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. 2010 Jul 21;151(9):4168–4177. doi: 10.1210/en.2010-0191

Curcumin Protects Hepatic Stellate Cells against Leptin-Induced Activation in Vitro by Accumulating Intracellular Lipids

Youcai Tang 1, Anping Chen 1
PMCID: PMC2940502  PMID: 20660066

Abstract

Obesity and type II diabetes mellitus are often associated with hyperleptinemia and commonly accompanied by nonalcoholic steatohepatitis, which could cause hepatic fibrosis. During hepatic fibrogenesis, the major effectors hepatic stellate cells (HSCs) become active, coupling with depletion of cellular lipid droplets and downexpression of genes relevant to lipid accumulation. Accumulating evidence supports the proposal that recovering the accumulation of lipids would inhibit HSC activation. We recently reported that leptin stimulated HSC activation, which was eliminated by curcumin, a phytochemical from turmeric. The current study was designed to explore the underlying mechanisms, focusing on their effects on the level of intracellular lipids. We hypothesized that one of the mechanisms by which leptin stimulated HSC activation was to stimulate the depletion of intracellular lipids, which could be abrogated by curcumin by inducing expression of genes relevant to lipid accumulation. In this report, we observed that leptin dose dependently reduced levels of intracellular fatty acids and triglycerides in passaged HSCs, which were eliminated by curcumin. The phytochemical abrogated the impact of leptin on inhibiting the activity of AMP-activated protein kinase (AMPK) in HSCs in vitro. The activation of AMPK resulted in inducing expression of genes relevant to lipid accumulation and increasing intracellular lipids in HSCs in vitro. In summary, curcumin eliminated stimulatory effects of leptin on HSC activation and increased AMPK activity, leading to inducing expression of genes relevant to lipid accumulation and elevating the level of intracellular lipids. These results provide novel insights into mechanisms of curcumin in inhibiting leptin-induced HSC activation.

Curcumin eliminated the stimulatory effects of leptin on hepatic stellate cell (HSC) activation by stimulating AMPK activity and inducing expression of genes relevant to lipogenesis as well as lipid uptake and metabolism in HSCs, leading to the accumulation of intracellular lipids.

The incidence of obesity and type II diabetes mellitus has sharply increased in recent years, making it one of the most urgent public health concerns worldwide. Obese and/or type II diabetes mellitus patients are often coupled with nonalcoholic fatty liver disease. Nonalcoholic steatohepatitis (NASH) is the advanced form of nonalcoholic fatty liver disease, featured by steatohepatitis (1,2). Approximately one third of NASH patients develop hepatic fibrosis and even cirrhosis (3).

Hepatic stellate cells (HSCs), previously called fat storing cells (4), are the key players in the development of hepatic fibrosis, regardless of etiology (5,6). During hepatic injury, quiescent HSCs undergo profound phenotypic changes, including enhanced cell proliferation, loss of lipid droplets, de novo expression of α-smooth muscle actin, and excessive production of extracellular matrix. This process is called HSC activation. Freshly isolated HSCs in culture gradually and spontaneously become fully activated (7), mimicking the process seen in vivo, which provides a good model for elucidating underlying mechanisms of HSC activation and studying potential therapeutic intervention of the process (5,6). Accumulating evidence supports the proposal that recovering the accumulation of lipids could inhibit HSC activation (8,9).

The hormone leptin is the product of an obese gene (ob) and circulates as a 16-kD protein in rodent and human plasma (10). It is mainly responsible for a negative feedback loop regulating energy homeostasis by acting on specific hypothalamic pathways (11). However, most obese human patients are leptin resistant, leading to hyperleptinemia, i.e. elevated levels of plasma leptin. In addition to its major functions, leptin was recently shown to stimulate HSC activation (12,13,14,15,16,17). Leptin-deficient mice fail to develop hepatic fibrosis during steatohepatitis or in response to chronic toxic liver injury (18). However, the restitution of physiological levels of circulating leptin by injecting exogenous leptin restores liver fibrosis caused by dietary manipulations in leptin-deficient mice (18). These observations collectively indicate the significance and necessity of leptin in the development of hepatic fibrosis. However, the underlying mechanisms remain elusive (19).

