Research Article Saturated Fatty Acid-Induced Cytotoxicity in Liver … · 2019. 7. 31. · fatty...

Hindawi Publishing CorporationJournal of Nutrition and MetabolismVolume 2013 Article ID 514206 8 pageshttpdxdoiorg1011552013514206

Research ArticleSaturated Fatty Acid-Induced Cytotoxicity inLiver Cells Does Not Involve Phosphatase and TensinHomologue Deleted on Chromosome 10

Dong Wang1 Yuren Wei1 Melinda Frye2

Christopher L Gentile1 and Michael J Pagliassotti1

1 Department of Food Science and Human Nutrition Colorado State University 234 Gifford Fort Collins CO 80523-1571 USA2Department of Biomedical Sciences Colorado State University Fort Collins CO 80523 USA

Correspondence should be addressed to Michael J Pagliassotti pagliasmcahscolostateedu

Received 5 December 2012 Revised 11 March 2013 Accepted 25 March 2013

Academic Editor Peter M Clifton

Copyright copy 2013 Dong Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Liver specific deletion of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) inducessteatosis and hypersensitivity to insulin Saturated fatty acids which induce endoplasmic reticulum stress and cell death appearto increase PTEN whereas unsaturated fatty acids which do not induce endoplasmic reticulum stress or cell death reduce thisprotein In the present study the role of PTEN in saturated fatty acid-induced cytotoxicity was examined in H4IIE and HepG2 livercells Palmitate and stearate increased the expression of PTEN whereas the unsaturated fatty acids oleate and linoleate reducedPTEN expression in both cell types SiRNA-mediated knockdown of PTEN did not increase liver cell triglyceride stores or reducepalmitate- or stearate-mediated ER stress or apoptosis These results suggest that PTEN does not play a significant role in saturatedfatty acid-induced cytotoxicity in these liver cell models and in the absence of insulin

1 Introduction

Nonalcoholic fatty liver disease (NAFLD) is a chronicmetabolic disorder characterized by an increase in the hep-atic triglyceride pool (steatosis) and in some individualsdevelopment of nonalcoholic steatohepatitis (NASH) [1]Accumulating data suggest that in addition to their role inthe development of hepatic steatosis [2] fatty acids play animportant role in disease progression Free fatty acids areelevated in patients with NASH and are positively correlatedwith disease severity [3 4] Suppression of circulating fattyacids improved hepatic insulin sensitivity and reduced liverenzymes in healthy individuals [5] Data such as these haveled to the emerging concept that elevated fatty acids andproducts of fatty acidmetabolism rather than the triglyceridepool per se promote hepatotoxicity and disease progressioninNAFLD Indeed hepatic triglycerides are higher in patients

with benign steatosis compared to those with NASH [6] andstimulation of lipogenesis andor fatty acid oxidation protectsagainst palmitate-induced cell death in 120573-cells [7]

The cytotoxic response to elevated fatty acids appears tobe specific to or made more severe by long-chain saturatedfatty acids [8ndash11] In hepatocytes saturated fatty acids pro-mote endoplasmic reticulum (ER) stress and apoptosis [1112] both of which are characteristic features of NAFLD[13 14] The toxic effects of saturated fatty acids may bedue to their inability relative to unsaturated fatty acids tobe esterified and incorporated into triglyceride [7 15ndash17]Lipid phosphatase and tensin homolog deleted on chromo-some 10 (PTEN) is a multifunctional phosphatase that con-verts phosphatidylinositol-345-triphosphate (PIP

3) to phos-

phatidylinositol bisphosphate [18] One function of PTEN isto antagonize insulin signaling by reducing the level of PIP

3

[19] PTEN also appears to be involved in the development

2 Journal of Nutrition and Metabolism

of hepatic steatosis and regulation of hepatic triglyceridestores [20] Liver specific deletion of PTEN resulted in hepaticsteatosis in mice [19] and PTEN expression was reduced inthe liver of rats with steatosis and high plasma levels of fattyacids as well as in human livers characterized by steatosis[20] In addition unsaturated fatty acids which are readilyincorporated into hepatocyte triglycerides reduce the expres-sion of PTEN in HepG2 cells [20 21] Therefore the aimof this study was to examine the role of PTEN in saturatedfatty acid-mediated ER stress and apoptosis in liver cells Wehypothesized that saturated fatty acid-mediated ER stress andapoptosis involved increased expression of PTEN

