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Curcumin Inhibits Aerobic Glycolysis in Hepatic Stellate Cells Associated with Activation of Adenosine Monophosphate-activated Protein Kinase

Naqi Lian1,2 Huanhuan Jin1,2 Feng Zhang1,2

Li Wu1,2
Jiangjuan Shao3 Yin Lu1,2
Shizhong Zheng1,2*

1Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China
2Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China

3Departemt of Pharmacy, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China

Summary

Activation of hepatic stellate cells (HSCs) is characterized by expression of extracellular matrix and loss of adipogenic phe- notype during liver fibrogenesis. Emerging evidence suggests that HSCs adopt aerobic glycolysis during activation. The pres- ent work aimed at investigating whether the anti-fibrogenic effects of curcumin was associated with interfering with gly- colysis in HSCs. Primary rat HSCs were cultured in vitro. We demonstrated that inhibition of glycolysis by 2-deoxyglucose or galloflavin reduced the expression of a-smooth muscle actin (a-SMA) and a1(I)procollagen at both mRNA and protein levels, and increased the intracellular lipid contents and upreg- ulated the gene and protein expression of adipogenic tran- scription factors C/EBPa and PPAR-c in HSCs. Curcumin at 20 lM produced similar effects. Moreover, curcumin decreased the expression of hexokinase (HK), phosphofructokinase-2

(PFK2), and glucose transporter 4 (glut4), three key glycolytic parameters, at both mRNA and protein levels. Curcumin also reduced lactate production concentration-dependently in HSCs. Furthermore, curcumin increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), but AMPK inhibitor BML-275 significantly abolished the curcumin downregulation of HK, PFK2, and glut4. In addi- tion, curcumin inhibition of a-SMA and a1(I)procollagen was rescued by BML-275, and curcumin upregulation of C/EBPa and PPAR-c was abrogated by BML-275. These results collec- tively indicated that curcumin inhibited glycolysis in an AMPK activation-dependent manner in HSCs. We revealed a novel mechanism for curcumin suppression of HSC activation impli- cated in antifibrotic therapy. VC 2016 IUBMB Life, 68(7):589–596, 2016

Research Communication

Curcumin Inhibits Aerobic Glycolysis in Hepatic Stellate Cells Associated with Activation of Adenosine Monophosphate-activated
Protein Kinase

Naqi Lian1,2 Huanhuan Jin1,2 Feng Zhang1,2
Li Wu1,2
Jiangjuan Shao3 Yin Lu1,2
Shizhong Zheng1,2*

1Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China
2Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China

3Departemt of Pharmacy, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China 

Keywords: liver fibrosis; hepatic stellate cell; curcumin; aerobic glycolysis; adenosine monophosphate-activated protein kinase

VC 2016 International Union of Biochemistry and Molecular Biology Volume 68, Number 7, July 2016, Pages 589–596

*Address correspondence to: Shizhong Zheng, Department of Pharmacol- ogy, School of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, Jiangsu, China. Tel: 186-25-86798154. Fax: 186-25-86798188.

E-mail: nytws@163.com
Received 3 February 2016; Accepted 16 May 2016 DOI 10.1002/iub.1518
Published online 9 June 2016 in Wiley Online Library (wileyonlinelibrary.com)

Introduction

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Hepatic fibrosis is caused by the net accumulation of extracel- lular matrix (ECM) in the liver following chronic liver disease. The hepatic stellate cells (HSCs), a type of nonparenchymal cells within the perisinusoidal space of Disse, are the central player in liver fibrosis (1). Chronic liver injury stimulates acti- vation of quiescent, lipid-containing HSCs into proliferative, contractile, and fibrogenic myofibroblasts. HSC activation is characterized by overexpression of a-smooth muscle actin (a- SMA) and collagens accompanied by dramatic loss of lipid droplets and adipogenic phenotype (2).

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Latest understanding of HSC biology reveals that HSCs undergo rapid energy reprogramming during activation simi- lar to the Warburg effect described in tumor cells, and their survival largely depends on aerobic glycolysis. This metabolic switch optimizes glucose consumption in HSCs and redirects them to support fibrogenic transdifferentiation (3). Glycolysis is a multistep process where glucose is imported into cells through glucose transporters (Glut) and converted to pyruvate by several rate-limiting enzymes including hexokinase (HK) and phosphofructokinase-2 (PFK2) (4). Moreover, adenosine monophosphate-activated protein kinase (AMPK) is a master metabolic switch that senses and decodes intracellular changes in energy status, playing an important role in the maintenance of cellular homeostasis. AMPK can regulate fatty acid oxidation, glucose uptake, and glycolysis in various cell types (5). Interestingly, activation of AMPK could negatively modulate the activated phenotype of HSCs (6) and mediate adi- ponectin inhibition of HSC proliferation (7). These data suggest that AMPK could be a therapeutic target for treatment of liver fibrosis.

