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ATGL pathway
Biochemical Pharmacology 84 (2012) 522–531
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Fenofibrate lowers lipid accumulation in myotubes by modulating the PPARa/AMPK/FoxO1/ATGL pathway Wei-Lu Chen a,b, Yu-Lin Chen d, Yu-Ming Chiang c, Shyang-Guang Wang d,**, Horng-Mo Lee a,b,d,* a
Graduate Institute of Medical Sciences, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan School of Medical Laboratory Sciences and Biotechnology, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan c Graduate Institute of Biomedical Materials and Engineering Research, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan d Institute of Pharmaceutical Sciences and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 April 2012 Accepted 31 May 2012 Available online 9 June 2012
Fenofibrate, a fibric acid derivative, is known to possess lipid-lowering effects. Although fenofibrate may activate peroxisome proliferator-activated receptor (PPAR)a and regulate the transcription of several genes, the underlying mechanisms are poorly understood. In this study, we demonstrated that incubation of C2C12 myotubes with fenofibrate increased adipose triglyceride lipase (ATGL) expression and suppressed fatty acid synthase (FAS) level, thereby decreasing intracellular triglyceride accumulation when cells were incubated at high-glucose condition. Fenofibrate increased the phosphorylation of AMP-activated protein kinase (AMPK), which subsequently increased fatty acid b-oxidation. AMPK phosphorylation was reduced by pretreatment with GW9662 (a PPARa inhibitor), suggesting that AMPK may be a downstream effector of PPARa. Pretreatment with compound C (an AMPK inhibitor) or GW9662 blocked fenofibrate-induced ATGL expression and the lipid-lowering effect. Our results suggest that AMPK is as an upstream regulator of ATGL. With further exploration, we demonstrated that fenofibrate stimulated FoxO1 translocation from the cytosol to nuclei by immunefluorescence assay, chromatin immuneprecipitation assay, and reporter assay. Furthermore, oral administration of fenofibrate ameliorated the body weight, visceral fat and serum biochemical indexes in db/db mice. Taken together, our results suggest that the lipid-lowering effect of fenofibrate was achieved by activating PPARa and AMPK signaling pathway that resulted in increasing ATGL expression, lipolysis, and fatty acid b-oxidation. ß 2012 Elsevier Inc. All rights reserved.
Keywords: AMP-activated protein kinase Adipose triglyceride lipase Fatty acid synthase Free fatty acid b-oxidation Lipid metabolism
1. Introduction Fenofibrate, an amphipathic carboxylic fibrate, has multiple blood lipid-modifying actions, including decreasing the blood triglyceride level and increasing the blood high-density lipoprotein (HDL) cholesterol level [1,2]. These effects are thought to be
Abbreviations: ATGL, adipose triglyceride lipase; FAS, fatty acid synthase; AMPK, AMP-activated protein kinase; PPARa, peroxisome proliferator-activated receptora; HDL, high-density lipoprotein; VLDL, very-low-density lipoprotein; ACC, acetyl-CoA carboxylase; CPT1, carnitine palmitoyltransferase 1; HSL, Hormonesensitive lipase. * Corresponding author at: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei 110, Taiwan. Tel.: +886 2 2736 1661x3316; fax: +886 2 2732 4510. ** Corresponding author at: Institute of Pharmaceutical Sciences and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan, No. 666 Buzih Road, Beitun District, Taichung City 40601, Taiwan. Tel: +886 4 22391647x3975; fax: +886 4 2239 4256. E-mail addresses: [email protected] (S.-G. Wang), [email protected] (H.-M. Lee). 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.05.022
mediated by activation of the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)a, which enhances peroxisomal b-oxidation [3] and activation of lipoprotein lipase [4]. After activating PPARa, fenofibrate stimulates lipoprotein lipase and decreases apoprotein C-III, a very-low-density lipoprotein (VLDL), to degrade triglyceride lipid droplets [5,6]. In a clinical survey, fenofibrate reduced the total plasma cholesterol level by 20–25% and the plasma triglyceride level by 40–45%, and raised the plasma HDL level by 10–30% [7]. Fenofibrate alone or in combination with atrovastatin was proved to be effective in treating hyperlipidemia in type 2 diabetes [8]. However, the molecular mechanisms underlying the lipid-lowering effect of fenofibrate are not completely understood. Obesity is a risk factor for type 2 diabetes mellitus, which results from an energy imbalance because of higher energy intake than energy expenditure [9]. Triglyceride accumulation in skeletal muscles increases in subjects with insulin resistance. The increase of triglyceride accumulation is a result of decreased mitochondrial fatty acid oxidation in cells. Fenofibrate was shown to prevent the development of diabetes in obese diabetes-prone rats, but the
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mechanism is not completely understood [10]. The cellular fuel gauge, 50 -AMP-activated protein kinase (AMPK), an energy sensor protein, is considered as a molecular target for treating type 2 diabetes. Fenofibrate was shown to activate AMPK in human umbilical vein endothelial cells and retinal endothelial cells [11,12], but whether fenofibrate regulates lipid metabolism through an AMPK pathway has not been investigated in C2C12 myotubes. Activation of AMPK is known to phosphorylate and inactivate the downstream protein, acetyl-CoA carboxylase (ACC). ACC phosphorylation results in decreased malonyl-CoA production and increased carnitine palmitoyltransferase (CPT)1 activity, which enhances the transportation of fatty acid into mitochondria for fatty acid b-oxidation [13,14]. ATGL, a recently discovered lipase, is responsible for triglyceride hydrolase activity in cells and is considered as a possible therapeutic target for dyslipidemia and fatty liver. Importantly, ATGL is a rate-limiting lipolytic enzyme in mammals, which initiates hydrolysis of triglyceride and produces diacylglycerol and fatty acids [15,16]. Hormone-sensitive lipase (HSL) is another major lipolytic enzyme that exhibits higher substrate affinity for diacylglycerol to form monoacylglycerol. Both enzymes are regulated by cAMP-mediated phosphorylation of perilipin [17,18]. ATGL expression is regulated by FoxO1 that is a class of forkhead proteins. Deprivation of nutrients may stimulate FoxO1 translocation from the cytosol to nuclei. FoxO1 may bind to the promoter region of the ATGL gene and enhances its transcription [19]. In the present study, we demonstrated that fenofibrate increased AMPK and ACC phosphorylation and enhanced fatty acid b-oxidation in C2C12 myotubes. We provided the evidence that fenofibrate-induced ATGL expression was mediated via an PPARa/ AMPK/FoxO1/ATGL pathway.