Lipid homeostasis is tightly controlled, via biosynthesis and cellular uptake, by a group of proteins. Several transcription factors, notably sterol regulatory element-binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor-γ (PPARγ), and CCAAT/enhancer-binding protein-α (C/EBPα) have emerged as master regulators in lipogenesis as well as in lipid uptake and metabolism (20). Interaction, cooperation, and cross talk have been observed among those regulators (21,22). It has been proposed that the process of HSC activation may be similar to that of adipocyte dedifferentiation, causally associated with transcriptional regulation of genes relevant to lipid accumulation (8,9).

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy homeostasis (23). It is activated by rising AMP and falling ATP by a complex mechanism that results in an ultrasensitive response. The activation of AMPK by pharmacological agents presents a unique challenge, given the complexity of the biology, but holds a considerable potential to reverse the metabolic abnormalities (24). In skeletal muscles, AMPK stimulates glucose transport and fatty acid (FA) oxidation. In the liver, it decreases glucose output, leading to lowering blood glucose levels in hyperglycemic individuals (25). AMPK may play a key role in regulating the activation of SREBP-1 and lipogenesis (26). The process of HSC activation is accompanied by depletion of intracellular lipid droplets, loss of lipid storage capacity, and suppression of expression of transcription factors, including SREBP-1, PPARγ, and C/EBPα (5,9).

Curcumin is one of the most studied natural compounds. Although the underlying mechanisms remain largely elusive, curcumin has shown its diverse and beneficial effects (27). Curcumin has received attention as a promising dietary supplement for liver protection (28). We previously showed that curcumin inhibited HSC activation by inhibiting cell proliferation, inducing gene expression of endogenous PPARγ and suppressing expression of genes closely relevant to the activation of HSCs and protected the liver from CCl4-caused fibrogenesis in vitro and in vivo (28,29,30,31,32). We recently reported that curcumin eliminated effects of leptin on the activation of HSCs in vitro by reducing the phosphorylation level of leptin receptor (Ob-R), stimulating PPARγ activity and attenuating oxidative stress, leading to the suppression of Ob-R gene expression and the interruption of leptin signaling (33). The purposes of this study were to further elucidate the underlying mechanisms, focusing on the effects of leptin and curcumin on regulating the levels of intracellular lipids. Results in this report supported our initial hypothesis that one of the mechanisms by which leptin stimulated HSC activation was to stimulate the depletion of intracellular lipids. The leptin stimulatory effect was eliminated by curcumin by activating AMPK activity, leading to the induction of expression of genes relevant to lipid accumulation, the elevation of the level of intracellular lipids, and the inhibition of HSC activation.

Materials and Methods

Materials and chemicals

Curcumin (purity >94%), leptin (recombinant, rat), 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine (Compound C), a specific AMPK inhibitor, and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an AMPK activator, and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2), a natural PPARγ agonist, were purchased from Sigma (St. Louis, MO). PGJ2 was unstable. As suggested by the manufacturer, a proper volume of the solvent dimethylsulfoxide was directly added to the vial containing the chemical and subaliquoted to avoid more than one freeze/thaw cycle. The chemical was stored at −20 C and used within 3 months. Full precaution should be exercised during handling PGJ2, because the pharmacological and toxicological properties of this chemical have not been fully investigated.

HSC isolation and cell culture

Male Sprague Dawley rats (200–250 g), purchased from the Harlan Laboratories, Inc. (Indianapolis, IN), were housed in a temperature-controlled animal facility (23 C) with a 12-h light, 12-h dark cycle and allowed free access to regular chew and water ad libitum. HSCs were isolated by the pronase-collagenase perfusion in situ before density gradient centrifugation, as we previously described (30). The animal protocol for the use of rats was approved by Institutional Animal Care and Use Committee of Saint Louis University. Primary cells were cultured in DMEM supplemented with 20% of fetal bovine serum (FBS). Cultured cells were passaged in DMEM with 10% of FBS. Semiconfluent HSCs with four to nine passages were used for experiments in this report. In some experiments, cells were cultured in serum-depleted media for 24 h before treatment, which rendered HSCs more sensitive to exogenous leptin. Cells were subsequently treated and cultured in serum-depleted media, which excluded the interference from other factors in FBS. Immortalized human hepatocytes (IHH) were kindly provided by Ratna Ray (Department of Pathology, Saint Louis University) (34). IHH were cultured in DMEM supplemented with glucose (450 mg/dl) and FBS (10%).

