Atorvastatin prevents carbohydrate response element binding protein activation in the fructose-fed rat by activating protein kinase A

Authors

  • Ricardo Rodríguez-Calvo,

    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
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  • Emma Barroso,

    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
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  • Lucía Serrano,

    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
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  • Teresa Coll,

    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
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  • Rosa M. Sánchez,

    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
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  • Manuel Merlos,

    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
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  • Xavier Palomer,

    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
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  • Juan C. Laguna,

    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
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  • Manuel Vázquez-Carrera

    Corresponding author
    1. Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III and Institut de Biomedicina de la Universidad de Barcelona (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
    • Unitat de Farmacologia, Facultat de Farmàcia, Diagonal 643, E-08028 Barcelona, Spain
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    • fax: (93)-4035982


  • Potential conflict of interest: Nothing to report.

Abstract

High fructose intake contributes to the overall epidemic of obesity and metabolic disease. Here we examined whether atorvastatin treatment blocks the activation of the carbohydrate response element binding protein (ChREBP) in the fructose-fed rat. Fructose feeding increased blood pressure (21%, P < 0.05), plasma free fatty acids (59%, P < 0.01), and plasma triglyceride levels (129%, P < 0.001) compared with control rats fed standard chow. These increases were prevented by atorvastatin. Rats fed the fructose-rich diet showed enhanced hepatic messenger RNA (mRNA) levels of glycerol-3-phosphate acyltransferase (Gpat1) (1.45-fold induction, P < 0.05), which is the rate-limiting enzyme for the synthesis of triglycerides, and liver triglyceride content (2.35-fold induction, P < 0.001). Drug treatment inhibited the induction of Gpat1 and increased the expression of liver-type carnitine palmitoyltransferase 1 (L-Cpt-1) (128%, P < 0.01). These observations indicate that atorvastatin diverts fatty acids from triglyceride synthesis to fatty acid oxidation, which is consistent with the reduction in liver triglyceride levels (28%, P < 0.01) observed after atorvastatin treatment. The expression of Gpat1 is regulated by ChREBP and sterol regulatory element binding protein-1c (SREBP-1c). Atorvastatin treatment prevented fructose-induced ChREBP translocation and the increase in ChREBP DNA-binding activity while reducing SREBP-1c DNA-binding activity. Statin treatment increased phospho-protein kinase A (PKA), which promotes nuclear exclusion of ChREBP and reduces its DNA-binding activity. Human HepG2 cells exposed to fructose showed enhanced ChREBP DNA-binding activity, which was not observed in the presence of atorvastatin. Furthermore, atorvastatin treatment increased the CPT-I mRNA levels in these cells. Interestingly, both effects of this drug were abolished in the presence of the PKA inhibitor H89. Conclusion: These findings indicate that atorvastatin inhibits fructose-induced ChREBP activity and increases CPT-I expression by activating PKA. (HEPATOLOGY > 2009;49:106-115.)

Since the introduction of fructose as a sweetener in foods and beverages in 1967, its consumption has risen steadily. There is now increasing evidence that high fructose intake contributes to the overall epidemic of obesity and metabolic disease.1 Unlike glucose, which is widely used by tissues throughout the body, fructose is metabolized mainly in the liver,2 the main organ responsible for the conversion of excess dietary carbohydrate into triglycerides. Digestion of carbohydrates yields simple sugars that are converted to pyruvate (glycolysis), which is either oxidized to provide energy or channeled into pathways for the synthesis of fatty acids (lipogenesis) when energy is available. Fatty acids are then incorporated into triglycerides, which serve as a long-term energy reservoir. Until recently, it was believed that glycolytic and lipogenic gene transcription was induced mainly by insulin through the sterol regulatory element binding protein-1c (SREBP-1c). The recognition that carbohydrates, independently of insulin, also induce glycolytic and lipogenic gene expression has led to the discovery of the carbohydrate response element binding protein (ChREBP), a transcription factor that binds to the carbohydrate-response element (ChoRE) binding site present in the promoters of its target genes.3 Under basal conditions, ChREBP is localized in the cytosol, and its nuclear translocation is rapidly induced at high carbohydrate concentrations. Nuclear translocation and activity of ChREBP are regulated by phosphorylation and dephosphorylation. The flux of carbohydrates promotes the formation of xylulose-5-phosphate, by the hexose monophosphate shunt pathway, which activates protein phosphatase 2A (PP2A) to dephosphorylate ChREBP and promote its nuclear localization and DNA binding. In contrast, protein kinase A (PKA)-dependent phosphorylation of Ser196 sequesters ChREBP in the cytosol, whereas phosphorylation of Thr666 by this kinase inhibits the DNA-binding capacity of ChREBP.4, 5 Similarly, adenosine monophosphate (AMP)-activated protein kinase (AMPK)-mediated phosphorylation of ChREBP inhibits its activity.5