The discovery of HSC activation has provided a fertile foundation for organizing approaches to antifibrotic therapies. Curcumin, the yellow pigment in curry from turmeric, is one of the most studied natural compounds and has received extensive attention as a promising dietary supplement for liver protection (8). Curcumin can inhibit HSC activation by disrupt- ing transforming growth factor-b (9) and platelet-derived growth factor pathways (10). We recently demonstrated that curcumin reduced liver fibrosis associated with suppression of cannabinoid receptor type-1 (11) and attenuation of hepatic pathological angiogenesis (12). However, the underlying mech- anisms remain elusive. The current studies aimed to investi- gate the effects of curcumin on aerobic glycolysis in HSCs and the role of AMPK in curcumin effects.

Materials and Methods

Reagents and Antibodies

The following compounds were used in this study: 2- deoxyglucose (2DG) and galloflavin (Tocris Bioscience, Bristol, UK); curcumin (Sigma, St Louis, MO); BML-275 (Santa Cruz Technology, Santa Cruz, CA). They were dissolved in dimethyl- sulfoxide (DMSO) for experiments. Treatment with DMSO alone was used as negative control throughout this study. The following primary antibodies were used in this study: a-SMA, a1(I)procollagen and fibronectin (Epitomics, San Francisco, CA); C/EBPa, HK, PFK2 and Glut 4 (Bioworld Technology, St. Louis Park, MN); PPAR-c, p-AMPK, AMPK and b-Actin (Cell Signaling Technology, Danvers, MA).

Cell Culture

Primary rat HSCs were obtained from Jiangyin CHI Scientific, Inc. (Wuxi, China). HSCs were cultured in Dulbecco’s modified eagle medium (DMEM; Invitrogen, Grand Island, NY) with 10% fetal bovine serum (FBS; Wisent Biotechnology Co., Ltd., Nanj-

ing, China), 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 378C. Cell morphology was assessed with an inverted microscope with a Leica Qwin System (Leica, Germany).

Oil Red O Staining

HSCs were seeded in six-well plates and cultured in DMEM with 10% FBS for 24 h, and then were treated with 2DG, gallo- flavin, or curcumin at indicated concentrations for 24 h. HSCs were stained with oil red O reagents (Nanjing Jiancheng Bio- engineering Institute, Nanjing, China) to visualize the lipids with a light microscope (1003 amplification). Lipids in HSCs were colored dark red by oil red O. Images were taken at ran- dom fields. Results were from triplicate experiments.

Measurement of Intracellular Lactate

HSCs were seeded in six-well plates and cultured in DMEM with 10% FBS for 24 h, and then were treated with curcumin at indicated concentrations for 24 h. Intracellular levels of lac- tate in lysates were determined using a kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufac- turers’ instructions. Results were from triplicate experiments.

Immunofluorescence Staining

HSCs were seeded in 24-well plates and cultured in DMEM with 10% FBS for 24 h, and then were treated with curcumin at indicated concentrations for 24 h. Immunofluorescent stain- ing with primary antibody against p-AMPK and with fluorescence-conjugated secondary antibodies (Wuhan Boster Biological Technology Ltd., Wuhan, China) in succession was performed as we previously described (13). The nucleus was stained with the Hoechst 33342 reagent (Beyotime Institute of Biotechnology, Haimen, China). Images were taken at random fields. Results were from triplicate experiments.