Western blot analyses

Whole-cell extracts were prepared from semiconfluent passaged HSCs. Protein concentrations were determined by using the BCA Protein Assay kit according to the protocol provided by the manufacturer (Pierce, Rockford, IL). Electrophoresis, transblotting, and immunodetection were conducted as previously described (30). Primary antibodies against PPARγ, SREBP1, C/EBPα, β-tubulin, β-actin, and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against AMPK and phosphorylated AMPK were purchased from Cell Signaling Technology, Inc. (Beverly, MA). β-Actin or β-tubulin was used as an internal control for equal loading. Densities of bands in Western blot analyses were normalized with the internal invariable control. Levels of target protein bands were densitometrically determined by using Quantity One 4.4.1 (Bio-Rad, Hercules, CA). Variations in the density were expressed as fold changes compared with the control in the blot (n = 3).

RNA isolation and real-time PCR

Preparation of total RNA and real-time PCR assays using SYBR green were carried out as we previously described (30). Total RNA was treated with deoxyribonuclease I before the synthesis of the first strand of cDNA. mRNA levels were expressed as fold changes after normalization with endogenous glyceraldehyde-3-phosphate dehydrogenase as suggested by Schmittgen et al. (35). Real-time PCR primers for PPARγ and glyceraldehyde-3-phosphate dehydrogenase (30) and for SREBP-1c (36) were previously described. The followings are the primers for C/EBPα: forward, 5′-AAG AAG TGG GTG GAT AAG AAC AG-3′ and reverse, 5′-GTT GCG CTG TTT GGC TTT ATC-3′.

Plasmids and transient transfection assays

The PPARγ activity luciferase reporter plasmid pPPRE-TK-Luc contains three copies of peroxisome proliferator response elements (PPREs) from the acyl-coenzyme A (CoA) oxidase gene linked to the herpes virus thymine kinase promoter (−105/+51) and a luciferase vector and was a gift from Kevin J. McCarthy (Louisiana State University Health Sciences Center in Shreveport). Semiconfluent HSC in six-well cell culture plates were transiently transfected using the LipofectAMINE reagent (Invitrogen Corp. Carlsbad, CA), as we previously described (30). Each sample was in triplicate in every experiment. Transfection efficiency was normalized by cotransfection of the β-galactosidase reporter plasmid, pSV-β-gal (0.5 μg/well) (Promega, Madison, WI). β-Galactosidase activities were measured by using a chemiluminescence assay kit (Tropix, Bedford, MA). Luciferase activities were presented in arbitrary units, i.e. optical densities per microgram of protein after normalization with β-galactosidase activities.

Intracellular FA and triglyceride (TG) assay

Levels of intracellular FAs and TGs were colorimetrically determined by using the Assay kits from BioVision, Inc. (Mountain View, CA), following the protocol provided by the manufacturer, as we previously described (36). Samples were quantitatively compared in the following formula: [(average FA/TG in treated HSCs − average FA/TG in control HSCs)/average FA/TG in control HSCs] × 100% (n = 3).

Statistical analysis

Differences between means were evaluated using an unpaired two-sided Student’s t test (P < 0.05 considered as significant). Where appropriate, comparisons of multiple treatment conditions with controls were analyzed by ANOVA with the Dunnett’s test for post hoc analysis.

Results

Leptin significantly reduced the levels of intracellular FAs and TGs in cultured HSCs, which was dose-dependently eliminated by curcumin

To evaluate the impact of leptin on the level of intracellular lipids and the role of curcumin in eliminating the impact, serum-starved HSC or IHH were treated with leptin at indicated doses plus or minus curcumin at different concentrations as indicated in serum-depleted media for 24 h. Cells were harvested for determining the levels of intracellular FAs and TGs. As shown in Fig. 1, leptin dose dependently reduced the levels of fatty acids (Fig. 1A) and TGs (Fig. 1B) in cultured HSCs and in IHH (the responding 1st–4th columns). For instance, compared with the untreated control (the corresponding 1st column), leptin at 100 ng/ml significantly reduced the levels of intracellular FA (Fig. 1A) and TG (Fig. 1B) by 71 and 47%, respectively (the corresponding 4th column). Curcumin dose dependently eliminated the impacts of leptin on the levels of FA (Fig. 1A) and TG (Fig. 1B) in HSCs (the corresponding 4th–8th columns). For instance, compared with leptin alone at 100 ng/ml (the corresponding 4th column), leptin plus curcumin at 20 μm increased the contents of intracellular FA (Fig. 1A) and TG (Fig. 1B) by 139 and 82%, respectively (the corresponding 7th column). It was of interest to observe that compared with the untreated control (the corresponding 1st column), curcumin alone at 20 μm significantly increased the levels of FA (Fig. 1A) and TG (Fig. 1B) by 38 and 48%, respectively (the corresponding last column). In contrast, curcumin had no apparent role in elevating the levels of cellular FA and/or TG in IHH treated with or without leptin at 100 ng/ml. Taken together, our results demonstrated that curcumin eliminated the effects of leptin and elevated the levels of intracellular FA and TG in activated HSC in vitro.

