Table Of ContentGuoetal.LipidsinHealthandDisease2012,11:10
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RESEARCH Open Access
O b
Cyanidin-3- - -glucoside regulates fatty acid
metabolism via an AMP-activated protein kinase-
dependent signaling pathway in human HepG2
cells
Honghui Guo1*, Guoling Liu1, Ruimin Zhong1, Yun Wang2, Duan Wang2 and Min Xia2
Abstract
Background: Hepatic metabolic derangements are key components in the development of fatty liver disease.
AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through
modulating the downstream acetyl CoA carboxylase (ACC) and carnitine palmitoyl transferase 1 (CPT-1) pathway. In
this study, cyanidin-3-O-b-glucoside (Cy-3-g), a typical anthocyanin pigment was used to examine its effects on
AMPK activation and fatty acid metabolism in human HepG2 hepatocytes.
Results: Anthocyanin Cy-3-g increased cellular AMPK activity in a calmodulin kinase kinase dependent manner.
Furthermore, Cy-3-g substantially induced AMPK downstream target ACC phosphorylation and inactivation, and
then decreased malonyl CoA contents, leading to stimulation of CPT-1 expression and significant increase of fatty
acid oxidation in HepG2 cells. These effects of Cy-3-g are largely abolished by pharmacological and genetic
inhibition of AMPK.
Conclusion: This study demonstrates that Cy-3-g regulates hepatic lipid homeostasis via an AMPK-dependent
signaling pathway. Targeting AMPK activation by anthocyanin may represent a promising approach for the
prevention and treatment of obesity-related nonalcoholic fatty liver disease.
Keywords: anthocyanin, AMP-activated protein kinase, acetyl CoA carboxylase, carnitine palmitoyl transferase 1,
fatty acid metabolism
Background major regulator of liver and whole body lipid homeosta-
Nonalcoholic fatty liver disease (NAFLD) is a serious sis [3]. AMPK activation in the liver results in the phos-
consequence of obesity, increasing the risk of liver can- phorylation and inactivation of acetyl-CoA carboxylase
cer or cirrhosis [1]. The origin of this disease is (ACC), a direct AMPK substrate, leading to decreased
unknown and probably multifactorial. Nevertheless, conversion of acetyl-CoA to malonyl CoA [4]. AMPK
because impaired lipid metabolism is recognized as an activation also results in phosphorylation and activation
associate and/or promoting mediator of the disease, of malonyl CoA decarboxylase (MCD), resulting in
management of hepatic metabolic disorders becomes an further lowering of malonyl CoA levels. Malonyl CoA
essential strategy for prevention and treatment of obe- allosterically inhibits carnitine palmitoyl-CoA transferase
sity-related NAFLD [2]. 1 (CPT-1), the enzyme responsible for transport of long
AMP-activated protein kinase (AMPK) is a key sensor chain acyl-CoAs into mitochondria for oxidation. Addi-
of cellular energy status and it is also recognized as a tionally, as malonyl CoA is required for de novo synth-
esis of fatty acids, decreased malonyl CoA leads to a
reduction in hepatic fatty acid synthesis [5]. Therefore,
*Correspondence:[email protected]
AMPK activation leads to a concomitant inhibition of
1DepartmentofFoodScience,YingdongCollegeofBioengineering,
ShaoguanUniversity,Shaoguan,GuangdongProvince,China fatty acid synthesis and activation of fatty acid oxidation.
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Guoetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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Anthocyanins are naturally occurring polyphenolic (IC = 1.6 μM, Figure 1D). These results strongly indi-
50
compounds in the plant foods and widely distributed in cate that anthocyanin stimulates AMPK activity in a
fruits, vegetables, and pigmented cereals, suggesting that manner that differs from AMP. To further address this
we can ingest significant amounts of anthocyanins from issue, we evaluated the additivity of AMP and anthocya-
plant-based daily diets [6]. Clinical investigations have nin mediated AMPK stimulation. Anthocyanin Cy-3-g
indicated that the moderate consumption of anthocya- increased AMPK activity in the presence of a saturating
nins through the intake of lyophilized grape powder or concentration of AMP (Figure 1E) and AMP stimulated
berry-derived anthocyanin supplements is associated AMPK in the presence of a maximally efficacious con-
with improved lipid profile [7,8]. In previous animal stu- centration of Cy-3-g (Figure 1F). This further supports
dies from us and other investigators, anthocyanins have the hypothesis that anthocyanin binds at a unique site
been shown to ameliorate dyslipidemia and hepatic stea- that differs from that of AMP binding.