Animals on a high-fructose diet have been used as models of hypertriglyceridemia and metabolic syndrome.6–10 Exposure to these diets induces metabolic dysfunction, which typically results in a rapid rise in serum triglycerides with a concomitant increase in blood pressure within 2 weeks after starting the diet. Exposure to this diet for longer periods leads to elevated free fatty acids and hyperinsulinemia at the expense of glycemic control.

Among the target genes involved in carbohydrate-induced hypertriglyceridemia, the glycerol-3-phosphate acyltransferase (Gpat) catalyzes the first committed step in phospholipid and triglyceride synthesis. The promoter of Gpat contains both ChoRE and sterol-response elements.11, 12 Gpat is located on the mitochondrial outer membrane,13, 14 thereby allowing reciprocal regulation with liver-type carnitine palmitoyltransferase 1 (L-Cpt-1). In fact, overexpression of Gpat in rat hepatocytes reduces fatty acid oxidation,15, 16 indicating competition for fatty acyl coenzymes A between Gpat and L-Cpt-I. Thus, Gpat can divert fatty acyl coenzymes A away from β-oxidation and toward triglyceride synthesis. The opposite may occur during Gpat inhibition, because Gpat knockout mice on a diet enriched in fat and sucrose have a lower hepatic triglyceride content.17, 18

Statins are inhibitors of hydroxymethyl glutaryl coenzyme A reductase, which reduce the plasma levels of cholesterol; however, they are also effective in reducing plasma triglyceride levels in patients with hypertriglyceridemia.19 Although the hypotriglyceridemic mechanisms of statins have not been fully characterized, they may influence the hepatic production and secretion of very-low-density lipoprotein, possibly through the reduction in cholesterol synthesis or through mechanisms of action on apolipoprotein B, such as reduced translocation across the endoplasmic reticulum membrane, increased intracellular degradation, and diminished lipoprotein assembly.20, 21 Furthermore, atorvastatin, a potent inhibitor of hydroxymethyl glutaryl coenzyme A reductase, enhances the susceptibility of newly synthesized apolipoprotein B to proteasome-mediated degradation20 and improves hepatic insulin sensitivity.7 Although these studies report some of the mechanisms of action of statins on hypertriglyceridemia, there is no information as to whether these drugs affect ChREBP activity and translocation in liver. In the current study, we used the fructose-fed rat to examine whether atorvastatin inhibits ChREBP activity. This rat model showed metabolic alterations that were prevented by atorvastatin treatment. Interestingly, drug treatment abolished ChREBP activation through a mechanism involving PKA activation. This new mechanism of action of atorvastatin may be responsible for the reduction in liver triglyceride content, an effect that may contribute to reversing the metabolic alterations in the fructose-fed rat.

Abbreviations

Acc, acetyl-CoA carboxylase; AMP, adenosine monophosphate; AMPK, adenosine monophosphate–activated kinase; Aprt, adenosyl phosphoribosyl transferase; ChoRE, carbohydrate-response element; ChREBP, carbohydrate response element binding protein; EMSA, electrophoretic mobility shift assay; Fas, fatty acid synthase; Gpat1, glycerol-3-phosphate acyltransferase; L-CPT-1, liver-type carnitine palmitoyltransferase 1; L-PK, liver-pyruvate kinase; LXR, liver X receptor; mRNA, messenger RNA; NE, nuclear protein extract; PKA, protein kinase A; PP2A, protein phosphatase 2A; RT-PCR, reverse transcription polymerase chain reaction; Scd-1, stearoyl-CoA desaturase; SEM, standard error of the mean; SREBP-1c, sterol regulatory element binding protein-1c.

Materials and Methods

Materials.

Atorvastatin was provided by Pfizer Laboratories. [γ-32P]dATP (3000 Ci/mmol) was purchased from Amersham Biosciences (Piscataway, NJ). All other chemicals were from Sigma.

Animals.