Real-Time PCR

Total RNA was prepared from treated HSCs using Trizol reagent (Sigma, St Louis, MO) following the protocol provided by the manu- facturer. Real-time PCR was performed as we described previ- ously (14). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the invariant control. Fold changes in the mRNA lev- els of target genes related to the invariant control GAPDH were calculated according to the suggested method (15). The following primers of genes (GenScript Corporation, Nanjing, China) were used: a-SMA: (forward) 5’-CCGACCGAATGCAGAAG GA-3’, (reverse) 5’-ACAGAGTATTTGCGCTCCGGA-3’; a(I)procolla- gen: (forward) 5’-CCTCAAGGGCTCCAACGAG-3’, (reverse) 5’- TCAATCACTGTCTTGCCCCA-3’; C/EBPa: (forward) 5’-TGAACA AGAACAGCAACGAG-3’ (reverse) 5’-TCACTGGTCACCTCCAGC AC-3’; PPAR-c: (forward) 5’-ATTCTGGCCCACCAACTTCGG-3’, (reverse) 5’-TGGAAGCCTGATGCTTTATCCCCA-3’; HK: (forward) 5’-CAACATTCTCATCGATTTCACGAA-3’, (reverse) 5’- GATGGC ACGAACCTGTAGCA-3’; PFK2: (forward) 5’- ACTGAAGGGCTCCC ACGGCA-3, (reverse) 5’-GGCCGCAGTTTCAGCCACCA-3’; Glut4: (forward) 5’-CTGCACTCCTTCTTCCCTTT-3’, (reverse) 5’- GCCTGCACTTGAGGAGGATTT-3’; GAPDH: (forward) 5’-GGCCCC

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Curcumin inhibits aerobic glycolysis in HSCs

FIG 1

Inhibition of glycolysis reduces fibrogenic marker expression in HSCs. HSCs were treated with vehicle DMSO, 2DG, or gallofla- vin, and curcumin at indicated concentrations for 24 h. (A) Real-time PCR analyses of profibrogenic markers. GAPDH was used as the invariant control. Significance: *P < 0.05 versus vehicle. (B) Western blot analyses of profibrogenic markers with densi- tometry after normalization to b-actin. Significance: *P < 0.05 versus vehicle, **P < 0.01 versus vehicle.

TCTGGAAAGCTGTG-3’, (reverse) 5’-CCGCCTGCTTCACCACCTTCT- 3’. Results were from triplicate experiments.

Western Blot Analyses

Whole cell protein extracts were prepared from treated HSCs. The protein levels were determined using a BCA assay kit (Pierce). Proteins (50 lg/well) were separated by SDS- polyacrylamide gel, transferred to a PVDF membrane (Milli- pore, Burlington, MA), blocked with 5% skim milk in Tris- buffered saline containing 0.1% Tween 20. Target proteins were detected by corresponding primary antibodies, and sub- sequently by horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using chemilumines- cence reagent (Millipore, Burlington, MA) by Bio-Rad Universal Hood II DOC Electrophoresis Imaging Cabinet. Equivalent load- ing was confirmed using an antibody against b-actin. The lev- els of target protein bands were densitometrically determined using Image Lab Software 3.0. The variation in the density of bands was expressed as fold changes compared to the control in the blot after normalization to b-actin or total protein in some experiments. Presented blots are representative of three independent experiments.

Statistical Analysis

Data were presented as mean 6 standard deviations, and ana- lyzed using GraphPad Prism 5 software (San Diego, CA). The significance of difference was determined by one-way ANOVA with the post hoc Dunnett’s test. Values of P < 0.05 were con- sidered to be statistically significant.

Results

Glycolysis Blockers and Curcumin Reduce Fibrogenic Marker Expression in HSCs
We initially examined the effects of glycolysis on HSC activa- tion. We used the compounds 2DG and galloflavin to block gly- colysis at different links. 2DG acts to competitively inhibit the production of glucose-6-phosphate from glucose at the phos- phoglucoisomerase level (16). Galloflavin is a lactate dehydro- genase inhibitor and blocks glycolysis at the lactate level (17). Real-time PCR analyses showed that both 2DG and galloflavin concentration-dependently reduced the mRNA expression of a- SMA and a1(I)procollagen and that curcumin at 20 lM also produced significant inhibitory effects on the mRNA expression of the two molecules (Fig. 1A). Moreover, the protein expres- sion of a-SMA and a1(I)procollagen was also suppressed by 2DG and galloflavin and by curcumin at 20 lM (Fig. 1B). Alto- gether, these results indicated that blockade of glycolysis could suppress the expression of fibrogenic markers in HSCs.

Glycolysis Blockers and Curcumin Restore Lipogenesis in HSCs
Given that loss of adipogenic phenotype is a hallmark of HSC activation, we next examined the regulation of glycolysis on intracellular lipids. As demonstrated by the oil red O staining assay, 2DG restored the lipid contents in HSCs and similar results were recaptured in galloflavin-treated HSCs (Fig. 2A). Curcumin at 20 lM also increased the lipid contents (Fig. 2A). It is known that transcriptional regulation is required for maintaining HSC adipogenic phenotype. The major