2 Materials and Methods

21 Cell Culture The rat hepatoma liver cell line H4IIEand the human hepatoma cell line HepG2 (American TypeCulture Collection Manassas VA USA) were cultured inDMEM containing 8mM glucose and supplemented with10 fetal bovine serum penicillin and streptomycin sulfateExperiments were performed in the absence (LG) or presenceof fatty acids that were complexed to bovine serum albuminat a 2 1 molar ratio [11] and were 16 h in duration Eachexperiment was performed in triplicate and a total of 6independent experiments were performed for each cell lineand treatment

22 Transfection PTEN SiRNAor scrambled SiRNA (50 nMCell Signaling Waverly MA USA) was transfected intoH4IIE or HepG2 liver cells using TransIT-siQuest transfec-tion reagent (Mirus Bio Madison WI USA) Experimentswere performed 24 h following transfection

23 Triglyceride Analysis Total lipids were extracted usingthe methods of Bligh and Dyer [22] Triglyceride concentra-tion was determined using a kit (Zen-Bio Research TrianglePark NC USA)

24 RNA Isolation and Analysis Total RNA was extractedwith Trizol reagent using the manufacturerrsquos protocol (Invit-rogen Carlsbad CA USA) Real Time PCR was per-formed following reverse transcription using 05 120583g ofDNAase-treated RNA Superscript II RnaseH and randomhexamers PCR reactions were performed using transcribedcDNA and IQ-SYBR green master mix (Bio Rad HeculaCA USA) as described in detail previously [11] Analysis ofspliced XBP1 was based on previously described methods[23] using the following primers Forward 51015840GTCTGC-TGAGTCCGCAGCAGG31015840 and Reverse 51015840GATTAGCAG-ACTCTGGGGAAG31015840

25 Immunoblot Analysis Cell processing and immunoblotanalysis were performed as described previously [11] Primaryantibodies included total and phosphorylated eukaryoticinitiation factor2120572 (eIF2120572 Cell Signaling) and glucose-reg-ulated protein-78 (GRP78 Santa Cruz Biotechnology SantaCruz CA USA) Proteins were detected using horseradishperoxidase-conjugated secondary antibodies and density was

determined using a UVP Bioimaging system (Upland CAUSA)

26 Analysis of Akt Total and phosphorylated (serine 473)Akt was determined using the STAR (Signal TransductionAssay Reaction) ELISA kit (Millipore) per themanufacturerrsquosinstructions This kit is based on a solid phase sandwichenzyme-linked immunosorbent assay in which 96-well platesare coated with a monoclonal Akt antibody and followingincubation with cell lysates Akt is detected using a specificrabbit anti-Akt1 antibody or phosphorylated Akt is detectedusing a specific rabbit anti-phospho-Akt (Ser473) antibody

27 Cell Viability Cell death was evaluated using the CellDeath Detection ELISA kit (Roche Diagnostics PenzbergGermany) This assay is based on the quantitative sandwich-enzyme-immunoassay principle using mouse monoclonalantibodies directed against DNA and histones Cell sur-vival was evaluated using 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assays (Promega IncMadison WI USA) based on the supplierrsquos protocolCaspase-3 activity was determined with the ColorimetricCaspase-3 Activation Assay which uses a caspase specificpeptide conjugated to the color reporter p-nitroaniline (RampDSystems Minneapolis MN USA)

28 Statistics Statistical comparisons were made based ona factorial design using 2-way ANOVA with Bonferronirsquospost hoc test or 1-way ANOVA with the least significantdifference testThe level of significancewas119875 lt 005 Data arereported as means plusmn standard deviations for 6 independentexperiments in each cell type

3 Results

31 Individual Fatty Acids Differentially Influence PTEN Ex-pression The unsaturated fatty acids oleate and linoleatereduced PTEN expression whereas the saturated fatty acidspalmitate and stearate increased PTEN expression in bothH4IIE and HepG2 liver cells (Figure 1(a))

32 SiRNA-Mediated Knockdown of PTEN Does Not IncreaseTriglyceride Stores or Mitigate Saturated Fatty Acid-InducedER Stress and Cell Death Previous studies demonstratedthat liver specific PTEN deletion increased hepatic steatosistherefore we hypothesized that increased expression of PTENmay account for saturated fatty acid-mediated cytotoxicityby restricting the incorporation of these fatty acids intothe triglyceride pool SiRNA for PTEN resulted in a 75reduction in the expression of PTEN (Figure 1(b)) and pre-vented the saturated fatty acid-induced increase in PTEN(data not shown) However PTEN knockdown did notincrease triglyceride concentrations in response to oleatelinoleate palmitate or stearate in H4IIE or HepG2 liver cells(Figure 1(c)) PTEN knockdown did not reduce markers ofER stress (Figure 2) or cell death (Figure 3) in response topalmitate or stearate