Male Sprague-Dawley rats (210-230 g) were maintained under standard conditions of illumination (12-hour light/dark cycle) and temperature (21°C ± 1°C). They were fed a standard diet (Harlan, Barcelona, Spain) for 5 days before the study began. The animals were randomly distributed into three experimental groups: those fed the standard diet, those fed a fructose-rich diet (referred to as fructose diet), that provided 50% of total calories as a fructose, and those fed fructose-rich diet supplemented with atorvastatin (approximately 30 mg/kg/day). After 30 days of treatment with the diets, tail-cuff pressure was evaluated using a Letica/Panlab (Barcelona, Spain) apparatus and animals were killed in nonfasting conditions. After collecting blood, we analyzed serum samples for triglycerides, glucose, cholesterol, free fatty acids (Wako, Japan), insulin (Amersham), and leptin (Linco, St. Charles, MO). The liver samples were frozen in liquid nitrogen and then stored at −80°C. The local committee on animal care approved the animal protocol.

Cell Culture.

HepG2 human hepatocarcinoma cells were obtained from the American Type Tissue Culture Collection (Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium with 1 g/L glucose containing 10% fetal bovine serum at 37°C with 5% CO2. Cells were incubated in serum-free Dulbecco's modified Eagle's medium for 16 hours before the treatments described.

Measurements of Messenger RNA.

Levels of messenger RNA (mRNA) were assessed by the reverse-transcription polymerase chain reaction (RT-PCR) as previously described.22 The sequences of the sense and antisense primers used for amplification were: liver-pyruvate kinase (L-Pk), 5′-TTTGCCTCCTTTGTGCGGAAA-3′ and 5′-TCTCCGCAGGGATCTCAATGG-3′; Acetyl-CoA carboxylase (Acc), 5′-CGAGGCCGCTCAGCAACAGTA-3′ and 5′-TGGGTTCCTCGGAGGCTTCTG-3′; Stearoyl-CoA desaturase, (Scd-1), 5′-GCTCATCGCTTGTGGAGCCAC-3′ and 5′-GGACCCCAGGGAAACCAGGAT-3′; fatty acid synthase (Fas), 5′-GTCTGCAGCTACCCACCCGTG-3′ and 5′-CTTCTCCAGGGTGGGGACCAG-3′; Gpat1 5′-ATCCGCAACGCTGAAATGGAA -3′ and 5′-GGCAAACATGCCCTTGTGGAC-3′; L-Cpt-I (rat) 5′-TATGTGAGGATGCTGCTT-3′ and 5′-CTCGGAGAGCTAAGCTTG-3′, Cpt-I (human) 5′-AGTCGGGAGGCCCTGAAACAC-3′ and 5′-CCCACTGTTGCCATGATGACG-3′; 18S 5′-CCAAAGTCTTTGGGTTCCGGG-3′ and 5′-GCTCAATCTCGG GTGGCTGAA-3′ and adenosyl phosphoribosyl transferase (Aprt), 5′-AGCTTCCCGGACTTCCCCATC-3′ and 5′-GACCACTTTCTGCCCCGGTTC-3′. Amplification of each gene yielded a single band of the expected size (L-Pk: 193 bp, Acc-1: 211 bp, Scd-1: 521 bp, Fas: 214 bp, Gpat1: 244 bp, L-Cpt-I (rat): 629 bp, Cpt-I (human): 143 bp, Aprt: 329 bp, and 18S: 337 bp). Preliminary experiments were carried out with various amounts of complementary DNA to determine nonsaturating conditions of PCR amplification for all the genes studied. Therefore, under these conditions, relative quantification of mRNA was assessed by the RT-PCR method used in this study.23 Radioactive bands were quantified by video-densitometric scanning (Vilbert Lourmat Imaging). The results for the expression of specific mRNAs are presented relative to the expression of the control gene (Aprt).

Isolation of Nuclear Extracts.

Nuclear extracts were isolated as previously described.22

Electrophoretic Mobility Shift Assay.

Electrophoretic mobility shift assay (EMSA) was performed using double-stranded oligonucleotides for the consensus binding site of ChREBP (5′-TCCTGCATGTGCCACAGGCGTGTCACC-3′) and SREBP-1c (5′-GATCCTGATCACGTGATCGAGGAG-3′) as previously described.22

Immunoblotting.