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myotubes was distinguished by their morphology. Myotubes were treated with various concentrations of indicated agents and incubated for the indicated time in a 5% CO2 humidified incubator at 37 8C. At the end of incubation, cells were lysed by adding lysis buffer containing 10 mM Tris–HCl (pH 7.5), 1 mM EGTA, 1 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1% mercaptoethanol, 0.5% Triton-X100, and the protease inhibitor cocktail (with final concentrations of 0.2 mM PMSF, 0.1% aprotinin, and 50 mg/ml leupeptin), then stored at 70 8C for further measurements. 2.3. Immunoprecipitation and western blotting Proteins from cell lysates were separated by SDS-PAGE and transferred to poly(vinylidene difluoride) membranes for immunoblotting. Membranes were blocked with blocking solution containing 3% BSA and 0.1% Tween 20 in PBS for 1 h at room temperature followed by incubation with the primary and secondary antibodies. For immunoprecipitation, the agarose beads were conjugated with antibody to LKB-1. Protein (500 mg) from cultured cells was incubated with cross-linked LKB-1 beads overnight, and the immunoprecipitates were boiled with sample loading buffer containing 0.5 mol/l Tris/HCl (pH 6.8), 4.4% (wt/v) SDS, 20% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol and bromophenol blue in distilled/deionised water for 5 min before SDSPAGE. Immunodetection was performed using a LumiGLO chemiluminescence kit (Amersham International, Amersham, UK). Levels of phosphorylation and abundance were quantified by scanning densitometry using a model GS-700 imaging densitometer (BioRad), normalized to levels of total protein. 2.4. Chromatin immunoprecipitation (Chip)
2. Materials and methods 2.1. Antibodies and reagents Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), glutamine, gentamycin, penicillin, and streptomycin were purchased from Life Technologies (Gaithersburg, MD, USA). 5Aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and antibodies specific for AMPK, phosphor-Thr172 AMPK, phosphor-Thr79 ACC, ATGL, phospho-Ser256 FoxO1, and FoxO1, were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific for sterol regulatory element-binding protein (SREBP), a-tubulin, and carnitine palmitoyltransferase-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for fatty acid synthase (FAS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Gene Tex (Irvine, CA, USA). A monoclonal antibody against RNA polymerase II was from Millipore (Bedford, MA, USA). 6-[4-(2Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine (Compound C) and 2-Chloro-5-nitrobenzanilide (GW9662) were obtained from Calbiochem (San Diego, CA, USA). Fenofibrate was purchased from Sigma–Aldrich (St. Louis, MO, USA). A horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody was purchased from Bio-Rad (Hercules, CA, USA). Protease inhibitor cocktail tablets were purchased from Boehringer Mannheim (Mannheim, Germany). 2.2. Culture of C2C12 cells and preparation of cell lysates C2C12 myoblast cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, and penicillin (100 U/ml)/ streptomycin (100 mg/ml). After reaching 80% confluency, C2C12 cells were induced to differentiate into myotubes by adding 2% horse serum. The differentiation status of C2C12
Chip assays were performed with a EZ-ChIP Assay kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. Briefly, protein–DNA complexes were cross-linked with 18.5% formaldehyde, lysed, and sonicated on ice seven times for 15 s each. FoxO1 proteins were then immunoprecipitated from precleared lysates. Protein–DNA complexes were eluted and treated with proteinase K to degrade the proteins. Purified DNA was subjected to polymerase chain reaction (PCR) amplification using forward (50 -ATCTTTAAAAGGCAATTAAGCTG-30 ) and reverse primers (50 -TAAGTCCAGGTCTTAGAAATGT-30 ) to amplify the ATGL promoter region (between 1004 and 1225) using 35 cycles of 94 8C for 20 s, 59 8C for 30 s, and 72 8C 30 s. For all PCRs, 10% input was analyzed along with the immunoprecipitated samples. 2.5. Immunofluorescence C2C12 cells were seeded on a cover glass (Deckgla¨ser, Germany) and incubated at 37 8C overnight before being treated. After a period of incubation, treated cells were washed with cold PBS and fixed with 4% paraformaldehyde for 10 min. Fixed cells were washed with PBS, permeabilized with 0.1% Triton-X100 for 5 min, blocked with PBS with 5% nonfat milk for 60 min, and then incubated with primary antibodies (1 mg/ml) at room temperature for 1 h in a moist container in the dark. The cover glass was washed with PBS three times, and then incubated with Alexa Fluor 555conjugated donkey anti-rabbit IgG secondary antibodies (Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h in a moist container in the dark. After three slow washes with PBS then once with 0.9% sodium chloride, the cover slips were stained with 40 ,6diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) at room temperature for 10 min. The cover slips were mounted using Fluoromount (Sigma Life Science, St. Louis, MO, USA) onto a slide and observed under a fluorescence microscope
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(Olympus IX71, Tokyo, Japan). Pictures were taken with the aid of the Image-Pro 4.5 program (Olympus, Tokyo, Japan). 2.6. Intracellular Oil red O staining C2C12 cells were seeded on a cover glass (Deckgla¨ser, Germany) and incubated at 37 8C overnight before being treated. After treatment, cells were fixed in 4% paraformaldehyde in PBS for 20 min, washed once with 60% isopropanol, and then stained with 60% Oil red O (Sigma, St. Louis, MO, USA) for 15 min at room temperature. Cover slips were then rinsed with water, stained with hematoxylin (Sigma Life Science, St. Louis, MO, USA) for 2 min, and washed thoroughly with distilled water. The cover slips were mounted onto a slide using Fluoromount and observed under a fluorescence microscope (Olympus IX71, Tokyo, Japan). 2.7. Measurement of palmitate b-oxidation After differentiation, cells were resuspended in medium supplemented with [9,10-3H]palmitate complexed to bovine serum albumin (BSA) by a mixture of palmitate and a 10% BSA solution at a 1:2 volume ratio. In total, 3.3 ml of [9,10-3H]palmitate and 6.7 ml of BSA were used per 1 ml of cell culture medium. Each sample used 0.5 106 cells in 1 ml of medium supplemented with the [9,10-3H]palmitate–BSA mixture and cultured for 24 h in 24well plates. After 24 h, the supernatant was applied to an ionexchange column (Dowex 1X8–200, Sigma, St. Louis, MO, USA), and tritiated water was recovered by elution with 2.5 ml of H2O. A 0.75 ml aliquot was then used for scintillation counting. 2.8. Plasmid construction and secreted alkaline phosphatase (SEAP) assay The mouse genomic DNA was prepared from C2C12 cells, in which the DNA fragments containing the nucleotides at positions 1004 to 1225 of the ATGL promoter were amplified by polymerase chain reaction (PCR) [19], with the primer set (50 ATCTTTAAAAGGTACCTAAGCTGGGGGCCTC-30 and 50 -AAGTCCAGGTCCTCGAGATGTGCCCAAGTACC-30 ), and then were subsequently cloned into KpnI/XhoI sites of pSEAP2-Control vector (BD Biosciences Clontech). The resultant DNA constructs were designated as pATGL. The correct orientation of all the DNA constructs was confirmed by restriction enzyme digestions, and the DNA sequences were verified by DNA sequencing. The plasmid DNAs were used to transfect the cells with the Lipofectamine according to the manufacturer’s instructions (Invitrogen, Grand Island, NY, USA). The transfected cells were differentiated and treated with 100 mM fenofibrate for 4 days. The relative amounts of SEAP expressed by the transfected cells were quantitatively determined based on the manufacturer’s instructions (AB Applied Biosystems). 2.9. Animals The db/db mice on a C57BL/6 background (male, 20 weeks old, 50 g) were gifts from the Development Center for Biotechnology of Taiwan. The animals were given free access to water and were fed on a standard diet. Fenofibrate (100 mg/kg, n = 4) or vehicle (n = 4) was administered orally in the afternoon (14:00–14:30 h). The serum biochemical profiles, including triglyceride, cholesterol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were determined with a Biochem-Immuno autoanalyser (Brea, CA, USA). The quality controls, calibrations and determining procedures were carried out according to the suppliers’ instructions.
2.10. Histology and immunohistochemistry Liver and muscle were fixed and embedded in tissue-freezing medium (Leica Microsystems, Wetzlar, Germany) and stored at 80 8C. The frozen tissue was cut into 7 mm-thick sections and placed on glass slides. The tissue sections were stained with haematoxylin and eosin, Oil Red or Sudan III. Oil Red staining and Sudan III staining were counterstained with haematoxylin to visualize lipid droplets. For immunohistochemical analysis, cryostat sections (7 mm) were fixed by incubation in ice cold methanol for 1 min at 4 8C. Afterwards, sections were washed three times with phosphate buffered saline (PBS), and stained using the ABC (avidin–biotin complex) staining kit (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), according to the manufacturer’s instructions. The following mouse specific primary rat antibodies were used for ATGL. The sections were counterstained with haematoxylin and examined by fluorescence microscope (Olympus IX71, Tokyo, Japan). 2.11. Statistical analysis All data are expressed as the mean standard error of the means (S.E.M.) for the number of experiments. Statistical significance (p < 0.05) between experimental groups was tested by a singlefactor analysis of variance (ANOVA) for multiple groups or an unpaired t-test for two groups. 3. Results 3.1. Fenofibrate regulates lipid metabolism-related genes and reduces lipid droplet accumulation in C2C12 myotubes To elucidate whether fenofibrate exerts a lipid-lowering effect via ATGL regulation, myotubes were treated with fenofibrate and the protein level of ATGL was examined by immunoblot. Fenofibrate increased the ATGL protein level in a concentration-dependent manner (Fig. 1A and B). In addition to the lipolytic protein, we also examined the influence of fenofibrate on the expression of lipogenic proteins, including FAS and the SREBP. Expression levels of these two proteins were elevated when cells were cultured in a high-glucose condition. Treatment of cells with a higher concentration of fenofibrate or AICAR decreased FAS and SREBP protein levels (Fig. 1C and D). Consistently, incubation of C2C12 myotubes in highglucose medium increased intracellular lipid droplet accumulation as detected by Oil red O staining. Treatment with fenofibrate reduced lipid droplet accumulation in myotubes (Fig. 1E). 3.2. Fenofibrate increases AMPK phosphorylation and enhances [9,10-3H] palmitate b-oxidation The AMPK signaling pathway is thought to be a natural response to reduce dyslipidemia and ameliorate insulin resistance. We next examined whether fenofibrate activated the AMPK/ACC pathway. As shown in Fig. 2A and B, AICAR, an AMPK activator, increased AMPK and ACC phosphorylation in C2C12 myotubes. Fenofibrate concentration-dependently increased AMPK and ACC phosphorylation in C2C12 myotubes. Fenofibrate is a well-known PPARa agonist. To further characterize the potential role of PPARa activation in regulating AMPK and its functional consequence, we examined the effect of GW9662 (a PPARa inhibitor) on AMPK and ACC phosphorylations. As shown in Fig. 2C and D, pretreatment with compound C (an AMPK inhibitor) or GW9662, suppressed fenofibrate-stimulated AMPK phosphorylation. We next determined whether fenofibrate induced CPT1 expression and whether fenofibrate stimulated fatty acid b-oxidation. Incubation of C2C12 myotubes with fenofibrate increased CPT1 protein level in a
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Fig. 1. Effects of fenofibrate on protein expressions of lipid-related gene and triglyceride accumulation. (A) and (B) Myotubes were incubated with fenofibrate (10–300 mM) or 1 mM AICAR (A) for 24 h. Cell lysates were immunoblotted with antibodies specific for ATGL and GAPDH. The results were representative of three independent experiments. Data are expressed as the mean SE; n = 3. Compared to the control (0 mmol/l): *p < 0.05 and **p < 0.01. (C) and (D) C2C12 myotubes were incubated for 24 h without or with increasing concentrations of fenofibrate in the absence or presence of 50 mM D-glucose (high glucose). Expressions of FAS and SREBP were detected by a western blotting analysis. Data are expressed as the mean SE; n = 3. Compared to the control (0 mmol/l): *p < 0.05 and **p < 0.01; #p < 0.05 and ##p < 0.01 vs. 50 mM glucose alone. (E) Images of cells treated with different concentrations of fenofibrate were taken by microscopy at an original magnification of 1000 by showing fat accumulation in cells stained with Oil red O.