tosis in different rodent models [9-12]. However, the
molecular mechanism under this action remains Intracellular Ca2+ and CaMKK-b mediates the
unknown and needs further investigation in cells. anthocyanin-induced activation of AMPK
Considering the key role of AMPK activation in regu- AMPK is also activated by calmodulin-dependent pro-
lating lipid metabolism and the potential capability of tein kinase kinase b (CaMKK-b) triggered by a rise in
anthocyanin in preventing hepatic steatosis, we observed intracellular calcium ions ([Ca2+]), without detectable
i
the influence of cyanidin-3-O-b-glucoside (Cy-3-g), the changes in the AMP/ATP ratio [14]. We next investi-
most abundant anthocyanin in plants [6], on AMPK gated whether the Ca2+ signaling was involved in the
activation and fatty acid metabolism in human HepG2 AMPK activation upon exposure of HepG2 cells to
hepatocytes. The signal pathway of the action involved anthocyanin. Anthocyanin exposure resulted in a rapid
in AMPK molecular activation was also investigated. and transient increase in [Ca2+] concentration (Figure
i
2A). Chelation of intracellular Ca2+ with BAPTA-AM
Results prevented the anthocyanin-induced phosphorylation of
Anthocyanin induces AMPK activation in HepG2 cells AMPK (Figure 2B), suggesting that intracellular Ca2+
To assess the impact of anthocyanin on AMPK activa- was involved in the signaling cascade initiated by the
tion, the human HepG2 cells were treated with Cy-3-g anthocyanin. Furthermore, preincubation of HepG2 cells
of 1~100 μM or with an AMPK activator-AICAR (1 with STO-609, a CaMKK-b inhibitor, decreased baseline
mM) for 1 h, respectively. We first determined the Thr- of AMPK activity, and anthocyanin did not increase
172 phosphorylation of AMPK because this site is an AMPK activation (Figure 2C), suggesting a role for
essential marker of AMPK activity. Cy-3-g significantly CaMKK-b. To confirm that CaMKK-b was the kinase
stimulated AMPK phosphorylation in a concentration- upstream of AMPK, a siRNA approach was utilized to
dependent fashion over the untreated control (Figure knock down CaMKK-b expression in HepG2 cells. Simi-
1A). Importantly, the effect of Cy-3-g at a concentration lar to the effects of STO-609, baseline AMPK activity
of 10 μM was comparable with AICAR. However, no was reduced and remained unchanged upon exposure to
change in the expression of endogenous total AMPK anthocyanin (Figure 2D).
protein was noted. To further confirm that the effect of
anthocyanin on AMPK, the kinase activity of AMPK Anthocyanin suppresses ACC activity, decreases malonyl
was assayed by using SAMS peptide as the substrate. CoA contents and stimulates CPT-1 expression in HepG2
Consistent with the increased AMPK phosphorylation, cells
Cy-3-g strongly stimulates AMPK kinase activity in a To determine whether AMPK activation affects its
concentration-dependent manner in HepG2 cells (Figure downstream target proteins, we examined the activity of
1B). ACC, a key enzyme in the regulation of fatty acid meta-
The enzymes fructose1,6-diphosphatase (FBPase) and bolism [5]. Anthocyanin suppressed ACC activity in a
glycogen phosphorylase (GPPase) are allosterically dose-dependent fashion in HepG2 cells (Figure 3A).