Journal of Nutrition and Metabolism 3

H4IIE HepG2

PTEN

tubu

linPTEN

Tubulin

PTEN

Tubulin3

25

2

15

1

05

0

PTEN

tubu

lin

3

25

2

15

1

05

0LG O250 L250 P250 S250 LG O250 L250 P250 S250

LG O250 L250 P250 S250 LG O250 L250 P250 S250

lowast+

lowast+

lowast+

lowast+

lowastlowast

lowastlowast

(a)

PTEN

Tubulin

PTEN

Tubulin

SiRNA SiRNA

PTEN SiRNA+++++minus +++++minus

+++++minus+++++minus

PTEN

tubu

lin

3

25

2

15

1

05

0

PTEN

tubu

lin

3

25

2

15

1

05

0

LG LG O25

0

L250

P250

S250

LG LG O25

0

L250

P250

S250

LG LG O250 L250 P250 S250LG LG O250 L250 P250 S250

lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

SiRNA

3

25

2

15

1

05

0

3

25

2

15

1

05

0

ControlSiRNAControl

LG O250 L250 P250 S250 LG O250 L250 P250 S250

lowastlowast

lowast lowastlowast lowast lowast lowast lowast lowast

lowast lowast lowastlowastlowastlowast

Trig

lyce

ride (

fold

chan

ge)

Trig

lyce

ride (

fold

chan

ge)

(c)

Figure 1 PTEN expression and effects of PTEN knockdown on triglyceride stores in liver cells PTEN protein expression following 16 hof incubations with control media (LG) or fatty acids (oleate (O) linoleate (L) palmitate (P) or stearate (S)) at 250120583M (a) in H4IIE (leftcolumn) or HepG2 (right column) liver cells (119899 = 6) Effectiveness of SiRNA-mediated knockdown of PTEN in liver cells (b) Effects ofSiRNA-mediated knockdown of PTEN on triglyceride stores in liver cells following 16 h of incubations with control media or fatty acids (c)lowast significantly (119875 lt 005) different from LG + significantly (119875 lt 005) different from O250 and L250


LG O250 P250 S250 LG O250 P250 S250


lowast

lowastlowast

lowast

Splic

ed X

BP1

mRN

A (a

u)

Splic

ed X

BP1

mRN

A (a

u)

0

1

2

3

4

5

0

1

2

3

4

5

H4IIE HepG2

(a)

SiRNA SiRNA+ +++ + +++minus minusminus minusminus minusminus minus

LG O250 P250 S250LG O250 P250 S250

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

0

02

04

06

08

1

12

0

02

04

06

08

1

12

p-eI

F2120572

eIF2120572

p-eI

F2120572

eIF2120572

p-eIF2120572

eIF2120572p-eIF2120572

eIF2120572

(b)

SiRNASiRNA

SiRNA SiRNA

+ +++ + +++minus minusminus minusminus minusminus minus

LG O250 P250 S250 LG O250 P250 S250

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

GRP

78tu

bulin

GRP

78tu

bulin

GRP78

TubulinGRP78

Tubulin

LG O P S LG O P S

0

05

2

1

15

25

0

02

04

06

08

1

12

14

Control Control

(c)

Figure 2 ER stress markers in H4IIE and HepG2 liver cells XBP1 mRNA splicing (a) phosphorylation of eIF2120572 (b) and GRP78 proteinexpression (c) in H4IIE (left column) and HepG2 (right column) liver cells following 16 h of incubations with control media (LG) or fattyacids (oleate (O) linoleate (L) palmitate (P) or stearate (S)) at 250 120583M in the absence (Control) or presence of PTEN knockdown (SiRNA)(119899 = 6) lowast significantly (119875 lt 005) different from LG and O250