concentration-dependent manner (Fig. 2E). In agreement, treatment with fenofibrate (10–300 mM) for 24 h increased b-oxidation in C2C12 myotubes (Fig. 2F). 3.3. Pharmacological inhibition of PPARa and AMPK attenuates lipid reduction in C2C12 myotubes To determine the roles of the AMPK and PPARa signaling pathway in ATGL induction, C2C12 myotubes were pretreated with
compound C (20 mM) or GW9662 (10 mM) respectively. Fenofibrate-induced ATGL expression was reduced by both inhibitors, suggesting that fenofibrate enhanced ATGL expression through both AMPK and PPARa signaling pathways (Fig. 3A and B). On the other hand, induction of FAS and SREBP expression by high glucose was suppressed by fenofibrate, and this effect was reversed by compound C and GW9662 (Fig. 3C). Oil red O staining also revealed that the reduction in lipid droplet accumulation by fenofibrate was reversed by compound C and GW9662 (Fig. 3D). Taken together,
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Fig. 2. Fenofibrate activates AMPK signaling and enhances b-oxidation. (A) C2C12 myotubes were treated with different concentrations (10–300 mM) of fenofibrate or 1 mM AICAR (A) for 1 h. The proteins of whole-cell lysate were examined by a western blotting analysis to check phosphorylation and total levels of AMPK and ACC. Similar results were obtained from triplicate experiments, and a representative experiment is shown. (B) Densitometric quantification of the phosphorylation of AMPK and ACC were normalized by total AMPK and ACC. *p < 0.05 and **p < 0.01 vs. the control (mean SE; n = 3). (C) and (D) C2C12 myotubes were pretreated with compound C (20 mM) and GW9662 (10 mM) for 1 h prior to the addition of fenofibrate (100 mM) for 1 h. The quantification of the phosphorylation of AMPK and ACC were normalized by total AMPK and ACC. Data are expressed as the mean SE; n = 3. **p < 0.01 vs. the control; #p < 0.05 vs. fenofibrate alone. (E) Cells were lysed after incubation with fenofibrate or 1 mM AICAR (A) for 24 h, and protein expression levels of CPT1 were detected with specific antibodies. (F) [3H]palmitate was added after treatment with fenofibrate for 24 h, and fatty acid b-oxidation was assayed as described in Section 2. Data are expressed as the mean SE; n = 3. Compared to the control (0 mmol/l): *p < 0.05.
these results suggest that fenofibrate may mediate the lipolytic effect through the PPARa or AMPK signaling pathway. 3.4. Fenofibrate stimulated FoxO1 translocation and binding to the ATGL promoter FoxO1 plays a pivotal role in regulating whole-body energy homeostasis [20,21]. As shown in Fig. 4A, FoxO1 was mostly
present in the cytosol when cells were treated with insulin (upper panel). However, when cells were treated with fenofibrate or Ly294002 (a PI3-kinase inhibitor), subcellular localization of FoxO1 was mostly in nuclei (Fig. 4A). The nuclear localization of FoxO1 by fenofibrate was suppressed by pretreating myotubes with compound C and with GW9662 (Fig. 4B), suggesting that the neclear translocation of FoxO1 may be mediated through both AMPK and PPARa pathways. While insulin stimulates FoxO1
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Fig. 3. Pharmacological inhibition of PPARa and AMPK attenuates lipid reduction in C2C12 myotubes. (A) and (B) C2C12 myotubes were pretreated with compound C (20 mM) or GW9662 (10 mM) for 1 h prior to the addition of fenofibrate (100 mM) for 1 h. Cell lysates were detected by ATGL- and GAPDH-specific antibodies. Data are expressed as the mean SE; n = 3. **p < 0.01 vs. the control; #p < 0.05 and ##p < 0.01 vs. fenofibrate alone. (C) and (D) Under a high-glucose condition, cells were incubated with fenofibrate for 24 h after treatment with compound C or GW9662 for 1 h. Extracted proteins were analyzed by western blotting to measure levels of FAS and SREBP, and images of lipid accumulation in cells were taken by microscopy at 1000 magnification (HG, high glucose; F10, 10 mM fenofibrate; F30, 30 mM fenofibrate; F100, 100 mM fenofibrate).
phosphorylation and exclusion from nuclei, cell starvation causes FoxO1 to be translocated from the cytosol to nuclei and promotes ATGL expression [19]. To determinate whether fenofibrate stimulated FoxO1 translocation in the presence of insulin, myotubes were treated with insulin prior to the addition of fenofibrate. Fig. 4C showed that fenofibrate stimulated FoxO1 translocation from the cytosol to nuclei even in the presence of insulin (Fig. 4C). To determine whether fenofibrate enhanced the binding of FoxO1 to ATGL promoter, a chromatin immunoprecipitation (Chip) assay was performed. Less ATGL promoter region was co-immunoprecipitated by anti-FoxO1 antibodies in myotubes in the absence of fenofibrate (Fig. 4D). However, when myotubes were treated with fenofibrate, the association between FoxO1 and the ATGL promoter was increased (Fig. 4D). To further confirm that FoxO1 translocation is associated with ATGL transcription, C2C12 myotube cells were transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. As shown in Fig. 4F, fenofibrate increased SEAP activity. These data support the notion that fenofibrate regulates FoxO1 translocation and binding to the ATGL promoter, which results in induction of ATGL transcription. Because activation of AMPK has been linked to FoxO1 deacetylation, we next investigated whether fenofibrate regulated the acetylation status of FoxO1. Immunoprecipitation using antiacetyl-lys antibody followed by blotting with anti-FoxO1 antibody revealed that both AICAR and fenofibrate treatments reduced the acetylation level of FoxO1 (Fig. 4E).
3.5. Fenofibrate ameliorated the metabolic syndrome of db/db mice To investigate the effects of fenofibrate on lipid metabolism in vivo, 20-week-old db/db mice were orally administered with vehicle (n = 4) or fenofibrate (100 mg/kg, n = 4) for 1 months. Compared with the control db/db mice that treated with vehicle only, the body size and visceral fat content of fenofibrate-treated db/db mice were significantly reduced (Fig. 5A). Fenofibrate caused 12.9% reduction in body weight in treated group (Table 1). In addition, the visceral fat and gonadal fat in fenofibrate-treated mice were reduced by 70.7% and 18.8%, respectively (Table 1). Serum triglyceride level was significantly lower in the fenofibratetreated group, but cholesterol was not changed (Table 2). Although the weight of liver (Table 1) in fenofibrate-treated mice showed no difference, the liver function index of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of fenofibrate-treated mice (123.00 27.79 and 107.00 13.01) were obviously improved compared with those of control mice (339.67 155.22 and 278.33 27.5) (Table 2). Consistent with the in vivo data, fenofibrate treatment decreased FAS production and increased phospho-AMPK and ATGL levels in db/db mice (Fig. 5B). Immunohistochemical analyses for the muscle section also revealed a marked increase in ATGL was seen in fenofibrate-treated group (Fig. 5C). Fewer lipid droplets were in fenofibrate-treated mice than in the untreated group as demonstrated by Oil Red-O and Sudan III staining of liver and muscle sections (Fig. 5D and E).
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Fig. 4. Fenofibrate regulates FoxO1 translocation to promote ATGL transcription. (A) Differentiated C2C12 myotubule cells were treated with 100 nM insulin, 100 mM fenofibrate (Feno), or 20 mM Ly294002 (Ly). Fixed cells were stained with a FoxO1 antibody and Alexa Fluor 555-conjugated donkey anti-rabbit IgG (red). 4,6-Diamidino-2phenylindole (DAPI) (blue) was incorporated in the mounting solution. (B) Differentiated C2C12 myotubule cells were pretreated with 20 mM of compound C (CC) or 10 mM GW9662 (GW), and then 100 mM fenofibrate was added. The staining method is shown in panel A. (C) Differentiated C2C12 myotubule cells were incubated in DMEM without serum (starvation), treated with 100 nM insulin, or pretreated with insulin prior to the addition of 100 mM fenofibrate. The staining method is shown in panel A. (D) Chip assays were performed on C2C12 myotubule cells incubated in either complete medium (NC) or DMEM without serum (Stav), or treated with 100 mM fenofibrate (Feno) or 100 nM insulin (Ins) for 3 h. Following cross-linking and sonication, genomic fragments were immunoprecipitated with an antibody against FoxO1 or rabbit IgG, and then 221-bp DNA fragment located between nucleotides 1004 and 1225 of the ATGL promoter was amplified by PCR, separated in a 3% agarose gel, and visualized by ethidium bromide staining (F, fenofibrate; CC, compound C; GW, GW9662). (E) C2C12 myotube cells were treated with AICAR(A) and fenofibrate(F) for 3 h. Then, total protein lysates were used for immunoprecipitation of FoxO1, and FoxO1 acetylation level was measured by western blotting analysis. (F) SEAP reporter assay was performed on C2C12 myotube cells. Fenofibrate was treated in 100 mM for 96 h. *p < 0.05 vs. the control (mean SE; n = 3). (For interpretation of the references to color in text, the reader is referred to the web version of the article.)