modulated by AMP. AMP activates GPPase, whereas it These effects are probably caused by phosphorylation of
inhibits FBPase [13]. To assess the specificity of antho- ACC by AMPK, as ACC serine-79 phosphorylation was
cyanin for AMPK activation, we determined their effects increased after anthocyanin treatment, whereas overall
on these two enzymes. Anthocyanin treatment had no ACC protein expression did not change (Figure 3B).
effect on GPPase activity at concentrations up to 1 mM, AMPK activation and ACC inhibition may lead to a
while the EC of AMP for the activation of GPPase was reduction in malonyl CoA levels and release of CPT-1
50
18.5 μM (Figure 1C). Similarly, anthocyanin showed no from allosteric inhibition [15,16]. We next investigated
significant inhibitory effect on FBPase at concentrations the net effect of these changes in malonyl CoA. Our
up to 1 mM, while AMP significantly inhibited FBPase results show that malonyl CoA levels decreased acutely
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Figure1AnthocyaninactivatesAMPKinculturedHepG2cells.(A)HepG2cellsweretreatedwithvariousconcentrationsofCy-3-gorAICAR
(1mM)for1h.Thecellswerethenlysed,and100μgofthecelllysatesunderwentSDS-PAGEfollowedbyWesternblotanalysisforAMPK
phosphorylation.(B)TwomicrogramofproteinextractsweresubjectedtoAMPKactivityassayswithSAMSpeptideassubstrates,resultsare
expressedasnmolesATP/mgprotein/min.*P<0.05or**P<0.01comparedtocontrol.(C)GPPaseactivitywasmeasuredinthepresenceof
glycogenusingaphosphoglucomutaseandglucose-6phosphatedehydrogenase-coupledspectrophotometricmethod.Effectsofanthocyanin
areexpressedasapercentofthemaximumstimulationachievedbyAMP.(D)FBPaseactivitywasdeterminedusingthesubstrateD-fructose-1,6-
bisphosphateandcolorimetricdetectionoffreephosphate.Resultsareexpressedaspercentinhibitionrelativetovehiclecontrol.(E)The
concentration-dependentAMPKactivationbyanthocyaninandadditiveeffectsinthepresenceof300μMAMP.*P<0.05comparedtovehicle.
(F)AMPdose-responsiveAMPKactivationandadditiveeffectsinthepresenceof100μMCy-3-g.*P<0.05comparedtovehicle.Theresults
presentedinpanels(B)through(F)aremeans±SEfromexperimentsrunintriplicateandarerepresentativeofatleastthreeindependent
experiments.
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Figure2Anthocyanin-inducedactivationofAMPKismediatedbyCa2+andCaMKK-b.(A)ArepresentativetraceofCa2+influxintoHepG2
cellsexposedtoanthocyaninisillustrated.Fura-2-loadedcellswereinitiallyincubatedwithmediaandthenswitchedtoanthocyanin.Changes
in[Ca2+] areexpressedasthenM.n=3.(B)HepG2cellswereexposedtomediaorCy-3-gfor30mininthepresenceorabsenceofBAPTA-AM
i
(20μM,20-minpreincubation).AMPKphosphorylationandtotalAMPKweredeterminedbyWesternblot.RepresentativeWesternblotofpAMPK
andtotalAMPKareshown.(C)HepG2cellswereexposedtoculturemedium(control)orCy-3-g(100μM)for30mininthepresenceorabsence
ofSTO-609(20μg/mL,30-minpreincubation).AMPKactivitywasdeterminedasindicated.Valuesareexpressedasfoldofcontrol.n=4.*P
<0.05betweenSTO-609andSTO-60plusCy-3-g.(D)HepG2cellsweretransfectedwithCaMKK-bsiRNA(CaMKK-bsi)orscrambledcontrolsiRNA
(Controlsi),and48hlatercellswereexposedtomediumorCy-3-g(100μM)for30min.Valuesareexpressedasfoldofcontrol.n=4.*P<0.05
betweenCy-3-gandCaMKKsiRNAplusCy-3-g.