Figure 1.

Figure 1

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Leptin significantly reduced the levels of intracellular FAs and TGs in cultured HSC, which was dose-dependently eliminated by curcumin. Serum-starved HSCs were treated with leptin at indicated concentrations plus or minus curcumin (Cur) (0–30 μm) in serum-free media for 24 h. Values were expressed as pmol/μG protein and presented as means ± sd (n = 3). The percentages were calculated in the formula: [(FA/TG in treated HSCs − FA/TG in control HSCs)/FA/TG in control HSCs] × 100% (n = 3). *, P < 0.05 vs. the untreated control cells (the corresponding 1st column); ‡, P < 0.05 vs. the cells treated with leptin alone at 100 ng/ml (the corresponding 4th column). A, Intracellular FA assays. B, Intracellular TG assays.

Curcumin stimulated the expression of genes relevant to lipid accumulation in cultured HSCs

We postulated that curcumin eliminated the effects of leptin and elevated the levels of intracellular FA and TG in activated HSC in vitro by inducing gene expression of proteins involved in lipid accumulation, including PPARγ, SREBP-1, and C/EBPα. To test the postulation, serum-starved HSCs were treated with or without leptin at 100 ng/ml in the presence of curcumin at indicated concentrations in serum-free media for 24 h. Total RNA and whole-cell extracts were prepared for real-time PCR and Western blot analyses. As shown in Fig. 2A, compared with the untreated control (the corresponding 1st column), leptin alone apparently reduced the mRNA levels of PPARγ, SREBP-1, and C/EBPα by 45, 36, and 44%, respectively (the corresponding 2nd column). The leptin impacts were dose-dependently eliminated by curcumin in a dose-dependent manner. For example, compared with leptin alone (the corresponding 2nd column), leptin plus curcumin at 20 μm significantly increased the mRNA levels of PPARγ, SREBP-1, and C/EBPα by 136, 84, and 135%, respectively (the corresponding 5th column). It was also observed that compared with the untreated control (the corresponding 1st column), curcumin alone (20 μm) significantly increased the mRNA levels of PPARγ, SREBP-1, and C/EBPα by 190, 180, and 200%, respectively (the corresponding last column).

Figure 2.

Figure 2

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Curcumin stimulated the expression of genes relevant to lipid accumulation in cultured HSCs. Serum-starved HSCs were treated with or without leptin at 100 ng/ml in the presence of curcumin (Cur) at indicated concentrations in serum-free media for 24 h. Total mRNA or whole-cell extracts were prepared. A, Real-time PCR analyses. Values were expressed as mRNA fold changes (means ± sd) (n = 3), compared with the untreated control (the corresponding 1st column). *, P < 0.05 vs. the untreated control cells (the corresponding 1st column); ‡, P < 0.05 vs. the cells treated with leptin only (the corresponding 2nd column). B, Western blot analyses. β-Tubulin was used as an invariant control for equal loading. Representatives were presented from three independent experiments. Italic numbers beneath blots were fold changes (means ± sd) in the densities of the bands compared with the control without treatment in the blot (n = 3), after normalization with the internal invariable control. C, HSCs were transiently transfected with the plasmid pPPRE-TK-Luc. After recovery, cells were serum-starved for 4 h and treated with or without leptin (100 ng/ml) in the presence of curcumin at indicated concentrations in serum-depleted DMEM with PGJ2 (5 μm) for 24 h. Luciferase activities were expressed as relative units after normalization with β-galactosidase and per microgram of proteins (means ± sd, n ≥ 6).

These observations were verified by Western blot analyses (Fig. 2B). Compared with the untreated control (the corresponding 1st well), leptin apparently reduced the protein contents of PPARγ, SREBP-1c, and C/EBPα (the corresponding 2nd well). The leptin effects were dose-dependently abrogated by curcumin (the corresponding 3rd–6th wells). It was further observed that curcumin alone (20 μm) (the corresponding last well) resulted in higher contents of these proteins, compared with the untreated control (the corresponding 1st well), or with leptin plus curcumin (20 μm) (the corresponding 5th well). SREBP-1c was the active form of its precursor SREBP-1 after proteolysis (37).