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Fig. 5. Fenofibrate ameliorates the metabolic syndrome in db/db mice. (A) Gross appearance of the whole body and abdomen of db/db mice treated with vehicle (50% alcohol, control, left; n = 4) or fenofibrate (100 mg/kg day1, right; n = 4). (B) Western blot analysis of lipid-related protein production in liver and muscle of db/db mice with or without treatment of fenofibrate. (C) Section of mice muscle was stained using ABC-DAB technique with ATGL primary antibody. The ATGL immunoreactivity was shown as brown bodies (black row). (D) and (E) Histopathological sections of muscle and liver from fenofibrate-treated and -untreated db/db mice were stained with Oil Red-O and Sudan III to detect the content of lipid droplets. H&E, haematoxylin and eosin.
4. Discussion ATGL is a newly identified triglyceride hydrolase, which initiates hydrolysis of triglyceride and produces diacylglycerol
and fatty acids [15,16]. In the present study, we tested whether the lipid-lowering effect of fenofibrate was through ATGL expression. We demonstrated that fenofibrate exerted a lipid-lowering effect through a PPARa/AMPK signaling pathway. We showed that AMPK
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Table 1 Effect of Fenofibrate on body, liver, and abdominal fat weight of db/db mice.
Body weight (g) Liver weight (g) Visceral fat weight (g) Gonadal fat weight (g)
Con
F100
53.27 3.73 5.22 0.34 7.47 0.63 2.92 0.17
46.36 4.37* 5.08 0.51 2.19 0.43* 2.37 0.39*
Values are mean S.E.M. (n = 4); F100, fenofibrate 100 mg/kg. * p < 0.05 compared with control mice.
Table 2 Effect of Fenofibrate on metabolic parameters of db/db mice.
Cholesterol (mg/dl) Triglyceride (mg/dl) AST (IU/l) ALT (IU/l)
Con
F100
183.67 23.57 190.00 32.74 339.67 155.22 278.33 27.5
187.00 11.02 61.00 7.8* 123.00 27.79* 107.00 13.01*
Values are mean SEM (n = 4); F100, fenofibrate 100 mg/kg. * p < 0.05 compared with control mice.
activation resulted in translocation of FoxO1 into nuclei and binding to the ATGL promoter, which in turn increased ATGL expression and decreased intracellular lipid droplet accumulation. These data agree with those of Gaidhu et al., who reported that AICAR induces AMPK activation, which promotes energy dissipation through induction of ATGL [22]. Triglyceride hydrolysis resulted in the release of free fatty acids, which were shown to cause insulin resistance. However, fenofibrate-stimulated AMPK activation may lead to phosphorylation and inhibition of ACC, which in turn increased fatty acid transport to mitochondria for boxidation. On the other hand, fenofibrate also induced CPT1 expression, which presumably would enhance fatty acid transport across mitochondrial inner membranes and facilitate fatty acid oxidation. Thus, free fatty acids released from fenofibratestimulated triglyceride hydrolysis may be transported to mitochondria and oxidized in a concerted manner. In addition, AMPK activation by fenofibrate also suppressed FAS expression. These findings are in accordance with results of previous studies showing that expression of the FAS gene was abrogated by treatment with AICAR in hepatocytes [23]. Fenofibrate is a well-known PPARa steroid nuclear receptor agonist, which has been used to lower serum triglyceride and
cholesterol in patients for decades [24]. However, the mechanism by which fenofibrate mediates the lipid-lowering effect is not completely understood. Skeletal muscles are the largest organ in the human body and a major site of glucose uptake and fatty acid b-oxidation in the body. Fasting- and exercise-regulated energy metabolism can be mimicked by AMPK activators and PPAR agonists to enhance running performance and muscle oxidative capacity [25], suggesting that both pathways are important in energy metabolism. We showed that fenofibrate may mediate the lipid-lowering effect through a PPARa/AMPK signaling pathway. AMPK is considered as a therapeutic target for treatment of diabetes and dyslipidemia [26,27]. These results agree with previous reports that fenofibrate activates AMPK in retinal endothelial cells [11] and in human umbilicalvein endothelial cells (11). Our results define a novel mechanism that lipidlowering agents may exert their effects though a PPARa/AMPKdependent pathway. FoxO1, a transcription factor that plays a critical role in metabolism, regulates expressions of genes involved in gluconeogenesis and lipid metabolism [28]. The FoxO1 signaling pathway is negatively regulated by the insulin/PI3K–Akt pathway, which excludes nuclear localization of FoxO1 and arrests its target gene transcription [29]. In the present study, we demonstrated that fenofibrate enhanced ATGL, a key triglyceride lipase, by stimulating FoxO1 translocation into nuclei. Consistently, Kamei et al. reported that overexpression of FoxO1 in C2C12 myocytes upregulates lipoprotein lipase expression [30]. Because the promoter of ATGL contains putative FoxO1-binding sites [19], it is possible that FoxO1 binds and regulates ATGL gene expression. Using a Chip assay, we demonstrated that fenofibrate enhanced FoxO1 binding to the ATGL promoter (Fig. 4D). AMPK regulated FoxO1 by decreasing its acetylation and increasing transcriptional activity [31]. In accordance, we demonstrated that fenofibrate deacetylated lysine residue of FoxO1 in C2C12 myotubes. Fenofibrate or PPAR-a agonists have been shown to lower muscle lipids and improve insulin sensitivity in high fat-fed rats [32,33]. Consistently, we found that oral administration of fenofibrate decreased body weight and viscerol fat content, and these effects were associated with increased ATGL and decreased FAS production in db/db mice. In conclusion, these results suggest that lipid-lowering agents may exert their effects through the PPARa/AMPK/FoxO1/ATGL pathway (Fig. 6).
Fig. 6. Schematic illustration of the signaling pathway involved in the action of fenofibrate. Fenofibrate increases AMPK phosphorylation by activating PPARa, which in turn phosphorylates ACC and promotes CPT1 expression. Elevated CPT1 recognizes free fatty acids and incorporates them into mitochondria for b-oxidation. On the other hand, fenofibrate also regulates FoxO1 localization by activating AMPK and PPARa. Once the level of nuclear FoxO1 increases, the FoxO1 increases the expression of ATGL by binding to its promoter region. ATGL, a key lipase, hydrolyzes triglyceride to form diglyceride and fatty acids. Finally, the fatty acids are transported for b-oxidation as mentioned above. A novel molecular mechanism underlies the potential therapeutic effects of fenofibrate on hyperlipidemia and obesity.