To obtain total protein, cultured cells and liver were homogenized in cold lysis buffer (5 mM Tris-HCl [pH 7.4], 1 mM ethylenediaminetetra-acetic acid, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 5.4 g/mL aprotinin). The homogenate was centrifuged at 16,700g for 30 minutes at 4°C. Protein concentration was measured by the Bradford method. Proteins (30 g) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% separation gels and transferred to Immobilon polyvinylidene fluoride membranes (Millipore, Bedford, MA). Western blot analysis was performed using antibodies against ChREBP, SREBP-1c, Oct-1 (Santa Cruz), Acc, the catalytic subunit of PP2A (52F8), total and phospho-PKA (Cell Signaling). Detection was achieved using the EZ-ECL chemiluminescence kit (Amersham). Size of detected proteins was estimated using protein molecular-mass standards (Invitrogen, Barcelona, Spain).

PKA Activity.

PKA activity was examined with a commercially available kit (Assay Designs, Ann Arbor, MI), following the instructions of the manufacturer.

Statistical Analyses.

Results are expressed as means ± standard error of the mean (SEM) of five separate experiments. Significant differences were established by one-way analysis of variance using the GraphPad Instat programme (GraphPad Software V2.03) (GraphPad Softwware Inc., San Diego, CA). When significant variations were found, the Tukey-Kramer multiple comparisons test was applied. Differences were considered significant at P < 0.05.

Results

Effect of Atorvastatin Treatment on Plasma Lipid Levels and on the Expression of Glycolytic and Lipogenic Genes in Fructose-Fed Rats.

None of the dietary treatments significantly affected body weight (control: 348 ± 13 g, fructose: 354 ± 15 g, and fructose supplemented with atorvastatin: 350 ± 12 g). Rats fed with the fructose diet for 30 days showed a 21% increase (P < 0.05) in blood pressure, an effect that was not observed in rats receiving the same diet supplemented with atorvastatin (Fig. 1). Nonfasting rats fed the fructose-rich diet showed no changes in the plasma levels of glucose, insulin, cholesterol, or leptin compared with controls (Fig. 1). In contrast, these rats had enhanced plasma levels of free fatty acids (59%, P < 0.01) and triglycerides (129%, P < 0.001), changes that were abolished by atorvastatin treatment (Fig. 1).

Figure 1.

Atorvastatin treatment prevents metabolic alterations in the fructose-fed rat. Rats were fed a standard chow or a fructose-rich diet with or without atorvastatin supplementation (approximately 30 mg/kg/day). After 30 days of treatment, blood samples were obtained under nonfasting conditions. Data are expressed as means ± SEM (six rats per group). *P < 0.05, **P < 0.01, and ***P < 0.001 versus control rats. #P < 0.05, ##P < 0.01, ###P < 0.001 versus fructose-fed rats.

To study the mechanisms involved in the effect of atorvastatin, we examined the expression of glycolytic (liver-pyruvate kinase, L-Pk) and lipogenic (acetyl-CoA carboxylase, Acc; stearoyl-CoA desaturase, Scd-1; and fatty acid synthase, Fas) genes in liver (Fig. 2). The fructose diet increased the mRNA levels of L-Pk (4.2-fold, P < 0.001), Acc (2.3-fold, P < 0.05), Scd-1 (2.5-fold, P < 0.01), and Fas (eightfold, P < 0.001); however, atorvastatin treatment significantly reduced the expression of only L-Pk (24% reduction, P < 0.05). Likewise, the fructose diet increased Acc protein levels, and atorvastatin did not reverse this induction (Fig. 2E). Once fatty acids are formed by the lipogenic pathway, they are either incorporated into triglycerides or oxidized by mitochondria. Triglycerides are synthesized through the action of enzymes such as diacylglycerol acyltransferase and Gpat. The expression of diacylglycerol acyltransferase 1 and 2 was not affected by either the fructose diet or atorvastatin treatment (data not shown). Unlike diacylglycerol acyltransferase, the mRNA levels of Gpat1, which catalyzes the first committed step in triacylglycerol synthesis and is believed to be rate limiting for the synthesis of these lipids,24 showed a 1.5-fold increase (P < 0.01) in rats fed the fructose diet compared with controls, whereas those rats receiving fructose plus atorvastatin had Gpat1 mRNA levels similar to those found in controls (Fig. 3A). Because Gpat1 and L-Cpt-I compete in liver for acyl coenzymes A and determine whether they are incorporated into triglycerides or oxidized by the mitochondrial β-oxidation process, we assessed whether atorvastatin treatment affected the expression of L-Cpt-I. Atorvastatin significantly enhanced L-Cpt-I mRNA levels (128% increase, P < 0.01) (Fig. 3B). In agreement with these changes, the fructose diet increased liver triglyceride levels (2.35-fold, P < 0.001) and atorvastatin significantly reduced this effect (28% reduction, P < 0.01) (Fig. 3C). Although fructose diet caused liver steatosis, this diet did increase neither the levels of apoptosis [Poly (ADP-ribose) polymerase, protein levels] nor fibrosis [transforming growth factor-beta1 (TGF-β), mRNA levels] markers in liver (data not shown).