after anthocyanin treatment in a dose-dependent fashion oxidation as Cy-3-g increased palmitate oxidation rates
(Figure 3C), accompanying with a statistically significant in HepG2 cells, with EC = 5.7 μM for Cy-3-g (Figure
50
and dose-responsive increase of CPT-1 mRNA expres- 4A). In addition, anthocyanin treatment significantly
sion in HepG2 cells (Figure 3D). inhibited the fatty acid synthesis as measured by incor-
poration of [14C]acetate into fatty acids, with IC = 6.3
50
Anthocyanin increases fatty acid oxidation and inhibits μM for Cy-3-g (Figure 4B). The effect of anthocyanin
lipogenesis in HepG2 cells on lipogenesis may be attributed to reduced fatty acid
Since AMPK activation inhibits ACC activity, further synthase (FAS) gene expression, as Cy-3-g treatment
leading to reduction of malonyl CoA and promotion of caused a dose-dependent inhibition of FAS mRNA
CPT-1 activation, one expected consequence is the expression in HepG2 cells (Figure 4C). Further measure-
increased fatty acid oxidation and reduced lipogenesis. ment showed that Cy-3-g treatment caused a dose-
To evaluate this hypothesis we studied the effects of dependent reduction of triglyceride (TG) content in
anthocyanin on hepatic fatty acid oxidation and synth- HepG2 cells (Figure 4D). Inhibition of mitochondrial
esis. Anthocyanin functionally enhanced fatty acid fatty acid oxidation with etomoxir was enough to
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Figure3AnthocyaninsuppressesACCactivity,decreaseMalonylCoAlevelsandpreventsFASexpressioninHepG2cells.(A)Western
blotanalysisofthephosphorylatedformofACCortotalACCincelllysatesfromHepG2cellstreatedfor1hwithCy-3-gorAICAR.(B)ACC
activityincellularextractswasmeasuredandexpressedasmeans±SE.(C-D)HepG2cellswereleftaloneortreatedwithvariousconcentrations
ofCy-3-gfor6h.Thencellswerecollectedandlysedformeasuring(C)malonylCoAlevelsand(D)CPT1mRNAexpressionasindicated.Results
areexpressedasmeans±SE.*P<0.05;**P<0.01comparedtocontrol.
hamper the decrease in triglyceride levels induced by effects by direct activation of AMPK activity and not as
anthocyanin (Figure 4E), indicating that AMPK- a consequence of cellular toxicity, or by altering the
mediated mitochondrial b-oxidation attributed for the AMP/ATP ratio.
major effects of anthocyanin. To exclude the possibility
that inhibition of fatty acid metabolism could be AMPK signaling mediates anthocyanin-induced reduction
mediated in part by cytotoxicity, we assessed the poten- of lipid levels in HepG2 cells
tial cellular cytotoxicity of anthocyanin. Treatment of To elucidate the AMPK pathway involved in regulating
human HepG2 cells with Cy-3-g at the concentrations hepatic fatty acid metabolism, we applied several inde-
up to 100 μM showed no measurable cytotoxicity, pendent approaches to inhibit AMPK activation. Pre-
whereas staurosporine, a known cytotoxic agent, signifi- treatment of HepG2 cells with compound C, a potent
cantly increased cellular cytotoxicity (Figure 4F). We and nonspecific inhibitor of AMPK [17], significantly
further probed the mechanism of action by assessing prevented anthocyanin-mediated inhibition of ACC
whether anthocyanin alters the cellular levels of AMP activity (Figure 5A) and FAS expression (Figure 5B) in
and ATP. Cy-3-g treatment at concentrations up to 100 HepG2 cells. To examine the critical role of AMPK in
μM caused no significant alterations in ATP and AMP anthocyanin-mediated hepatic fatty oxidation, we used
levels in HepG2 cells (Table 1). Taken together, these the specific siRNA against AMPK. Expression of endo-
results indicate the anthocyanin exert their cellular genous AMPK protein was remarkably suppressed by
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Figure4Anthocyaninincreasesfattyacidoxidation inHepG2cells.(A)Fattyacid oxidationratewasdetermined bycotreatmentwith
variousconcentrationsofCy-3-ginthepresenceof[14C]palmitate.Resultsareexpressedasfoldofcontrolfromatleastthreeindependent