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Conflict of interest The authors declare no conflict of interest. Acknowledgments H.M.L and his laboratory were supported by grants (NSC993112-B-166-001 and NSC100-2320-B-166-001) from the National Science Council of Taiwan. References [1] Miura Y, Hosono M, Oyamada C, Odai H, Oikawa S, Kondo K. Dietary isohumulones, the bitter components of beer, raise plasma HDL-cholesterol levels and reduce liver cholesterol and triacylglycerol contents similar to PPARalpha activations in C57BL/6 mice. Br J Nutr 2005;93:559–67. [2] van der Hoogt CC, de Haan W, Westerterp M, Hoekstra M, Dallinga-Thie GM, Romijn JA, et al. Fenofibrate increases HDL-cholesterol by reducing cholesteryl ester transfer protein expression. J Lipid Res 2007;48:1763–71. [3] Pruimboom-Brees IM, Brees DJ, Shen AC, Keener M, Francone O, Amacher DE, et al. Using laser scanning cytometry to measure PPAR-mediated peroxisome proliferation and beta oxidation. Toxicol Pathol 2005;33:86–91. [4] Ziouzenkova O, Perrey S, Asatryan L, Hwang J, MacNaul KL, Moller DE, et al. Lipolysis of triglyceride-rich lipoproteins generates PPAR ligands: evidence for an antiinflammatory role for lipoprotein lipase. Proc Natl Acad Sci U S A 2003;100:2730–5. [5] Bijland S, Pieterman EJ, Maas AC, van der Hoorn JW, van Erk MJ, van Klinken JB, et al. Fenofibrate increases very low density lipoprotein triglyceride production despite reducing plasma triglyceride levels in APOE*3-Leiden CETP mice. J Biol Chem 2010;285:25168–75. [6] Ginsberg HN. Changes in lipoprotein kinetics during therapy with fenofibrate and other fibric acid derivatives. Am J Med 1987;83:66–70. [7] Brown WV. Review of clinical studies of fenofibrate in combination with currently approved lipid-lowering drugs. Cardiology 1989;76(Suppl. 1):S45–51. discussion 2–4. [8] Athyros VG, Papageorgiou AA, Athyrou VV, Demitriadis DS, Kontopoulos AG. Atorvastatin and micronized fenofibrate alone and in combination in type 2 diabetes with combined hyperlipidemia. Diabetes Care 2002;25:1198–202. [9] Lang A, Froelicher ES. Management of overweight and obesity in adults: behavioral intervention for long-term weight loss and maintenance. Eur J Cardiovasc Nurs 2006;5:102–14. [10] Motawi TM, Hashem RM, Rashed LA, El-Razek SM. Comparative study between the effect of the peroxisome proliferator activated receptor-alpha ligands fenofibrate and n-3 polyunsaturated fatty acids on activation of 50 -AMPactivated protein kinase-alpha1 in high-fat fed rats. J Pharm Pharmacol 2009;61:1339–46. [11] Kim J, Ahn JH, Kim JH, Yu YS, Kim HS, Ha J, et al. Fenofibrate regulates retinal endothelial cell survival through the AMPK signal transduction pathway. Exp Eye Res 2007;84:886–93. [12] Murakami H, Murakami R, Kambe F, Cao X, Takahashi R, Asai T, et al. Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC. Biochem Biophys Res Commun 2006;341:973–8. [13] Linher-Melville K, Zantinge S, Sanli T, Gerstein H, Tsakiridis T, Singh G. Establishing a relationship between prolactin and altered fatty acid betaOxidation via carnitine palmitoyl transferase 1 in breast cancer cells. BMC Cancer 2011;11:56. [14] Folmes CD, Lopaschuk GD. Role of malonyl-CoA in heart disease and the hypothalamic control of obesity. Cardiovasc Res 2007;73:278–87.
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Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Fenofibrate lowers lipid accumulation in myotubes by modulating the PPARa/AMPK/FoxO1/ATGL pathway Wei-Lu Chen a,b, Yu-Lin Chen d, Yu-Ming Chiang c, Shyang-Guang Wang d,**, Horng-Mo Lee a,b,d,* a
Graduate Institute of Medical Sciences, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan School of Medical Laboratory Sciences and Biotechnology, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan c Graduate Institute of Biomedical Materials and Engineering Research, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan d Institute of Pharmaceutical Sciences and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 April 2012 Accepted 31 May 2012 Available online 9 June 2012
Fenofibrate, a fibric acid derivative, is known to possess lipid-lowering effects. Although fenofibrate may activate peroxisome proliferator-activated receptor (PPAR)a and regulate the transcription of several genes, the underlying mechanisms are poorly understood. In this study, we demonstrated that incubation of C2C12 myotubes with fenofibrate increased adipose triglyceride lipase (ATGL) expression and suppressed fatty acid synthase (FAS) level, thereby decreasing intracellular triglyceride accumulation when cells were incubated at high-glucose condition. Fenofibrate increased the phosphorylation of AMP-activated protein kinase (AMPK), which subsequently increased fatty acid b-oxidation. AMPK phosphorylation was reduced by pretreatment with GW9662 (a PPARa inhibitor), suggesting that AMPK may be a downstream effector of PPARa. Pretreatment with compound C (an AMPK inhibitor) or GW9662 blocked fenofibrate-induced ATGL expression and the lipid-lowering effect. Our results suggest that AMPK is as an upstream regulator of ATGL. With further exploration, we demonstrated that fenofibrate stimulated FoxO1 translocation from the cytosol to nuclei by immunefluorescence assay, chromatin immuneprecipitation assay, and reporter assay. Furthermore, oral administration of fenofibrate ameliorated the body weight, visceral fat and serum biochemical indexes in db/db mice. Taken together, our results suggest that the lipid-lowering effect of fenofibrate was achieved by activating PPARa and AMPK signaling pathway that resulted in increasing ATGL expression, lipolysis, and fatty acid b-oxidation. ß 2012 Elsevier Inc. All rights reserved.
Keywords: AMP-activated protein kinase Adipose triglyceride lipase Fatty acid synthase Free fatty acid b-oxidation Lipid metabolism
1. Introduction Fenofibrate, an amphipathic carboxylic fibrate, has multiple blood lipid-modifying actions, including decreasing the blood triglyceride level and increasing the blood high-density lipoprotein (HDL) cholesterol level [1,2]. These effects are thought to be
Abbreviations: ATGL, adipose triglyceride lipase; FAS, fatty acid synthase; AMPK, AMP-activated protein kinase; PPARa, peroxisome proliferator-activated receptora; HDL, high-density lipoprotein; VLDL, very-low-density lipoprotein; ACC, acetyl-CoA carboxylase; CPT1, carnitine palmitoyltransferase 1; HSL, Hormonesensitive lipase. * Corresponding author at: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei 110, Taiwan. Tel.: +886 2 2736 1661x3316; fax: +886 2 2732 4510. ** Corresponding author at: Institute of Pharmaceutical Sciences and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan, No. 666 Buzih Road, Beitun District, Taichung City 40601, Taiwan. Tel: +886 4 22391647x3975; fax: +886 4 2239 4256. E-mail addresses: [email protected] (S.-G. Wang), [email protected] (H.-M. Lee). 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.05.022
mediated by activation of the nuclear receptor, peroxisome proliferator-activated receptor (PPAR)a, which enhances peroxisomal b-oxidation [3] and activation of lipoprotein lipase [4]. After activating PPARa, fenofibrate stimulates lipoprotein lipase and decreases apoprotein C-III, a very-low-density lipoprotein (VLDL), to degrade triglyceride lipid droplets [5,6]. In a clinical survey, fenofibrate reduced the total plasma cholesterol level by 20–25% and the plasma triglyceride level by 40–45%, and raised the plasma HDL level by 10–30% [7]. Fenofibrate alone or in combination with atrovastatin was proved to be effective in treating hyperlipidemia in type 2 diabetes [8]. However, the molecular mechanisms underlying the lipid-lowering effect of fenofibrate are not completely understood. Obesity is a risk factor for type 2 diabetes mellitus, which results from an energy imbalance because of higher energy intake than energy expenditure [9]. Triglyceride accumulation in skeletal muscles increases in subjects with insulin resistance. The increase of triglyceride accumulation is a result of decreased mitochondrial fatty acid oxidation in cells. Fenofibrate was shown to prevent the development of diabetes in obese diabetes-prone rats, but the
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mechanism is not completely understood [10]. The cellular fuel gauge, 50 -AMP-activated protein kinase (AMPK), an energy sensor protein, is considered as a molecular target for treating type 2 diabetes. Fenofibrate was shown to activate AMPK in human umbilical vein endothelial cells and retinal endothelial cells [11,12], but whether fenofibrate regulates lipid metabolism through an AMPK pathway has not been investigated in C2C12 myotubes. Activation of AMPK is known to phosphorylate and inactivate the downstream protein, acetyl-CoA carboxylase (ACC). ACC phosphorylation results in decreased malonyl-CoA production and increased carnitine palmitoyltransferase (CPT)1 activity, which enhances the transportation of fatty acid into mitochondria for fatty acid b-oxidation [13,14]. ATGL, a recently discovered lipase, is responsible for triglyceride hydrolase activity in cells and is considered as a possible therapeutic target for dyslipidemia and fatty liver. Importantly, ATGL is a rate-limiting lipolytic enzyme in mammals, which initiates hydrolysis of triglyceride and produces diacylglycerol and fatty acids [15,16]. Hormone-sensitive lipase (HSL) is another major lipolytic enzyme that exhibits higher substrate affinity for diacylglycerol to form monoacylglycerol. Both enzymes are regulated by cAMP-mediated phosphorylation of perilipin [17,18]. ATGL expression is regulated by FoxO1 that is a class of forkhead proteins. Deprivation of nutrients may stimulate FoxO1 translocation from the cytosol to nuclei. FoxO1 may bind to the promoter region of the ATGL gene and enhances its transcription [19]. In the present study, we demonstrated that fenofibrate increased AMPK and ACC phosphorylation and enhanced fatty acid b-oxidation in C2C12 myotubes. We provided the evidence that fenofibrate-induced ATGL expression was mediated via an PPARa/ AMPK/FoxO1/ATGL pathway.