To confirm the role of curcumin in stimulating the trans-activation activity of PPARγ, passaged HSCs were transfected with the PPARγ activity luciferase reporter plasmid pPPRE-TK-Luc. After recovery, cells were serum-starved for 4 h. Cells were treated with or without leptin at 100 ng/ml in the presence of curcumin at indicated concentrations for 24 h in serum-depleted media with PGJ2 at 5 μm. Because the serum-depleted media had no PPARγ agonists, exogenous natural PPARγ agonist PGJ2 at 5 μm was added to the media to bind to PPARγ induced by curcumin and to activate its signaling. Results from luciferase activity assays in Fig. 2C revealed that compared with the untreated control (the 1st column), leptin significantly reduced luciferase activity by 40% (the 2nd column), which was dose-dependently reversed by curcumin (the 3rd-6th columns). For instance, compared with leptin alone (the corresponding 2nd column), leptin plus curcumin at 20 μm significantly increased luciferase activity by 33%. Curcumin alone (20 μm) dramatically increased, as expected, luciferase activity by 30% (the last column), compared with the untreated control (the 1st column). These results indicated that curcumin indeed abrogated the inhibitory impact of leptin and increased the trans-activation activity of PPARγ in activated HSCs in vitro. Taken together, these results demonstrated that curcumin induced expression of genes relevant to lipid accumulation in cultured HSCs, which likely led to the elevation of the level of intracellular lipids and to the inhibition of HSC activation.

Leptin reduced the level of phosphorylated AMPK in HSCs in vitro, which was abolished by curcumin in a dose-dependent manner

We assumed that leptin inhibited AMPK activity, leading to the suppression of the expression of genes relevant to lipid accumulation, which could be eliminated by curcumin. To begin to test the assumption and to evaluate the impact of leptin and/or curcumin on the activity of AMPK, serum-starved HSCs were treated with or without curcumin at indicated doses plus or minus leptin at various concentrations for 30 min in serum-depleted media. We have shown that leptin rapidly activates its signaling in HSCs and reaches its peak within 20 to 30 min (33). As shown in Fig. 3A, leptin dose dependently reduced the level of phosphorylated AMPK in HSCs in vitro (the 1st–3rd wells), indicating that leptin inhibited the activity of AMPK. The inhibitory impact of leptin was dose-dependently abrogated by curcumin by increasing the phosphorylation levels of AMPK (the 3rd–7th wells). Compared with the untreated control (the 1st well), curcumin alone (20 μm) dramatically increased the level of phosphorylated AMPK (the last well), confirming the role of curcumin in activating AMPK.

Figure 3.

Figure 3

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Leptin inhibited AMPK activity in HSCs in vitro, which was abrogated by curcumin in a dose-dependent manner. Serum-starved HSCs were treated with or without curcumin (Cur) at indicated concentrations plus or minus leptin (A), or the selective AMPK inhibitor Compd C (B), at indicated concentrations, in serum-depleted media for 30 min. On the other hand, serum-starved HSCs were treated with curcumin alone at 20 μm, or with the specific AMPK activator AICAR alone at 2 mm, or with the combination of AICAR at 0.5 mm and curcumin at indicated concentrations in serum-depleted media for 30 min. C, Whole-cell extracts of the cells were prepared for evaluating the levels of phosphorylated AMPK by Western blot analyses. Total AMPK was used as an invariant control for equal loading. Representatives were presented from three independent experiments. Italic numbers beneath blots were fold changes (means ± sd) in the densities of the bands compared with the control without treatment in the blot (n = 3), after normalization with the internal invariable control.

To verify the role of curcumin in increasing the activity of AMPK, serum-starved HSCs were treated with or without curcumin at indicated concentrations plus or minus the selective AMPK inhibitor Compound C (Compd C) at indicated doses for 30 min in serum-depleted media. As shown in Fig. 3B by Western blot analyses, compared with the untreated control (the 1st well), Compd C at 20 μm apparently reduced the phosphorylation level of AMPK (the 2nd well), confirming its role in inhibiting the activity of AMPK. Curcumin at 20 μm significantly increased, as expected, the level of phosphorylated AMPK (the 3rd well). It was further observed that the effect of curcumin on elevating the level of phosphorylated AMPK was dose-dependently diminished by Compd C (the 3rd–7th wells).