experiments.(B)Fattyacidsynthesiswasmeasuredasincorporationof14C-acetateintofattyacidsinHepG2cellsduringa6-htreatmentwith
increasingconcentrationsofCy-3-gorAICAR,respectively.Resultsaregivenaspercentinhibitionoffattyacidsynthesisrelativetothecontrol
andaremeans±SEfromtriplicateanalysis.(C)Dose-dependentinhibitionofFASmRNAexpressionbyCy-3-g.Theresultsareexpressedas
percentofcontrolfromexperimentsrunintriplicateofthreeindependentexperiments.(D)IntracellularTGcontentsweremeasuredincell
lysatesbyspectrophotometicmethods.Resultsaremeans±SEfromtriplicatemeasurements.*P<0.05;**P<0.01comparedwithcontrol.(E)
HepG2cellspre-incubatedwithvehicleoretomoxir(50mM)for1hweretreatedwitheithervehicle(control)orCy-3-g(100μM).Then,
Intracellulartriglyceridecontentsweredetermined.*P<0.05betweenCy-3-gandCy-3-gplusetomoxir.(F)Thecellularcytotoxityassaysrunin
HepG2cellsusingMTSreagentscomparingaknowncytotoxin-staurosporineandCy-3-g.Dataarefromaminimumof4experimentsrunin
triplicateandisdemonstratedasmeans±SE.
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Table 1Nucleotide levels following treatment ofHepG2 downstream effector in regulation of lipid metabolism
withCy-3-g by anthocyanin.
Cy-3-g AMP ATP AMP/ATP
concentration (nM/106 (nM/106 ratio Discussion
cells) cells)
This study demonstrates that Cy-3-g, a typical anthocya-
Untreatedcontrol 0.348±0.021 6.236±0.160 0.056±0.003
nin pigment, stimulates AMPK activation via CaMKK in
1μM 0.336±0.044 6.525±0.129 0.051±0.007
HepG2 cells. The increase in AMPK activation by
10μM 0.398±0.018 6.271±0.087 0.064±0.002
anthocyanin suppresses ACC activity, causes the reduc-
100μM 0.354±0.011 6.633±0.310 0.054±0.003
tion in malonyl CoA levels and stimulation of CPT-1,
1mM 0.347±0.021 6.412±0.235 0.054±0.006 leading to enhanced fatty acid b-oxidation and finally
inhibiting the lipid accumulation in HepG2 cells. Thus,
AMPK siRNA (Figure 6A), suggesting that the knock- the novel findings from the present study is that antho-
down effect is specific. Compared to control siRNA, cyanin beneficially regulates hepatic fatty acid metabo-
AMPK siRNA transfection reversed the phosphorylation lism through AMPK-ACC-malonyl CoA-CPT1 pathway.
of ACC induced by Cy-3-g (Figure 6B), indicating that Previous studies had shown that Cy-3-g activated
ACC activity is mainly dependent on the presence of AMPK and enhanced adipocytokine secretion and adi-
AMPK. In line with this, direct measurement of palmi- pocyte-specific gene expression in isolated adipocytes
tate oxidation (Figure 6C) confirmed that the effects of [18,19]. Recently, Takikawa et al. have demonstrated
anthocyanin on CPT-1 expression (Figure 6D) and mal- that anthocyanin-rich bilberry extract effectively amelio-
onyl CoA levels (Figure 6E) were blunted in HepG2 rates hyperglycemia and insulin sensitivity via activation
cells where AMPK expression was knocked down. The of AMPK in diabetic mice [20]. The results of these stu-
lack of AMPK hence alters the long-term actions of dies suggest that anthocyanin may be a potential AMPK
anthocyanin on lipid metabolism (Figure 6F). activator. However, the impact of anthocyanin on hepa-
To further rule out that AMPK could participate to tic AMPK and its-mediated physiological consequences
sustain the action of anthocyanin, adenoviral overex- on fatty acid metabolism is still undefined. In this study,
pression of dominant-negative AMPK mutants (Ad-DN- we identified that anthocyanin Cy-3-g is a potent activa-
AMPK) was infected into HepG2 cells. The lack of tor of AMPK in hepatocytes. Meanwhile, GPPase and
AMPK significantly compromised the anthocyanin- FBPase activity were not altered in response to antho-
mediated reduction of malonyl CoA levels (Figure 7A). cyanin, implying that anthocyanin activated AMPK via a
As a consequence, the DN-AMPK completely blunted unique pathway unlike AICAR/AMP.