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myotubes was distinguished by their morphology. Myotubes were treated with various concentrations of indicated agents and incubated for the indicated time in a 5% CO2 humidified incubator at 37 8C. At the end of incubation, cells were lysed by adding lysis buffer containing 10 mM Tris–HCl (pH 7.5), 1 mM EGTA, 1 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1% mercaptoethanol, 0.5% Triton-X100, and the protease inhibitor cocktail (with final concentrations of 0.2 mM PMSF, 0.1% aprotinin, and 50 mg/ml leupeptin), then stored at 70 8C for further measurements. 2.3. Immunoprecipitation and western blotting Proteins from cell lysates were separated by SDS-PAGE and transferred to poly(vinylidene difluoride) membranes for immunoblotting. Membranes were blocked with blocking solution containing 3% BSA and 0.1% Tween 20 in PBS for 1 h at room temperature followed by incubation with the primary and secondary antibodies. For immunoprecipitation, the agarose beads were conjugated with antibody to LKB-1. Protein (500 mg) from cultured cells was incubated with cross-linked LKB-1 beads overnight, and the immunoprecipitates were boiled with sample loading buffer containing 0.5 mol/l Tris/HCl (pH 6.8), 4.4% (wt/v) SDS, 20% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol and bromophenol blue in distilled/deionised water for 5 min before SDSPAGE. Immunodetection was performed using a LumiGLO chemiluminescence kit (Amersham International, Amersham, UK). Levels of phosphorylation and abundance were quantified by scanning densitometry using a model GS-700 imaging densitometer (BioRad), normalized to levels of total protein. 2.4. Chromatin immunoprecipitation (Chip)
2. Materials and methods 2.1. Antibodies and reagents Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), glutamine, gentamycin, penicillin, and streptomycin were purchased from Life Technologies (Gaithersburg, MD, USA). 5Aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and antibodies specific for AMPK, phosphor-Thr172 AMPK, phosphor-Thr79 ACC, ATGL, phospho-Ser256 FoxO1, and FoxO1, were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific for sterol regulatory element-binding protein (SREBP), a-tubulin, and carnitine palmitoyltransferase-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for fatty acid synthase (FAS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Gene Tex (Irvine, CA, USA). A monoclonal antibody against RNA polymerase II was from Millipore (Bedford, MA, USA). 6-[4-(2Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine (Compound C) and 2-Chloro-5-nitrobenzanilide (GW9662) were obtained from Calbiochem (San Diego, CA, USA). Fenofibrate was purchased from Sigma–Aldrich (St. Louis, MO, USA). A horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody was purchased from Bio-Rad (Hercules, CA, USA). Protease inhibitor cocktail tablets were purchased from Boehringer Mannheim (Mannheim, Germany). 2.2. Culture of C2C12 cells and preparation of cell lysates C2C12 myoblast cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, and penicillin (100 U/ml)/ streptomycin (100 mg/ml). After reaching 80% confluency, C2C12 cells were induced to differentiate into myotubes by adding 2% horse serum. The differentiation status of C2C12
Chip assays were performed with a EZ-ChIP Assay kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. Briefly, protein–DNA complexes were cross-linked with 18.5% formaldehyde, lysed, and sonicated on ice seven times for 15 s each. FoxO1 proteins were then immunoprecipitated from precleared lysates. Protein–DNA complexes were eluted and treated with proteinase K to degrade the proteins. Purified DNA was subjected to polymerase chain reaction (PCR) amplification using forward (50 -ATCTTTAAAAGGCAATTAAGCTG-30 ) and reverse primers (50 -TAAGTCCAGGTCTTAGAAATGT-30 ) to amplify the ATGL promoter region (between 1004 and 1225) using 35 cycles of 94 8C for 20 s, 59 8C for 30 s, and 72 8C 30 s. For all PCRs, 10% input was analyzed along with the immunoprecipitated samples. 2.5. Immunofluorescence C2C12 cells were seeded on a cover glass (Deckgla¨ser, Germany) and incubated at 37 8C overnight before being treated. After a period of incubation, treated cells were washed with cold PBS and fixed with 4% paraformaldehyde for 10 min. Fixed cells were washed with PBS, permeabilized with 0.1% Triton-X100 for 5 min, blocked with PBS with 5% nonfat milk for 60 min, and then incubated with primary antibodies (1 mg/ml) at room temperature for 1 h in a moist container in the dark. The cover glass was washed with PBS three times, and then incubated with Alexa Fluor 555conjugated donkey anti-rabbit IgG secondary antibodies (Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h in a moist container in the dark. After three slow washes with PBS then once with 0.9% sodium chloride, the cover slips were stained with 40 ,6diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) at room temperature for 10 min. The cover slips were mounted using Fluoromount (Sigma Life Science, St. Louis, MO, USA) onto a slide and observed under a fluorescence microscope
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(Olympus IX71, Tokyo, Japan). Pictures were taken with the aid of the Image-Pro 4.5 program (Olympus, Tokyo, Japan). 2.6. Intracellular Oil red O staining C2C12 cells were seeded on a cover glass (Deckgla¨ser, Germany) and incubated at 37 8C overnight before being treated. After treatment, cells were fixed in 4% paraformaldehyde in PBS for 20 min, washed once with 60% isopropanol, and then stained with 60% Oil red O (Sigma, St. Louis, MO, USA) for 15 min at room temperature. Cover slips were then rinsed with water, stained with hematoxylin (Sigma Life Science, St. Louis, MO, USA) for 2 min, and washed thoroughly with distilled water. The cover slips were mounted onto a slide using Fluoromount and observed under a fluorescence microscope (Olympus IX71, Tokyo, Japan). 2.7. Measurement of palmitate b-oxidation After differentiation, cells were resuspended in medium supplemented with [9,10-3H]palmitate complexed to bovine serum albumin (BSA) by a mixture of palmitate and a 10% BSA solution at a 1:2 volume ratio. In total, 3.3 ml of [9,10-3H]palmitate and 6.7 ml of BSA were used per 1 ml of cell culture medium. Each sample used 0.5 106 cells in 1 ml of medium supplemented with the [9,10-3H]palmitate–BSA mixture and cultured for 24 h in 24well plates. After 24 h, the supernatant was applied to an ionexchange column (Dowex 1X8–200, Sigma, St. Louis, MO, USA), and tritiated water was recovered by elution with 2.5 ml of H2O. A 0.75 ml aliquot was then used for scintillation counting. 2.8. Plasmid construction and secreted alkaline phosphatase (SEAP) assay The mouse genomic DNA was prepared from C2C12 cells, in which the DNA fragments containing the nucleotides at positions 1004 to 1225 of the ATGL promoter were amplified by polymerase chain reaction (PCR) [19], with the primer set (50 ATCTTTAAAAGGTACCTAAGCTGGGGGCCTC-30 and 50 -AAGTCCAGGTCCTCGAGATGTGCCCAAGTACC-30 ), and then were subsequently cloned into KpnI/XhoI sites of pSEAP2-Control vector (BD Biosciences Clontech). The resultant DNA constructs were designated as pATGL. The correct orientation of all the DNA constructs was confirmed by restriction enzyme digestions, and the DNA sequences were verified by DNA sequencing. The plasmid DNAs were used to transfect the cells with the Lipofectamine according to the manufacturer’s instructions (Invitrogen, Grand Island, NY, USA). The transfected cells were differentiated and treated with 100 mM fenofibrate for 4 days. The relative amounts of SEAP expressed by the transfected cells were quantitatively determined based on the manufacturer’s instructions (AB Applied Biosystems). 2.9. Animals The db/db mice on a C57BL/6 background (male, 20 weeks old, 50 g) were gifts from the Development Center for Biotechnology of Taiwan. The animals were given free access to water and were fed on a standard diet. Fenofibrate (100 mg/kg, n = 4) or vehicle (n = 4) was administered orally in the afternoon (14:00–14:30 h). The serum biochemical profiles, including triglyceride, cholesterol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were determined with a Biochem-Immuno autoanalyser (Brea, CA, USA). The quality controls, calibrations and determining procedures were carried out according to the suppliers’ instructions.