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FIG 2

Inhibition of glycolysis upregulates lipogenesis in HSCs. HSCs were treated with vehicle DMSO, 2DG, or galloflavin, and curcu- min at indicated concentrations for 24 h. (A) Oil red O staining for evaluating lipids. Images were taken at random fields. Repre- sentative views with positive staining are shown (1003 magnification). (B) Real-time PCR analyses of adipogenic transcription factors. GAPDH was used as the invariant control. Significance: *P<0.05 versus vehicle. (C) Western blot analyses of adipo- genic transcription factors with densitometry after normalization to b-actin. Significance: *P<0.05 versus vehicle, **P<0.01 versus vehicle.

transcription factors involved in HSC adipocyte differentiation include CCAAT/enhancer binding protein a (C/EBPa) and per- oxisome proliferator-activated receptor-c (PPAR-c) (18). We found that both the mRNA and protein expression of C/EBPa and PPAR-c was elevated significantly by the two glycolytic blockers concentration-dependently and by curcumin at 20 lM (Fig. 2B,C). Consistently, these data suggested that inhibition of glycolysis could restore lipogenesis in HSCs.

Curcumin Inhibits Aerobic Glycolysis in HSCs

The above examinations have established a profibrogenic role for aerobic glycolysis and suggested that blockade of glycolysis could inhibit HSC activation. We next explored whether curcu- min could affect this process. The key molecules HK, PFK2, and glut4 implicated in glycolysis were selected as the primary parameters. We found that curcumin downregulated their mRNA expression concentration-dependently in HSCs (Fig. 3A). Similarly, curcumin decreased the protein abundance of HK, PFK2, and glut4 (Fig. 3B). Moreover, we determined the lactate levels in curcumin-treated HSCs and demonstrated that curcumin reduced the lactate production in a concentration- dependent manner in HSCs (Fig. 3C). Taken together, these

data revealed that curcumin blocked aerobic glycolysis in HSCs.

Activation of AMPK is Involved in Curcumin Inhibition of Aerobic Glycolysis and Reduction of Activation of HSCs
We finally explored the role for AMPK in curcumin blockade of HSC glycolysis. Western blot assays showed that curcumin pro- moted phosphorylation of AMPK concentration-dependently in HSCs (Fig. 4A), suggesting curcumin activation of AMPK. The subsequent immunofluorescence assays yielded consistent results, showing that curcumin at 20 lM considerably upregu- lated AMPK phosphorylation (Fig. 4B). We further used AMPK specific inhibitor BML-275 to explore the relationship between activation of AMPK and suppression of aerobic glycolysis. Real-time PCR data demonstrated that curcumin reduction in the mRNA expression of HK, PFK2, and glut4 was abolished by BML-275 significantly in HSCs (Fig. 4C). Additionally, BML- 275 concentration-dependently rescued curcumin inhibition of mRNA expression of a-SMA and a1(I)procollagen and abro- gated curcumin upregulation of mRNA expression of C/EBPa and PPAR-c (Fig. 4D,E). Similar results were recaptured at the

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Curcumin inhibits aerobic glycolysis in HSCs

FIG 3

Curcumin inhibits aerobic glycolysis in HSCs. HSCs were treated with vehicle DMSO, and curcumin at indicated concentrations for 24 h. (A) Real-time PCR analyses of key molecules in the glycolysis pathway. GAPDH was used as the invariant control. Sig- nificance: *P < 0.05 versus vehicle, **P < 0.01 versus vehicle. (B) Western blot analyses of key molecules in the glycolysis path- way with densitometry after normalization to b-actin. Significance: *P<0.05 versus vehicle, **P<0.01 versus vehicle. (C) Measurement of intracellular levels of lactate by ELISA. Significance: *P < 0.05 versus vehicle.

protein level shown by Western blot analyses (Fig. 4F–H). Col- lectively, these results indicated that activation of AMPK was required for curcumin blockade of aerobic glycolysis and reduction of activation in HSCs.

Discussion

There has been evidence that metabolic reprogramming takes place in activated HSCs, which redirects them to aerobic gly- colysis as a primary source of energy (3). These metabolic per- turbations lead to lactate accumulation and suggest that aero- bic glycolysis must provide advantages during HSC activation. Glycolysis is inefficient to generate adenosine triphosphate (ATP), as it produces only two ATP molecules per molecule of glucose, whereas complete oxidation of one glucose molecule by oxidative phosphorylation can produce up to 36 ATP mole- cules. Despite so, glycolysis is able to generate more ATP than oxidative phosphorylation by producing ATP at a faster rate. Under the condition of abundant glucose supply, glycolysis as a faster pathway for ATP production may be preferred to meet the high demands of rapid cell proliferation (19). It was observed that a series of glycolytic and gluconeogenic enzymes were reciprocally altered during HSC activation, suggesting that glucose catabolism for energy supply is primarily depend- ent on aerobic glycolysis (3). It could be inferred that this met- abolic switch could promote the global reprogramming of gene expression to maintain the fibrogenic properties of HSCs.