the effect of anthocyanin to increase CPT-1 mRNA In mammals, LKB1 and CaMKK-b have been identi-
expression (Figure 7B) and fatty acid oxidation (Figure fied as upstream kinases of AMPK, since both kinases
7C), reversing the inhibition of triglyceride accumulation can phosphorylate Thr172 and thus activate AMPK
by anthocyanin (Figure 7D). Together, these data indi- [14,21]. The phosphorylation of Thr172 by LKB1
cate that, to a large extent, AMPK functions as a novel requires an increase in AMP, while phosphorylation of
Figure5AMPKinhibitionabrogatesanthocyanin-mediatedincreaseofACCphosphorylationanddecreaseofFASexpression.HepG2
cellsweretreatedwithCy-3-g(100μM)intheabsenceorpresenceofAMPKinhibitorcompoundC(10mM).(A)ACCactivityand(B)FAS
mRNAexpressionwereanalyzedasindicated.Resultsareexpressedasmeans±SE.*P<0.05betweenCy-3-gandCy-3-gpluscompoundC.
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Figure6AMPKmediatesanthocyanin-inducedlipidreductioninHepG2cells.(A)HepG2cellweretransfectedwithAMPKsiRNA.Control
siRNAwasusedascontrol.AMPKproteinexpressionwasdeterminedbyWesternblotanalysis.Representativeblotsfromthreeindependent
assayswereshown.(B-E)HepG2cellsweretransfectedwitheithercontrolsiRNA(Controlsi)orAMPKspecificsiRNA(AMPKsi),followedby
treatmentwithCy-3-g(100μM).Afterthen,(B)ACCphosphorylation,(C)fattyacidoxidation,(D)CPT1mRNAexpression,(E)malonylCoAand(F)
hepatictriglycerideweremeasuredandresultsareshownasmeans±SEfromtriplicatemeasurementsfromatleastthreeindependent
experiments.*P<0.05betweenCy-3-gandCy-3-gplusAMPKsiRNA.
Thr172 by CaMKK-b is independent of AMP levels and baseline AMPK activity and prevented activation of the
is triggered by a rise in intracellular Ca2+. We found AMPK upon anthocyanin exposure. Moreover, when
that exposing HepG2 cells to anthocyanin initiated a transfected with CaMKK-b siRNA, both baseline and
rapid and transient influx of Ca2+ into the cells and that anthocyanin-induced activity of AMPK were inhibited.
AMPK activation by anthocyanin was prevented by the These data suggest that the anthocyanin-induced activa-
intracellular Ca2+-chelating agent, BAPTA-AM, suggest- tion of AMPK was mediated by CaMKK-b indepen-
ing that the anthocyanin-induced signaling pathway was dently of LKB1 signaling pathway.
