and in vivo. Both GW0742 treatment and PPARδ overexpression induced Insig-1 at mRNA and protein levels, which points to a PPARδ-specific effect. This finding is further supported by promoter reporter assay results showing that GW501516, another specific PPARδ agonist, has a similar effect. In contrast, the PPARδ agonist had little effect on Insig-2, the other isoform of Insig. Thus, Insig-1 is primarily responsible for the antilipogenesis effect of PPARδ in vivo. ChIP assay and gel shift assays revealed a direct binding of PPARδ to a noncanonical PPRE motif at −769/−757 bp upstream of the transcription initiation site of human Insig-1. The disruption of this site diminished the induction of Insig-1, which suggests that Insig-1 is a direct PPARδ target in hepatocytes. A recent study showed that the PPARγ agonist rosiglitazone can induce Insig-1 in mouse white adipose tissue, and PPARγ also binds this PPRE.28 In hepatocytes, we have also observed a slight increase of Insig-1 by rosiglitazone. In light of the powerful adipogenic ability of PPARγ,29 the functional role of PPARγ inducing Insig-1 remains to be examined. In rat liver, treatment with PPARα agonists increased Insig-1 and decreased the active form of SREBP-2, resulting in a reduction in cholesterol synthesis.30 Therefore, expression of Insig-1 might be regulated by different PPAR isoforms in a tissue-specific and context-specific way. Because PPARγ is less abundant in the liver than PPARδ, which is ubiquitously expressed, PPARδ may be the major isoform in the liver to be possibly and actively involved in lipogenesis via SREBP-1 under a physiological or pathological condition. However, it remains interesting to examine whether PPARγ agonists have an additive or synergistic effect with the agonists for other isoforms.
Insig regulates lipid synthesis by binding to the ER protein SCAP in a sterol-dependent fashion, thus playing a central role in the feedback control of lipid synthesis. Binding of Insig to SCAP retains the SCAP–SREBP complex in the ER, thus preventing SREBPs from entering the Golgi apparatus for proteolytic generation of their transcriptionally active forms, thereby limiting the induction of SREBP target genes. Thus, up-regulation of Insig would reduce lipid synthesis. Our results showed that PPARδ activation did not affect the protein expression of SREBP-1, but rather reduced the cleaved mature form of SREBP-1 and its target genes, as well as fat accumulation in vitro and in vivo. These results are consistent with previous findings that hepatic overexpression of Insig-1 reduces lipogenesis.16 SREBP-1c mainly regulates the transcription of lipogenic genes, including FAS, ACC, SCD-1, and GPAT, whereas SREBP-2 predominantly regulates genes controlling cholesterol homeostasis, such as low-density lipoprotein receptor, HMG coenzyme A reductase, and squalene synthase.14, 15 In our hepatocytes, PPARδ down-regulated multiple SREBP-1c targets, including FAS, SCD-1, and GPAT, but did not affect the expression of low-density lipoprotein receptor and HMG coenzyme A reductase (data not shown), which indicates that the PPARδ–Insig-1 mechanism may preferentially inhibit the SREBP-1c–mediated processes. FAS catalyzes the conversion of acetyl-CoA and malonyl-CoA in the presence of reduced form of nicotinamide adenine dinucleotide phosphate into long-chain saturated fatty acids.31 SCD-1, an iron-containing enzyme, catalyzes the desaturation of stearic acids into oleic acids, a rate-limiting step in the synthesis of unsaturated fatty acids and required for the onset of diet-induced hepatic insulin resistance.32 GPAT catalyzes the first committed step in glycerolipid biosynthesis and is predicted to play a pivotal role in the regulation of cellular triacylglycerol and phospholipid levels.33 Hepatic mRNA levels of these key lipogenic enzymes are increased in obesity, insulin resistance, and dyslipidemia.34 Thus, suppressing the expression of these genes may contribute to PPARδ inhibiting lipogenesis in hepatocytes. In obese db/db mice, overexpression of PPARδ ameliorated hepatic steatosis. In a recent observation, GW501516 and bezafibrate, a pan-PPARδ agonist, inhibited the increase in hepatic triglyceride level and fatty droplets in the diet-induced mouse model for nonalcoholic steatohepatitis.35
In conclusion, we have shown for the first time that PPARδ prevents the proteolytic processing and activity of SREBP-1, likely via the induction of Insig-1, and ameliorated hepatic steatosis in obese diabetic db/db mice. Thus, PPARδ modulators may have therapeutic potential in the treatment of steatotic liver associated with obesity and type 2 diabetes.