Figure 2.

Atorvastatin treatment does not affect the expression of several lipogenic genes. Effects of atorvastatin on the expression of L-Pk (A), Acc (B), Scd-1 (C), and Fas (D) in the livers of rats fed a standard chow or a fructose-rich diet with or without atorvastatin supplementation. Total RNA was isolated and analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the Aprt mRNA levels are shown. Data are expressed as means ± SEM (6 rats per group). (E) Analysis of Acc protein levels by immunoblotting of total protein extracts from livers. The blot data are representative of three separate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control rats. #P < 0.05 versus fructose-fed rats.

Figure 3.

Atorvastatin prevents fructose-induced Gpat1 up-regulation and induces the expression of L-Cpt-I. Effects of atorvastatin on the mRNA levels of (A) Gpat1 and (B) L-Cpt-I in the livers of rats fed a standard chow or a fructose-rich diet with or without atorvastatin supplementation. Total RNA was isolated and analyzed by RT-PCR. A representative autoradiogram and the quantification normalized to the Aprt mRNA levels are shown. (C) Analysis of liver triglyceride levels. Data are expressed as means ± SEM (six rats per group). **P < 0.01 and ***P < 0.001 versus control rats. #P < 0.05 and ###P < 0.001 versus fructose-fed rats.

Atorvastatin Prevents ChREBP Translocation in the Liver of Fructose-Fed Rats.

Given that the promoter of Gpat1 contains both ChoRE and sterol-responsive elements,11, 12 atorvastatin may reduce the expression of this gene by influencing the activity of these transcription factors. An analysis of the nuclear protein levels of SREBP-1c showed an increase in the livers of fructose-fed rats. Atorvastatin treatment did not reverse this effect (Fig. 4A), indicating that this drug does not affect the content of this transcription factor. We then assessed the DNA-binding activity of SREBP1c by EMSA (Fig. 4B). SREBP1c formed two complexes (I and II) with nuclear proteins. Specificity of the complexes was assessed by adding an excess of unlabeled SREBP1c oligonucleotide. DNA-binding activity of SREBP1c was reduced in the livers of rats fed the fructose diet supplemented with atorvastatin compared with those fed the fructose diet alone. These findings indicate that although nuclear SREBP1c protein levels are increased in atorvastatin-treated rats, its activity is reduced.

Figure 4.

Atorvastatin does not affect fructose-induced SREBP-1c protein levels, but reduces SREBP-1c DNA-binding activity. (A) Analysis of SREBP-1c protein levels by immunoblotting of nuclear protein extracts from livers of rats fed a standard diet or a fructose-rich diet with or without atorvastatin supplementation. Oct-1 protein levels are shown as a control to demonstrate equal loading. The blot data are representative of three separate experiments. (B) Autoradiograph of EMSA performed with a 32P-labeled SREBP-1c nucleotide and crude nuclear protein extract (NE). Two specific complexes (I and II), based on competition with a molar excess of unlabeled probe, are shown. The blot data are representative of three separate experiments.