2.10. Histology and immunohistochemistry Liver and muscle were fixed and embedded in tissue-freezing medium (Leica Microsystems, Wetzlar, Germany) and stored at 80 8C. The frozen tissue was cut into 7 mm-thick sections and placed on glass slides. The tissue sections were stained with haematoxylin and eosin, Oil Red or Sudan III. Oil Red staining and Sudan III staining were counterstained with haematoxylin to visualize lipid droplets. For immunohistochemical analysis, cryostat sections (7 mm) were fixed by incubation in ice cold methanol for 1 min at 4 8C. Afterwards, sections were washed three times with phosphate buffered saline (PBS), and stained using the ABC (avidin–biotin complex) staining kit (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), according to the manufacturer’s instructions. The following mouse specific primary rat antibodies were used for ATGL. The sections were counterstained with haematoxylin and examined by fluorescence microscope (Olympus IX71, Tokyo, Japan). 2.11. Statistical analysis All data are expressed as the mean standard error of the means (S.E.M.) for the number of experiments. Statistical significance (p < 0.05) between experimental groups was tested by a singlefactor analysis of variance (ANOVA) for multiple groups or an unpaired t-test for two groups. 3. Results 3.1. Fenofibrate regulates lipid metabolism-related genes and reduces lipid droplet accumulation in C2C12 myotubes To elucidate whether fenofibrate exerts a lipid-lowering effect via ATGL regulation, myotubes were treated with fenofibrate and the protein level of ATGL was examined by immunoblot. Fenofibrate increased the ATGL protein level in a concentration-dependent manner (Fig. 1A and B). In addition to the lipolytic protein, we also examined the influence of fenofibrate on the expression of lipogenic proteins, including FAS and the SREBP. Expression levels of these two proteins were elevated when cells were cultured in a high-glucose condition. Treatment of cells with a higher concentration of fenofibrate or AICAR decreased FAS and SREBP protein levels (Fig. 1C and D). Consistently, incubation of C2C12 myotubes in highglucose medium increased intracellular lipid droplet accumulation as detected by Oil red O staining. Treatment with fenofibrate reduced lipid droplet accumulation in myotubes (Fig. 1E). 3.2. Fenofibrate increases AMPK phosphorylation and enhances [9,10-3H] palmitate b-oxidation The AMPK signaling pathway is thought to be a natural response to reduce dyslipidemia and ameliorate insulin resistance. We next examined whether fenofibrate activated the AMPK/ACC pathway. As shown in Fig. 2A and B, AICAR, an AMPK activator, increased AMPK and ACC phosphorylation in C2C12 myotubes. Fenofibrate concentration-dependently increased AMPK and ACC phosphorylation in C2C12 myotubes. Fenofibrate is a well-known PPARa agonist. To further characterize the potential role of PPARa activation in regulating AMPK and its functional consequence, we examined the effect of GW9662 (a PPARa inhibitor) on AMPK and ACC phosphorylations. As shown in Fig. 2C and D, pretreatment with compound C (an AMPK inhibitor) or GW9662, suppressed fenofibrate-stimulated AMPK phosphorylation. We next determined whether fenofibrate induced CPT1 expression and whether fenofibrate stimulated fatty acid b-oxidation. Incubation of C2C12 myotubes with fenofibrate increased CPT1 protein level in a
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Fig. 1. Effects of fenofibrate on protein expressions of lipid-related gene and triglyceride accumulation. (A) and (B) Myotubes were incubated with fenofibrate (10–300 mM) or 1 mM AICAR (A) for 24 h. Cell lysates were immunoblotted with antibodies specific for ATGL and GAPDH. The results were representative of three independent experiments. Data are expressed as the mean SE; n = 3. Compared to the control (0 mmol/l): *p < 0.05 and **p < 0.01. (C) and (D) C2C12 myotubes were incubated for 24 h without or with increasing concentrations of fenofibrate in the absence or presence of 50 mM D-glucose (high glucose). Expressions of FAS and SREBP were detected by a western blotting analysis. Data are expressed as the mean SE; n = 3. Compared to the control (0 mmol/l): *p < 0.05 and **p < 0.01; #p < 0.05 and ##p < 0.01 vs. 50 mM glucose alone. (E) Images of cells treated with different concentrations of fenofibrate were taken by microscopy at an original magnification of 1000 by showing fat accumulation in cells stained with Oil red O.
concentration-dependent manner (Fig. 2E). In agreement, treatment with fenofibrate (10–300 mM) for 24 h increased b-oxidation in C2C12 myotubes (Fig. 2F). 3.3. Pharmacological inhibition of PPARa and AMPK attenuates lipid reduction in C2C12 myotubes To determine the roles of the AMPK and PPARa signaling pathway in ATGL induction, C2C12 myotubes were pretreated with
compound C (20 mM) or GW9662 (10 mM) respectively. Fenofibrate-induced ATGL expression was reduced by both inhibitors, suggesting that fenofibrate enhanced ATGL expression through both AMPK and PPARa signaling pathways (Fig. 3A and B). On the other hand, induction of FAS and SREBP expression by high glucose was suppressed by fenofibrate, and this effect was reversed by compound C and GW9662 (Fig. 3C). Oil red O staining also revealed that the reduction in lipid droplet accumulation by fenofibrate was reversed by compound C and GW9662 (Fig. 3D). Taken together,
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Fig. 2. Fenofibrate activates AMPK signaling and enhances b-oxidation. (A) C2C12 myotubes were treated with different concentrations (10–300 mM) of fenofibrate or 1 mM AICAR (A) for 1 h. The proteins of whole-cell lysate were examined by a western blotting analysis to check phosphorylation and total levels of AMPK and ACC. Similar results were obtained from triplicate experiments, and a representative experiment is shown. (B) Densitometric quantification of the phosphorylation of AMPK and ACC were normalized by total AMPK and ACC. *p < 0.05 and **p < 0.01 vs. the control (mean SE; n = 3). (C) and (D) C2C12 myotubes were pretreated with compound C (20 mM) and GW9662 (10 mM) for 1 h prior to the addition of fenofibrate (100 mM) for 1 h. The quantification of the phosphorylation of AMPK and ACC were normalized by total AMPK and ACC. Data are expressed as the mean SE; n = 3. **p < 0.01 vs. the control; #p < 0.05 vs. fenofibrate alone. (E) Cells were lysed after incubation with fenofibrate or 1 mM AICAR (A) for 24 h, and protein expression levels of CPT1 were detected with specific antibodies. (F) [3H]palmitate was added after treatment with fenofibrate for 24 h, and fatty acid b-oxidation was assayed as described in Section 2. Data are expressed as the mean SE; n = 3. Compared to the control (0 mmol/l): *p < 0.05.
these results suggest that fenofibrate may mediate the lipolytic effect through the PPARa or AMPK signaling pathway. 3.4. Fenofibrate stimulated FoxO1 translocation and binding to the ATGL promoter FoxO1 plays a pivotal role in regulating whole-body energy homeostasis [20,21]. As shown in Fig. 4A, FoxO1 was mostly
present in the cytosol when cells were treated with insulin (upper panel). However, when cells were treated with fenofibrate or Ly294002 (a PI3-kinase inhibitor), subcellular localization of FoxO1 was mostly in nuclei (Fig. 4A). The nuclear localization of FoxO1 by fenofibrate was suppressed by pretreating myotubes with compound C and with GW9662 (Fig. 4B), suggesting that the neclear translocation of FoxO1 may be mediated through both AMPK and PPARa pathways. While insulin stimulates FoxO1
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Fig. 3. Pharmacological inhibition of PPARa and AMPK attenuates lipid reduction in C2C12 myotubes. (A) and (B) C2C12 myotubes were pretreated with compound C (20 mM) or GW9662 (10 mM) for 1 h prior to the addition of fenofibrate (100 mM) for 1 h. Cell lysates were detected by ATGL- and GAPDH-specific antibodies. Data are expressed as the mean SE; n = 3. **p < 0.01 vs. the control; #p < 0.05 and ##p < 0.01 vs. fenofibrate alone. (C) and (D) Under a high-glucose condition, cells were incubated with fenofibrate for 24 h after treatment with compound C or GW9662 for 1 h. Extracted proteins were analyzed by western blotting to measure levels of FAS and SREBP, and images of lipid accumulation in cells were taken by microscopy at 1000 magnification (HG, high glucose; F10, 10 mM fenofibrate; F30, 30 mM fenofibrate; F100, 100 mM fenofibrate).
phosphorylation and exclusion from nuclei, cell starvation causes FoxO1 to be translocated from the cytosol to nuclei and promotes ATGL expression [19]. To determinate whether fenofibrate stimulated FoxO1 translocation in the presence of insulin, myotubes were treated with insulin prior to the addition of fenofibrate. Fig. 4C showed that fenofibrate stimulated FoxO1 translocation from the cytosol to nuclei even in the presence of insulin (Fig. 4C). To determine whether fenofibrate enhanced the binding of FoxO1 to ATGL promoter, a chromatin immunoprecipitation (Chip) assay was performed. Less ATGL promoter region was co-immunoprecipitated by anti-FoxO1 antibodies in myotubes in the absence of fenofibrate (Fig. 4D). However, when myotubes were treated with fenofibrate, the association between FoxO1 and the ATGL promoter was increased (Fig. 4D). To further confirm that FoxO1 translocation is associated with ATGL transcription, C2C12 myotube cells were transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. As shown in Fig. 4F, fenofibrate increased SEAP activity. These data support the notion that fenofibrate regulates FoxO1 translocation and binding to the ATGL promoter, which results in induction of ATGL transcription. Because activation of AMPK has been linked to FoxO1 deacetylation, we next investigated whether fenofibrate regulated the acetylation status of FoxO1. Immunoprecipitation using antiacetyl-lys antibody followed by blotting with anti-FoxO1 antibody revealed that both AICAR and fenofibrate treatments reduced the acetylation level of FoxO1 (Fig. 4E).
3.5. Fenofibrate ameliorated the metabolic syndrome of db/db mice To investigate the effects of fenofibrate on lipid metabolism in vivo, 20-week-old db/db mice were orally administered with vehicle (n = 4) or fenofibrate (100 mg/kg, n = 4) for 1 months. Compared with the control db/db mice that treated with vehicle only, the body size and visceral fat content of fenofibrate-treated db/db mice were significantly reduced (Fig. 5A). Fenofibrate caused 12.9% reduction in body weight in treated group (Table 1). In addition, the visceral fat and gonadal fat in fenofibrate-treated mice were reduced by 70.7% and 18.8%, respectively (Table 1). Serum triglyceride level was significantly lower in the fenofibratetreated group, but cholesterol was not changed (Table 2). Although the weight of liver (Table 1) in fenofibrate-treated mice showed no difference, the liver function index of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of fenofibrate-treated mice (123.00 27.79 and 107.00 13.01) were obviously improved compared with those of control mice (339.67 155.22 and 278.33 27.5) (Table 2). Consistent with the in vivo data, fenofibrate treatment decreased FAS production and increased phospho-AMPK and ATGL levels in db/db mice (Fig. 5B). Immunohistochemical analyses for the muscle section also revealed a marked increase in ATGL was seen in fenofibrate-treated group (Fig. 5C). Fewer lipid droplets were in fenofibrate-treated mice than in the untreated group as demonstrated by Oil Red-O and Sudan III staining of liver and muscle sections (Fig. 5D and E).