dependent upon intracellular Ca2+. Since CaMKK-b- In the liver, AMPK coordinates the changes in the
mediated stimulation of AMPK required an increase in activity of enzymes of lipid metabolism and regulates
intracellular Ca2+ levels, we hypothesized that CaMKK-b the partitioning of fatty acid both in oxidative and bio-
may be the upstream kinase induced by anthocyanin. synthetic pathways [22]. In accordance with this, we
Indeed, the CaMKK inhibitor STO-609 decreased the observed that anthocyanin treatment induced ACC
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Figure 7 AMPK depletionabolishes the effects of anthocyaninonhepatic fatty acidmetabolism.HepG2cells were infected with a
controlnulladenovirus(Ad-Null)ordominant-negativeAMPK(Ad-DNAMPK)eitherwithanthocyanin(Cy-3-g,100μM)orwithoutanthocyanin
(Control).(A)malonylCoAlevels,(B)CPT-1mRNAexpression,(C)fattyacidoxidation,and(D)TGlevelsweredeterminedasindicated,
respectively.Resultsacquiredfromthreeindependentexperimentsareexpressedasmeans±SE.*P<0.05comparedtoAd-Nullcontrol.
phosphorylation, depressed its enzymetic activity in AMPK in the control of lipid metabolism and accumula-
HepG2 cells. Inhibition of ACC by AMPK leads to a fall tion, possibly through increased fatty acid oxidation.
in malonyl CoA content and a subsequent decrease in
fatty acid synthesis and increase in mitochondrial fatty Conclusion
acid oxidation via the allosteric regulation of CPT-1, In summary, our present study provide the mechanistic
which catalyses the entry of long-chain fatty acyl-CoA data that activation of hepatic AMPK by anthocyanin
into mitochondria [14]. Our results show that malonyl actsupstreamonlipidmetabolismbyactivatingthepath-
CoA is robustly decreased after anthocyanin treatment, wayscontrollingtheoxidationoffattyacids.Althoughit
as a result of AMPK and ACC phosphorylation. This appears hard to get the concentration of anthocyanin
resulted in a decrease in malonyl CoA levels associated showninthisstudyfromdailyconsumedfoods,ourfind-
with increased CPT-1 expression, which resulted in ingsprovideanovelinsightintothetherapeuticimplica-
increased fatty acid oxidation. The ability of anthocyanin tionsofanthocyanininobesity-relatedliverdisorders.
to stimulate AMPK and reduce hepatocellular lipid
accumulation was abolished by pharmacological inhibi- Methods
tion or knockdown of AMPK. Thus, anthocyanin-depen- Reagents
dent AMPK activation functionally inhibits ACC and Anthocyanin cyanidin-3-O-b-glucoside (Cy-3-g; HPLC
FAS which are two key downstream regulators of grade) was provided by Polyphenol AS (Sandnes,
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Norway). Dulbecco’s modified Eagle’s medium (DMEM), Fructose 1,6-bis-phosphatase assay
fetal bovine serum (FBS), and antibiotic mixture (peni- D-fructose-1,6 diphosphatase (FBPase, 0.01 U/mL final
cillins-treptomycin) were purchased from the Gibco concentration) in 2× assay buffer, 100 mM Tris (pH
BRL (Grand Island, NY). 5-aminoimidazole-4-carboxa- 7.0), 4 mM MgCl , 300 mM NaCl, 0.2 mg/mL BSA and
2
mide 1-ribofuranoside (AICAR), anti-phospho-AMPK 6 mM DTT, was added to test compounds to give final
Thr-172, anti-phospho-ACC Ser-79 and anti-b-actin concentrations as indicated. Substrate, D-fructose-1,6-
were purchased from Cell Signaling Technology, Inc. diphosphate, at a final concentration of 0.1 mM was
(Danvers, MA). AMPK inhibitor compound C and cal- added to the reaction and the reaction mix was incu-
modulin-dependent kinase kinase (CaMKK) inhibitor bated at 30°C for 20 min. Following the incubation 2
STO-609 were obtained from Calbiochem (San Diego, volumes of Malachite Green solution (Upstate Inc., Wal-
CA). The specific AMPK-targeted SAMS peptide used tham, MA) containing 0.001% Tween 20 was added and
in AMPK activity assay was purchased from GenScript absorbance at 640 nm read immediately [24].