In current study, we mainly investigated aerobic glycolysis regulation of profibrogenic properties of HSCs in terms of

fibrotic marker expression and lipogenesis, two major charac- teristics of HSC activation. In order to obtain reliable results, we herein employed two distinct inhibitors to probe the glycol- ysis pathway at different levels. Compound 2DG interferes with the early stage of glycolysis, while galloflavin blocks the final stage of lactate generation. We found that both the compounds could effectively downregulate the expression of fibrogenic markers in HSCs, confirming the essential role of aerobic gly- colysis in maintaining HSC activation. Moreover, interference with glycolysis restored the contents of intracellular lipids and increased the expression of adipogenic transcription factors C/ EBPa and PPAR-c. C/EBPa plays an important role during adi- pocyte terminal differentiation (20). PPAR-c is a master regula- tor of adipogenesis and adipocyte differentiation and is estab- lished as a switch molecule of HSC activation with antifibrotic functions (21). Concomitant upregulation of C/EBPa and PPAR- c resulted from glycolysis inhibition presumably contributed to the restoration of lipids shown in the oil red O staining. These data indicated that aerobic glycolysis exerted repressive effects on HSC lipogenesis and adipogenic phenotype associated with affecting the adipogenic transcription pattern.

This metabolism-centric mechanism opens a new perspec- tive to identify antifibrotic agents with therapeutic implications for hepatic fibrosis. It has recently been summarized that cur- cumin is able to halt HSC activation in vitro and in vivo through targeting multiple pathways (22). In the present study, we discovered a novel mechanism for curcumin’s antifibrotic activity. Curcumin could disrupt the process of glycolysis by downregulation of HK and PFK2, and reduce the glucose

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FIG 4

Activation of AMPK is required for curcumin inhibition of aerobic glycolysis and reduction of activation of HSCs. HSCs were treated with vehicle DMSO, curcumin, and/or BML-275 at indicated concentrations for 24 h. (A) Western blot analyses of AMPK and its phosphorylation with densitometry after normalization to total-AMPK. Significance: *P < 0.05 versus vehicle. (B) Immu- nofluorescence staining using antibody against p-AMPK. The Hochest reagent was used to stain the nucleus. Images were taken at random fields. Representative views with positive staining are shown (1003 magnification). (C–E) Real-time PCR analy- ses of key molecules in the glycolysis pathway (C), profibrogenic markers (D), and adipogenic transcription factors (E) with densitometry. GAPDH was used as the invariant control. Significance: *P<0.05 versus vehicle, **P<0.01 versus vehicle, #P < 0.05 versus curcumin at 20 lM alone, ##P < 0.01 versus curcumin at 20 lM alone. (F–H) Western blot analyses of key mole- cules in the glycolysis pathway (F), profibrogenic markers (G), and adipogenic transcription factors (H) with densitometry after normalization to b-actin. Significance: *P<0.05 versus vehicle, **P<0.01 versus vehicle, #P<0.05 versus curcumin at 20 lM alone, ##P < 0.01 versus curcumin at 20 lM alone.

supply for glycolysis by downregulation of glut4 in HSCs, resulting in decreased intracellular lactate levels. These find- ings could be reasonably associated with previous data that curcumin reduced ECM gene expression in HSCs (23) and that

curcumin induced the expression of genes relevant to lipid accumulation and elevated intracellular lipids levels (24). Con- sistently, two latest investigations showed that curcumin inhib- ited glycolysis and induced apoptosis in human colorectal

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Curcumin inhibits aerobic glycolysis in HSCs

FIG 5

A schematic diagram illustrating an AMPK activation- dependent mechanism by which curcumin inhibits aerobic glycolysis leading to reduced HSC activation.

Fig. 5). These findings uncovered a novel mechanism for cur- cumin reduction of HSC activation for antifibrotic therapy.

Acknowledgements

This work was supported by the National Natural Science Foun- dation of China (81270514, 31401210, and 31571455), the Youth Natural Science Foundation of Jiangsu Province (BK20140955), the Natural Science Research General Program of Jiangsu Higher Education Institutions (14KJB310011), the Open Project Program of Jiangsu Key Laboratory for Pharmacol- ogy and Safety Evaluation of Chinese Materia Medica (JKLPSE 201502), and the Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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