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Fig. 4. Fenofibrate regulates FoxO1 translocation to promote ATGL transcription. (A) Differentiated C2C12 myotubule cells were treated with 100 nM insulin, 100 mM fenofibrate (Feno), or 20 mM Ly294002 (Ly). Fixed cells were stained with a FoxO1 antibody and Alexa Fluor 555-conjugated donkey anti-rabbit IgG (red). 4,6-Diamidino-2phenylindole (DAPI) (blue) was incorporated in the mounting solution. (B) Differentiated C2C12 myotubule cells were pretreated with 20 mM of compound C (CC) or 10 mM GW9662 (GW), and then 100 mM fenofibrate was added. The staining method is shown in panel A. (C) Differentiated C2C12 myotubule cells were incubated in DMEM without serum (starvation), treated with 100 nM insulin, or pretreated with insulin prior to the addition of 100 mM fenofibrate. The staining method is shown in panel A. (D) Chip assays were performed on C2C12 myotubule cells incubated in either complete medium (NC) or DMEM without serum (Stav), or treated with 100 mM fenofibrate (Feno) or 100 nM insulin (Ins) for 3 h. Following cross-linking and sonication, genomic fragments were immunoprecipitated with an antibody against FoxO1 or rabbit IgG, and then 221-bp DNA fragment located between nucleotides 1004 and 1225 of the ATGL promoter was amplified by PCR, separated in a 3% agarose gel, and visualized by ethidium bromide staining (F, fenofibrate; CC, compound C; GW, GW9662). (E) C2C12 myotube cells were treated with AICAR(A) and fenofibrate(F) for 3 h. Then, total protein lysates were used for immunoprecipitation of FoxO1, and FoxO1 acetylation level was measured by western blotting analysis. (F) SEAP reporter assay was performed on C2C12 myotube cells. Fenofibrate was treated in 100 mM for 96 h. *p < 0.05 vs. the control (mean SE; n = 3). (For interpretation of the references to color in text, the reader is referred to the web version of the article.)
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Fig. 5. Fenofibrate ameliorates the metabolic syndrome in db/db mice. (A) Gross appearance of the whole body and abdomen of db/db mice treated with vehicle (50% alcohol, control, left; n = 4) or fenofibrate (100 mg/kg day1, right; n = 4). (B) Western blot analysis of lipid-related protein production in liver and muscle of db/db mice with or without treatment of fenofibrate. (C) Section of mice muscle was stained using ABC-DAB technique with ATGL primary antibody. The ATGL immunoreactivity was shown as brown bodies (black row). (D) and (E) Histopathological sections of muscle and liver from fenofibrate-treated and -untreated db/db mice were stained with Oil Red-O and Sudan III to detect the content of lipid droplets. H&E, haematoxylin and eosin.
4. Discussion ATGL is a newly identified triglyceride hydrolase, which initiates hydrolysis of triglyceride and produces diacylglycerol
and fatty acids [15,16]. In the present study, we tested whether the lipid-lowering effect of fenofibrate was through ATGL expression. We demonstrated that fenofibrate exerted a lipid-lowering effect through a PPARa/AMPK signaling pathway. We showed that AMPK
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Table 1 Effect of Fenofibrate on body, liver, and abdominal fat weight of db/db mice.
Body weight (g) Liver weight (g) Visceral fat weight (g) Gonadal fat weight (g)
Con
F100
53.27 3.73 5.22 0.34 7.47 0.63 2.92 0.17
46.36 4.37* 5.08 0.51 2.19 0.43* 2.37 0.39*
Values are mean S.E.M. (n = 4); F100, fenofibrate 100 mg/kg. * p < 0.05 compared with control mice.
Table 2 Effect of Fenofibrate on metabolic parameters of db/db mice.
Cholesterol (mg/dl) Triglyceride (mg/dl) AST (IU/l) ALT (IU/l)
Con
F100
183.67 23.57 190.00 32.74 339.67 155.22 278.33 27.5
187.00 11.02 61.00 7.8* 123.00 27.79* 107.00 13.01*
Values are mean SEM (n = 4); F100, fenofibrate 100 mg/kg. * p < 0.05 compared with control mice.
activation resulted in translocation of FoxO1 into nuclei and binding to the ATGL promoter, which in turn increased ATGL expression and decreased intracellular lipid droplet accumulation. These data agree with those of Gaidhu et al., who reported that AICAR induces AMPK activation, which promotes energy dissipation through induction of ATGL [22]. Triglyceride hydrolysis resulted in the release of free fatty acids, which were shown to cause insulin resistance. However, fenofibrate-stimulated AMPK activation may lead to phosphorylation and inhibition of ACC, which in turn increased fatty acid transport to mitochondria for boxidation. On the other hand, fenofibrate also induced CPT1 expression, which presumably would enhance fatty acid transport across mitochondrial inner membranes and facilitate fatty acid oxidation. Thus, free fatty acids released from fenofibratestimulated triglyceride hydrolysis may be transported to mitochondria and oxidized in a concerted manner. In addition, AMPK activation by fenofibrate also suppressed FAS expression. These findings are in accordance with results of previous studies showing that expression of the FAS gene was abrogated by treatment with AICAR in hepatocytes [23]. Fenofibrate is a well-known PPARa steroid nuclear receptor agonist, which has been used to lower serum triglyceride and
cholesterol in patients for decades [24]. However, the mechanism by which fenofibrate mediates the lipid-lowering effect is not completely understood. Skeletal muscles are the largest organ in the human body and a major site of glucose uptake and fatty acid b-oxidation in the body. Fasting- and exercise-regulated energy metabolism can be mimicked by AMPK activators and PPAR agonists to enhance running performance and muscle oxidative capacity [25], suggesting that both pathways are important in energy metabolism. We showed that fenofibrate may mediate the lipid-lowering effect through a PPARa/AMPK signaling pathway. AMPK is considered as a therapeutic target for treatment of diabetes and dyslipidemia [26,27]. These results agree with previous reports that fenofibrate activates AMPK in retinal endothelial cells [11] and in human umbilicalvein endothelial cells (11). Our results define a novel mechanism that lipidlowering agents may exert their effects though a PPARa/AMPKdependent pathway. FoxO1, a transcription factor that plays a critical role in metabolism, regulates expressions of genes involved in gluconeogenesis and lipid metabolism [28]. The FoxO1 signaling pathway is negatively regulated by the insulin/PI3K–Akt pathway, which excludes nuclear localization of FoxO1 and arrests its target gene transcription [29]. In the present study, we demonstrated that fenofibrate enhanced ATGL, a key triglyceride lipase, by stimulating FoxO1 translocation into nuclei. Consistently, Kamei et al. reported that overexpression of FoxO1 in C2C12 myocytes upregulates lipoprotein lipase expression [30]. Because the promoter of ATGL contains putative FoxO1-binding sites [19], it is possible that FoxO1 binds and regulates ATGL gene expression. Using a Chip assay, we demonstrated that fenofibrate enhanced FoxO1 binding to the ATGL promoter (Fig. 4D). AMPK regulated FoxO1 by decreasing its acetylation and increasing transcriptional activity [31]. In accordance, we demonstrated that fenofibrate deacetylated lysine residue of FoxO1 in C2C12 myotubes. Fenofibrate or PPAR-a agonists have been shown to lower muscle lipids and improve insulin sensitivity in high fat-fed rats [32,33]. Consistently, we found that oral administration of fenofibrate decreased body weight and viscerol fat content, and these effects were associated with increased ATGL and decreased FAS production in db/db mice. In conclusion, these results suggest that lipid-lowering agents may exert their effects through the PPARa/AMPK/FoxO1/ATGL pathway (Fig. 6).
Fig. 6. Schematic illustration of the signaling pathway involved in the action of fenofibrate. Fenofibrate increases AMPK phosphorylation by activating PPARa, which in turn phosphorylates ACC and promotes CPT1 expression. Elevated CPT1 recognizes free fatty acids and incorporates them into mitochondria for b-oxidation. On the other hand, fenofibrate also regulates FoxO1 localization by activating AMPK and PPARa. Once the level of nuclear FoxO1 increases, the FoxO1 increases the expression of ATGL by binding to its promoter region. ATGL, a key lipase, hydrolyzes triglyceride to form diglyceride and fatty acids. Finally, the fatty acids are transported for b-oxidation as mentioned above. A novel molecular mechanism underlies the potential therapeutic effects of fenofibrate on hyperlipidemia and obesity.
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