The fructose diet enhanced the protein levels of ChREBP in the nuclear fraction, an induction that was blocked in the liver of fructose-fed rats supplemented with atorvastatin (Fig. 5A). To test whether this inhibition resulted in a reduction in ChREBP activity, we performed EMSA. ChREBP formed two complexes with nuclear proteins (complexes I and II) (Fig. 5B), whose specificity was assessed in competition experiments by adding an excess of unlabeled ChREBP oligonucleotide. ChREBP binding activity, mainly of specific complex I, increased in nuclear extracts from livers of fructose-fed rats. In contrast, in the livers of rats fed a fructose diet supplemented with atorvastatin, the DNA-binding activity of this nuclear receptor decreased. Addition of antibody against ChREBP formed a super-shifted band, thereby indicating that ChREBP was present in the complexes. Because ChREBP activity depends on its nuclear translocation,5 these findings indicate that atorvastatin prevents either ChREBP translocation to the nucleus or promotes its nuclear exclusion. This nuclear translocation process of ChREBP is controlled by phosphorylation and dephosphorylation.4, 5 Thus, PP2A dephosphorylates ChREB, thereby promoting its translocation into the nucleus, and enhanced DNA-binding activity. Atorvastatin treatment did not alter the protein levels of the catalytic subunit of PP2A, suggesting that this drug does not affect ChREBP translocation by modulating the protein levels of this phosphatase (Fig. 5C). In contrast, PKA-mediated ChREBP phosphorylation promotes nuclear exclusion of ChREBP and reduces its DNA-binding activity.4, 5 Given that statins activate the PKA pathway25 and that this kinase up-regulates the expression of L-Cpt-I26 and may suppress SREBP-1c expression,27 we next evaluated whether atorvastatin treatment led to enhanced PKA phosphorylation and activity. Atorvastatin strongly enhanced the phospho-protein levels (Thr197) of the catalytic subunit of PKA (Fig. 5D), which is required for full catalytic activity of this kinase.28 Accordingly, atorvastatin treatment caused a 2.3-fold induction (P < 0.05) in PKA activity compared with rats fed the fructose diet (Fig. 5E).

Figure 5.

Atorvastatin prevents ChREBP activation in the fructose-fed rat. (A) Analysis of ChREBP by immunoblotting of nuclear protein extracts from livers of rats fed a standard diet or a fructose-rich diet with or without atorvastatin supplementation. Oct-1 protein levels are shown as a control to demonstrate equal loading. (B) Autoradiograph of EMSA performed with a 32P-labeled ChREBP nucleotide and crude NE. Two specific complexes (I and II), based on competition with a molar excess of unlabeled probe, are shown. A supershift analysis performed by incubating NE with an antibody directed against ChREBP is also shown. Analysis of (C) PP2A, α-actin, and (D) total and phospho-PKA by immunoblotting of total protein extracts. The blot data are representative of three separate experiments. (E) Liver extracts from rats fed a standard diet or a fructose-rich diet with or without atorvastatin supplementation were used for measuring PKA activity as mentioned in experimental procedures. **P < 0.01 versus control rats. #P < 0.05 versus fructose-fed rats.

Atorvastatin Blocks Fructose-Induced ChREBP Translocation by Activating PKA.

To demonstrate the involvement of PKA activation in atorvastatin-mediated inhibition of ChREBP, we exposed HepG2 cells to fructose in the presence or in the absence of atorvastatin and the PKA inhibitor H89. Cells exposed to fructose showed enhanced ChREBP DNA-binding activity, whereas this effect was prevented in the presence of atorvastatin (Fig. 6A). Interestingly, in the presence of the PKA inhibitor H89, the reduction in ChREBP-DNA-binding activity caused by atorvastatin was reversed, indicating that PKA activation was involved in the effects of this statin. Furthermore, atorvastatin treatment strongly increased the expression of CPT-I in HepG2 cells, whereas in control and fructose-exposed cells the expression of this gene was not detected (Fig. 6B). Again, this effect of atorvastatin was inhibited by the PKA inhibitor, demonstrating that statin treatment induced the expression of CPT-I through the activation of this kinase. In agreement with the involvement of PKA activation after atorvastatin treatment, this statin enhanced phospho-PKA protein levels, an effect that was abolished by H89 (Fig. 6C), and PKA activity (Fig. 6D). Interestingly, AMPK regulates both Gpat and Cpt-I29, 30 expression. Although we did not observe changes in the phospho-protein levels of this kinase in vivo (data not shown), HepG2 cells exposed to atorvastatin showed enhanced phospho-AMPK protein levels (Fig. 6E), suggesting that this mechanism may also contribute to the changes observed after short statin treatment.

Figure 6.