To further confirm the role of curcumin in increasing the activity of AMPK, serum-starved HSCs were treated with curcumin alone at 20 μm, or with the specific AMPK activator AICAR alone at 2 mm, or with the combination of curcumin (0–30 μm) and AICAR at a lower concentration of 0.5 mm in serum-depleted media for 30 min. As shown in Fig. 3C by Western blot analyses, compared with the untreated control (the 1st well), AICAR at 2.0 mm alone (the 2nd well), like curcumin (20 μm) (the 3rd well), significantly elevated the level of phosphorylated AMPK, verifying the role of AICAR in activating AMPK. It was understandable that, compared with AICAR at 2 mm (the 2nd well), a lower dose of AICAR at 0.5 mm would cause a weaker increase in the phosphorylation level of AMPK (the 4th well). It was of interest to observe that the combination of AICAR (0.5 mm) and curcumin increased the levels of phosphorylated AMPK in a curcumin dose-dependent manner (the 4th–7th wells). These results suggested an additive impact of AICAR and curcumin on the activation of AMPK. Taken together, these observations demonstrated that leptin, like Compd C, inhibited the activity of AMPK. Curcumin, like AICAR, activated AMPK in HSCs in vitro and abrogated the inhibitory impact of leptin in a dose-dependent manner.

The alterations in AMPK activity resulted in the changes in the expression of the genes relevant to lipid accumulation in activated HSCs in vitro

To further test our aforesaid assumption, passaged HSCs were treated with the specific AMPK activator AICAR, or the selective AMPK inhibitor Compd C, at indicated doses, in the presence or absence of curcumin (20 μm) for 24 h. Total RNA or whole-cell extracts were prepared from the cells for real-time PCR or for Western blot analyses. As shown in Fig. 4, A and B, like curcumin (the corresponding 1st column or well), AICAR dose dependently increased mRNA levels (Fig. 4A) and protein contents (Fig. 4B) of PPARγ, SREBP-1, or C/EBPα (the corresponding 2nd–6th columns or wells), suggesting that the activation of AMPK induced the expression of the genes relevant to lipid accumulation in activated HSCs in vitro. On the other hand, as shown in Fig. 4, C and D, the curcumin-elevated levels of mRNA (Fig. 4C) or proteins (Fig. 4D) of PPARγ, SREBP-1, or C/EBPα (the corresponding 2nd columns or wells) were dose-dependently diminished by Compd C (the corresponding 3rd–6th columns or wells), suggesting that the inhibition of AMPK activity by Compd C eliminated the effect of curcumin and suppressed the expression of the genes relevant to lipid accumulation in activated HSCs in vitro. Taken together, our results indicated that the alterations in AMPK activity resulted in the changes in the expression of the genes relevant to lipid accumulation in activated HSCs in vitro.

Figure 4.

Figure 4

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The alterations in AMPK activity resulted in the changes in the expression of the genes relevant to lipid accumulation in activated HSCs in vitro. Passaged HSCs were treated with curcumin (Cur) (20 μm), or AICAR, or Compd C, at indicated concentrations for 24 h. Total mRNA and whole-cell extracts were prepared. A and C, Real-time PCR analyses of cells treated with AICAR (A) or Compd C (C). Values were expressed as mRNA fold changes and presented as means ± sd (n = 3). *, P < 0.05 vs. the untreated control; ‡, P < 0.05 vs. the cells treated with curcumin only (the corresponding 2nd column in C). B and D, Western blot analyses of cells treated with AICAR (B) or Compd C (D). β-Actin was used as an invariant control for equal loading. Representatives were presented from three independent experiments. Italic numbers beneath blots were fold changes (means ± sd) in the densities of the bands compared with the control without treatment in the blot (n = 3), after normalization with the internal invariable control.