(Piscataway, NJ); siRNA against CaMKK-b was from
Qiagen, Inc. (Valencia, CA); and all other chemicals, Measurement of intracellular ATP and AMP levels
unless otherwise specified, from Sigma-Aldrich (St. To measure adenine nucleotide concentrations in
Louis, MO). HepG2 cells, each well of HepG2 cells in 6-well plates
was harvested in 300 μL of lysis buffer containing 5%
Cell culture (v/v) perchloric acid. After sonication for 5 s, the sam-
HepG2 cells (human hepatoma cell line, ATCC #HB ples were centrifuged at 16,000 × g for 10 min at 4°C
8065, American Type Culture Collection) were cultured twice to remove acid-insoluble material. The superna-
in DMEM supplemented with 10% FBS and antibiotics tant neutralized with KOH and analyzed by HPLC
at 37°C in a humidified, 5% CO /95% air atmosphere. (Waters, Milford, MA) with an LC-18T reverse-phase
2
After reaching ~80% confluence, cells were serum- column (Supelco, Bellefonte, PA) at a flow rate of 1 mL/
starved for 24 h in medium containing 0.5% FBS. min. Nucleotides were detected by their absorbance at
254 nm and compared with external AMP and ATP
Adenoviruses standards to confirm its identity [25].
The adenoviral vector expressing a dominant-negative
AMPKa mutant (Ad-DN-AMPK) was kindly donated by Live cell intracellular calcium measurements
Dr J. Ha (Department of Molecular Biology, Kyung Hee The fluorescent calcium indicator, fura-2-acetoxymethyl
University, College of Medicine, Seoul, Korea). ester (fura-2-AM; Molecular Probes, Eugene, OR), was
used to measure changes in intracellular free calcium
AMPK activity ([Ca2+]). Cells were incubated at 25°C for 60 min with 1
i
AMPK activity was measured by monitoring phosphory- μM fura-2-AM in modified Krebs Ringer buffer [(in
lation of the SAMS peptide substrate as previously mM) 120 NaCl, 5 KCl, 1.2 CaCl , 0.7 MgSO , 15 N-2-
2 4
described [13]. To determine whether compound- hydroxyethylpiperazine-N’-2-ethanesulfonic acid
induced AMPK activation occurs in a reversible manner, (HEPES), and 1.8 g/L glucose (pH 7.4)]. After dye load-
AMP or Cy-3-g were preincubated with rat liver AMPK ing, cells were washed three times in the same medium
for 10 min at 20 times standard assay concentrations and kept in the dark for at least 30 min before single-
prior to dilution and measurement of AMPK activity. cell [Ca2+] measurements. Changes in [Ca2+] were
i i
determined ratiometrically (340 nm/380 nm excitation,
Glycogen phosphorylase assay 512 nm emission) in 1-mL aliquots using a spectrofluo-
The rabbit glycogen phosphorylase (GPPase) at the con- rometer. Calcium concentrations were calculated using
centration of 1.5 μg/mL of was added to a reaction mix- the equation: [Ca2+] = K (F /F ) (R - R )/
i d 380 max 380 min min
ture containing 20 mM Na HPO (pH 7.2), 2 mM (R - R) [26]. A dissociation constant (K ) value of
2 4 max d
MgSO , 1 mM b-NADP (b-nicotinamide adenine dinu- 224 nM was assumed for the binding of calcium to
4
cleotide phosphate), 1.4 U/mL G-6-PDH (glucose-6- fura-2-AM. R and R were determined in each
max min
phosphate-dehydrogenase) and 3 U/mL PGM (phospho- experimental group by the consecutive addition of 30
glucomutase). AMP or Cy-3-g was added to the assay μM digitonin (R ) and 50 mM EGTA (R ). R
max min max
medium at the specified concentrations followed by the and R are the maximum and minimum F /F
min 340 380
addition of glycogen (final concentration 1 mg/mL) to ratios, respectively. F /F = the ratio of fluor-
380 max 380 min
initiate the reaction. After incubating 10 min at 25°C, escence emission intensity at 380-nm excitation in Ca2
GPPase activity was assessed by measuring absorbance +-depleting (F ) and Ca2+-saturating (F )
380 max 380 min
at 340 nm [23]. conditions.