Atorvastatin prevents fructose-induced ChREBP activation and induced CPT-I mRNA levels through PKA. (A) Autoradiograph of EMSA performed with a 32P-labeled ChREBP nucleotide and NE from HepG2 cells incubated for 24 hours with 30 mM fructose. When indicated, cells were co-incubated in the presence or in the absence of atorvastatin (10 μM) or the PKA inhibitor H89 (10 μM). One specific complex, based on competition with a molar excess of unlabeled probe, is shown. The autoradiograph data are the result of three separate experiments. (B) Messenger RNA levels of Cpt-I were analyzed in HepG2 cells incubated for 24 hours with 30 mM fructose. When indicated cells were co-incubated in the presence or in the absence of atorvastatin (10 μM) or the PKA inhibitor H89 (10 μM). Total RNA was analyzed by RT-PCR. A representative autoradiogram and quantification normalized to the 18S mRNA levels are shown. Data are expressed as mean ± SD of four experiments.**P < 0.01 versus control and fructose-exposed cells. ##P < 0.01 versus fructose-exposed cells in the presence of atorvastatin. Analysis of total and phospho-PKA protein levels (C), PKA activity (E), and total and phospho-AMPK (E) protein levels in HepG2 cells incubated for 24 hours with 30 mM fructose in the presence or in the absence of atorvastatin (10 μM). The blot data are representative of three separate experiments.

Discussion

Hypertriglyceridemia reflects an imbalance between the rates of very-low-density lipoprotein–triglyceride production and clearance. The main factor influencing hepatic triglyceride secretion is fatty acid availability.30 High fructose intake can enhance fatty acid availability by increasing hepatic de novo lipogenesis through the activation of several transcription factors. ChREBP is determinant for the induction of lipogenic genes by carbohydrates,31 whereas SREBP-1c is responsible for the insulin-mediated induction of lipogenic enzymes in liver. In addition to these transcription factors, liver X receptor (LXR) directly regulates the expression of several lipogenic genes (Fas and Scd-1)32 and, indirectly, through the transcriptional control of SREBP-1c, plays a central role in insulin-stimulated lipogenesis.33

Statins prevent the development of fructose-induced hypertriglyceridemia through several mechanisms,7 but little is known about the effects of these drugs on ChREBP activity. Here we provide the first report that atorvastatin treatment reduces nuclear ChREBP levels by activating PKA (Fig. 7). Additional studies are required to elucidate whether this mechanism contributes to the hypotriglyceridemic effect of statins. However, the observation that liver-specific inhibition of ChREBP improves hepatic steatosis and insulin resistance in obese ob/ob mice by significantly decreasing the rate of lipogenesis points to ChREBP as a modulator of fatty acid content in liver.34

Figure 7.

A schematic of the de novo lipogenesis pathway is shown. Glycolysis in liver provides carbons from carbohydrates for lipogenesis. Atorvastatin may prevent ChREBP translocation and DNA-binding activity in the fructose-fed rat through a mechanism that involves PKA activation. Furthermore, PKA activation by atorvastatin leads to enhanced CPT-I expression. As a result of these changes, fatty acids synthesized by lipogenesis might be diverted from triglyceride synthesis to mitochondrial β-oxidation, thereby resulting in a reduced liver triglyceride content. FK, fructokinase; X-5-P, xylulose-5-phosphate; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; MTTP, microsomal triglyceride transfer protein; ATV, atorvastatin.

In the current study, we used high fructose feeding rats as a model of hypertriglyceridemia and other metabolic alterations. Because it has been previously reported that under fasted conditions ChREBP was not located in the nucleus,33 in our study animals were killed under nonfasting conditions. Exposure of rats to a 50% fructose diet for 30 days augmented serum triglycerides with a concomitant increase in blood pressure. In addition, the increase in plasma free fatty acids observed in the fructose-fed rats indicates that insulin resistance leads to increased lipolysis in white adipose tissue. Atorvastatin treatment blocked all these metabolic alterations caused by fructose intake. The reduction in plasma free fatty acids caused by statin treatment indicates that this drug reverts insulin resistance, as previously reported.7 Indeed, atorvastatin improves insulin signaling in the fructose-fed Syrian golden hamster by reducing the content of protein tyrosine phosphatase-1B, which is involved in the development of insulin resistance.7 This improvement in insulin signaling was not mediated by a change in hepatic triglyceride accumulation because no significant difference was observed in liver triglyceride levels.7 Because insulin suppresses very-low-density lipoprotein production by reducing plasma free fatty acids, in our study the improvement in insulin sensitivity may have contributed to the reduction in plasma triglyceride levels observed after atorvastatin treatment. However, we observed that atorvastatin treatment led to a decrease in the hepatic content of triglycerides, thereby suggesting that additional mechanisms are also involved in the effect of this drug.