The activation of AMPK elevated the trans-activation activity of PPARγ in activated HSCs in vitro

To verify the role of the activation of AMPK activity in stimulating the trans-activity of PPARγ, passaged HSCs were transfected with the PPARγ activity luciferase reporter plasmid pPPRE-TK-Luc. After recovery, cells were treated with curcumin (20 μm), or AICAR or Compd C at indicated doses in DMEM with 10% of FBS for 24 h. Prior experiments have suggested that 10% of FBS in the medium contains enough agonists to activate PPARγ in HSCs (8,30,31). Luciferase activities were analyzed. As shown in Fig. 5A, compared with the untreated control (the 2nd column), curcumin, as expected, caused a significant increase in luciferase activity (the 1st column). Like curcumin, AICAR resulted in a dose-dependent increase in luciferase activities (the 3rd–6th columns), suggesting increases in the trans-activation activity of PPARγ. On the other hand, the AMPK inhibitor Compd C caused a dose-dependent reduction in luciferase activities (the 3rd–6th columns in Fig. 5B), suggesting decreases in the trans-activation activity of PPARγ. These results collectively demonstrated that the activation of AMPK elevated the trans-activation activity of PPARγ in activated HSCs in vitro.

Figure 5.

Figure 5

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The activation of AMPK elevated the trans-activation activity of PPARγ in activated HSCs in vitro. HSCs were transiently transfected with the plasmid pPPRE-TK-Luc. After recovery, cells were treated with curcumin (Cur) (20 μm), or AICAR at indicated doses (A), or Compd C, at indicated doses (B), in DMEM with 10% of FBS for 24 h. Luciferase activities were expressed as relative units after β-galactosidase and protein normalization (means ± sd, n ≥ 6). *, P < 0.05 vs. the untreated control (the corresponding 2nd column). The floating schema denoted the plasmid in use and the application of AICAR or Compd C to the system.

The alterations in the activity of AMPK resulted in the changes in the levels of intracellular FAs and TGs in activated HSCs in vitro

To further evaluate the effects of the alterations in AMPK activity on lipid accumulation, passaged HSCs were treated with AICAR, or Compd C, at indicated concentrations, or with curcumin at 20 μm, in DMEM with 10% FBS for 24 h. Cells were harvested for FA and TG assays, respectively. As shown in Fig. 6, A and B, like curcumin (the corresponding 1st column), the AMPK activator AICAR dose dependently elevated the levels of intracellular FA and TG (the corresponding 3rd–6th columns), compared with the untreated control (the corresponding 2nd column), suggesting that the activation of AMPK stimulated the accumulation of intracellular FAs and TGs in cultured HSCs. In consistence, the inhibition of AMPK activity by Compd C dose dependently reduced the levels of intracellular FAs and TGs (the corresponding 3rd–6th columns). Taken together, our results demonstrate that the alterations in the activity of AMPK resulted in the changes in the levels of intracellular FAs and TGs in cultured HSCs.

Figure 6.

Figure 6

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The alterations in the activity of AMPK resulted in the changes in the levels of intracellular FAs and TGs in activated HSCs in vitro. Passaged HSCs were treated with curcumin (Cur) (20 μm) or AICAR (A and B), or with Compd C (C and D), at indicated concentrations for 24 h. Assays were conducted for intracellular FAs (A and C) or intracellular TGs (B and D). Values were expressed as pmol/μG protein and presented as means ± sd (n = 3). *, P < 0.05 vs. the untreated control (the corresponding 2nd column).

Discussion

We recently showed that curcumin eliminated the effect of leptin on the activation of HSCs in vitro (33). The underlying mechanisms remain, however, largely undefined. In the current report, we demonstrated that leptin significantly reduced the levels of FAs and TGs in cultured HSCs. Curcumin eliminated the impact of leptin by stimulating AMPK activity, leading to the induction of gene expression of PPARγ, SREBP-1, and C/EBPα, the accumulation of lipids, and the inhibition of HSC activation. Leptin at 50 ng/ml or above could be found in obese human and animals (38,39), although the precise range of plasma leptin could vary. Leptin at 100 ng/ml was chosen for most of our in vitro experiments. However, it bears emphasis that because the in vivo system is multifactorial, directly extrapolating in vitro conditions and results, e.g. effective concentrations, to the in vivo system might be misleading.