To examine the potential new mechanisms of action of atorvastatin in the fructose-fed rat, we assessed its effects on the expression of several lipogenic genes. Although the fructose-induced expression of several lipogenic genes (Acc, Fas, and Scd1) was not affected by atorvastatin, drug treatment prevented the increase in Gpat1 expression after fructose exposure. Gpat1 is crucial for directing the metabolic fate of fatty acids in hepatocytes.16 It competes for fatty acids with L-Cpt-I, the rate-limiting enzyme for fatty acid oxidation, thus determining whether fatty acids are either used for triglyceride synthesis or oxidized. Therefore, the increase in Gpat1 mRNA levels is consistent with the enhanced content of hepatic triglyceride in the fructose-fed rat. In agreement with the pivotal role of Gpat1 in controlling the balance between storage and fatty acid oxidation, liver-specific overexpression of Gpat1 in lean mice results in hepatic steatosis, increased triglyceride secretion, and reduced fatty acid oxidation.35 Likewise, Gpat1 knockout mice have lower liver triglyceride levels and show changes that suggest increased β-oxidation.18 These observations are consistent with the enhanced expression in L-Cpt-I that we observed after atorvastatin treatment. Thus, taken together, these findings suggest that the mechanisms that reduce Gpat1 expression also lead to the increase in L-Cpt-I. In fact, Gpat1 and L-Cpt-I are reciprocally regulated by AMP-activated kinase,29, 36 although we did not observe changes in the total and phospho-AMPK levels in livers of rats exposed to either fructose or fructose supplemented with atorvastatin (data not shown), HepG2 cells exposed to atorvastatin showed enhanced phospho-AMPK protein levels, suggesting that this mechanism may also contribute in short treatments. Overall, the reduction of Gpat1 together with the concomitant increase in L-Cpt-I may reduce the availability of fatty acids to form triglycerides, thereby diverting fatty acids from these lipids to mitochondrial β-oxidation. This hypothesis is supported by the reduction in the content of liver triglycerides observed after atorvastatin treatment compared with rats receiving the fructose diet alone. In addition, this effect of atorvastatin on Cpt-I expression was reproduced in the human HepG2 cell line at a low concentration (10 μM), suggesting that this effect is not limited to rodents.

In fed rats, the expression of the lipogenic genes is under the control of the combined action of ChREBP, SREBP-1c, and LXR. Interestingly, the activity of these three transcription factors is regulated by PKA. In regard to energy metabolism, PKA is activated by glucagon and adrenalin and, therefore, is classically recognized as a fasting signal to activate gluconeogenesis and β-oxidation and to oppose triglyceride synthesis and glucose utilization. Thus, it has been reported that activation of this kinase reduces the translocation and the DNA-binding activity of ChREBP4, 5 and suppresses SREBP-1c expression via phosphorylation of LXR in the liver.37 Moreover, the PKA-mediated increase in the β-oxidation of fatty acids involves the enhanced expression of Cpt-I.26 In this study, we show that atorvastatin treatment activates PKA, which is consistent with the findings of previous studies.25 PKA is activated by cyclic AMP, produced by adenylate cyclase. It is possible, therefore, to speculate that statins may somehow activate adenylate cyclase, increasing cyclic AMP and activating PKA.

In agreement with the activation of PKA after exposure to atorvastatin, we found that this drug prevented fructose-induced ChREBP translocation into the nucleus and the binding of this protein to DNA. Atorvastatin treatment also reduced SREBP-1c binding activity. The PKA-mediated regulation of SREBP-1c is controversial because there are reports showing that PKA phosphorylates this transcription factor in vitro, thereby enhancing its DNA-binding activity.38 However, other studies report that PKA activation suppresses SREBP-1c expression by reducing LXR activity.37 Our results do not support the latter, because we did not observe a reduction in the nuclear protein levels of SREBP-1c after atorvastatin treatment. Thus, additional studies are required to elucidate the potential mechanisms by which atorvastatin reduces the DNA-binding activity of SREBP-1c. Finally, it remains to be established whether the effects reported here are a class-family effect of statins or specific to atorvastatin.

In summary, we report that atorvastatin prevents ChREBP translocation and DNA-binding activity in the fructose-fed rat through a mechanism that involves PKA activation. As a result of these changes, fatty acids synthesized by lipogenesis might be diverted from triglyceride synthesis to mitochondrial β-oxidation, thereby resulting in a reduced liver triglyceride content. This new mechanism of action of atorvastatin might contribute to preventing metabolic alterations in the fructose-fed rat and humans.

Acknowledgements

The authors thank the University of Barcelona's Language Advisory Service for its help. CIBER de Diabetes y Enfermedades Metabólicas Asociadas is an ISCIII project.

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