SREBP-1 is synthesized as inactive, membrane-bound precursor and is proteolytically activated when cellular sterol concentration is low. The active SREBP-1c is subsequently transported into the nucleus and bind to SREs in promoter regions of target genes, facilitating transcription of the target genes (37). We previously showed that curcumin induced the activation of SREBP-1 in cultured HSCs by increasing the abundance of nuclear SREBP-1c (36). Our results in this report demonstrated that curcumin not only eliminated the inhibitory effect of leptin on gene expression of PPARγ but also stimulated the activity of PPARγ in activated HSCs in vitro. We previously showed that curcumin interrupted MAPK signaling pathways in HSCs (40,41), which might lead to the reduction in the level of phosphorylated PPARγ and the increase in its activity (42,43). The accumulation of cellular lipids could result from an increase in lipogenese and lipid uptake, as well as a reduction in lipid catabolism. Substantial evidence has indicated that lipogenesis is controlled by a large family of transcription factors. Among them, PPARγ, SREBP-1, and C/EBPα play a critical role in lipogenesis in addition to lipid uptake and metabolism (44). They cross talk, interact, regulate mutual expression, and synergistically stimulate lipogenesis (44). The transcription of PPARγ is regulated by SREBP-1c (45). PPARγ activates C/EBPα, which, in turn, exerts a positive feedback on the elevation and maintenance of PPARγ expression (44). To verify the role of curcumin in stimulating lipogenesis in HSCs, additional experiments are required to evaluate activities of key enzymes involved in the process of lipogenesis, including stearoyl CoA desaturase, acetyl CoA carboxylase, and diacylglycerol acyltransferase. Furthermore, the determination of the C16:1/C16:0 or C18:1/C18:0 ratios would be necessary to show the endogenous lipogenesis process.

It has been reported that the expression of PPARγ and C/EBPα are regulated by AMPK (46,47). In this report, we demonstrated that leptin inhibited the activity of AMPK in HSCs in vitro, which was dose-dependently eliminated by curcumin (Fig. 3). Our observations were supported by other reports (48,49,50). In the current report, we further demonstrated that the alterations in AMPK activity by AICAR, or by Compd C, resulted in the changes in the expression of genes relevant to lipid accumulation (Fig. 4), PPARγ trans-activation activity (Fig. 5), and the levels of FAs and TGs (Fig. 6) in HSCs. These results are consistent with other studies (47).

A critical concern was raised during conducting these experiments whether curcumin would stimulate the accumulation of lipids in hepatocytes and/or muscle cells. If true, curcumin would exacerbate hepatosteatosis. It is noteworthy that curcumin might have distinct effects on regulating expression of genes depending on cell types (51). Curcumin induced gene expression of low-density lipoprotein (LDL) receptor in hepatoma cell line HepG2 (52). However, curcumin suppressed gene expression of LDL receptor in cultured HSCs, which attenuated the stimulatory impact of LDL in the activation of HSCs (36). Other reports revealed that the activation of AMPK by curcumin down-regulated the expression of PPARγ in adipocytes (53). In addition, curcumin might exert different functions dependent on cell types. In this study, we reported that curcumin eliminated the impacts of leptin and elevated the levels of intracellular FAs and TGs in cultured HSCs. However, curcumin had no apparent role in changing the level of intracellular FAs and TGs in IHH (Fig. 1). Moreover, curcumin suppressed lipid synthesis in adipocytes and reduced body fat and body weight, preventing obesity in a mouse model (54). These observations collectively suggested that curcumin might show distinct effects on regulating gene expression and on lipid accumulation depending on cell types. Although beyond the scope of this project, additional experiments are necessary to elucidate the mechanisms by which curcumin activates AMPK and shows distinct effects in different cell types.

Based on the current observations, we propose that one of the mechanisms by which curcumin eliminates the effects of leptin on inducing HSC activation is to activate AMPK activity, leading to the induction of the expression of genes relevant to lipid accumulation and to the elevation of the levels of intracellular FAs and TGs. It bears emphasis that our observations do not exclude any additional mechanisms by which curcumin eliminated the effects of leptin on the activation of HSCs. Our results in this report shed novel insights into mechanisms of curcumin in the inhibition of leptin-induced HSC activation and provide a therapeutic candidate for the treatment of NASH-associated hepatic fibrogenesis in obese patients.

Footnotes

This work was supported by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1 DK 047995 (to A.C.).

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 21, 2010

Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; AMPK, AMP-activated protein kinase; C/EBPα, CCAAT/enhancer-binding protein-α; CoA, coenzyme A; Compd C, Compound C; Compound C, 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine; FA, fatty acid; FBS, fetal bovine serum; HSC, hepatic stellate cell; IHH, immortalized human hepatocytes; LDL, low-density lipoprotein; NASH, nonalcoholic steatohepatitis; PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; PPARγ, peroxisome proliferator-activated receptor-γ; PPRE, peroxisome proliferator response element; SREBP-1c, sterol regulatory element-binding protein-1c; TG, triglyceride.

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