33 SiRNA-Mediated Knockdown of PTEN Increases Basaland Insulin-Stimulated Phosphorylation of Akt To examinewhether the inability of PTEN knockdown to influencetriglyceride stores or saturated fatty acid-induced ER stressand cell death was due to a general ineffectiveness of the

knockdown we examined phosphorylation of Akt PTENknockdown increased the phosphorylation of Akt in theabsence and presence of insulin (Figure 4) These resultssuggest that our observations with respect to PTEN inthe context of saturated fatty acid-induced ER stress and


lowast

HepG2H4IIE

LG O250 L250 P250 S250 LG O250 L250 P250 S250

300

250

200

150

100

50

0

300

250

200

150

100

50

0

lowast

lowastlowast

lowastlowast

lowast lowast

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

(a)

LG O250 L250 P250 S250LG O250 L250 P250 S250

120

100

80

60

40

20

0

120

100

80

60

40

20

0

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)lowast

lowastlowast

lowast

lowast lowastlowast

lowast

(b)

Scrambled SiRNASiRNA


LG O250 L250 P250 S250 LG O250 L250 P250 S250

08

06

04

02

0

08

06

04

02

0

lowast

lowastlowast

lowast

lowast lowastlowastlowast

Casp

ase-

3 (U

middotmg

prot

einminus

1)

Casp

ase-

3 (U

middotmg

prot

einminus

1)

(c)

Figure 3 Cell viability in H4IIE and HepG2 liver cells ELISA-based cell death (a) MTT assay (b) and caspase-3 activity (c) in H4IIE (leftcolumn) and HepG2 (right column) liver cells following 16 h of incubations with control media (LG) or fatty acids (oleate (O) linoleate (L)palmitate (P) or stearate (S)) at 250120583M in the absence (Control) or presence of PTEN knockdown (SiRNA) (119899 = 6) lowast significantly (119875 lt 005)different from LG O250 and L250


+InsulinminusInsulin +InsulinminusInsulin

Tota

l Akt

(OD

at4

50

nm)

Tota

l Akt

(OD

at4

50

nm)

0

05

1

15

2

0

05

1

15

2H4IIE HepG2

(a)

Phos

pho-

Akt

(OD

at450

nm)

+InsulinminusInsulin +InsulinminusInsulin0

05

1

15

2

Phos

pho-

Akt

(OD

at450

nm)

0

05

1

15

2

LG scrambled SiRNALG SiRNAO250 scrambled SiRNA

O250 SiRNAP250 scrambled SiRNAP250 SiRNA



lowast lowast lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowastlowast

(b)

Figure 4 Effects of PTEN knockdown on phosphorylation of Akt in liver cells Total Akt (a) and phosphorylated Akt (b)) in H4IIE (leftcolumn) and HepG2 (right column) liver cells following 16 h of incubations with control media (LG) or fatty acids (oleate (O) or palmitate(P)) at 250120583M Following incubations cells were either treated (+ insulin) or not treated (minus insulin) with 1 nM insulin for 30 minutes (119899 = 6)lowast significantly (119875 lt 005) different from corresponding scrambled SiRNA group

cell death were not due to a general ineffectiveness of theknockdown

4 Discussion

Lipid phosphatase and tensin homologue deleted on chro-mosome 10 (PTEN) converts phosphatidylinositol-345-tri-phosphate (PIP

3) to phosphatidylinositol bisphosphateThus

PTEN antagonizes insulin signaling in the liver by reducingthe levels of PIP

3 Hepatocyte specific deletion of PTEN

results in accelerated lipogenesis and hepatic steatosis Aprevious study demonstrated that the unsaturated fatty acidsoleate and palmitoleate but not saturated fatty acids reducedthe expression of PTEN We hypothesized that the inabilityof saturated fatty acids to downregulate PTEN might restrictdiversion into the triglyceride pool and therefore result in ER

stress and cell death We first examined the effects of unsat-urated and saturated fatty acids on PTEN expression Theunsaturated fatty acids oleate and linoleate reduced PTENwhereas the saturated fatty acids palmitate and stearateincreased PTEN expression in H4IIE and HepG2 liver cellsHowever knockdown of PTEN did not result in an increaseof triglyceride stores nor did it reduce ER stress or cell deathin response to palmitate or stearate These data suggest thatPTEN expression is not a critical determinant of saturatedfatty acid-mediated ER stress or cell death in these liver cellmodels

High plasma free fatty acid and triglyceride concentra-tions can lead to increased import of free fatty acids into bothadipose tissue and nonadipose tissues such as liver heartand pancreas and lead to intracellular lipid accumulation[24] The accumulation of triglyceride in liver cells is a char-acteristic feature of nonalcoholic fatty liver disease and has


been proposed as an initiating event in disease progression[1] Accumulating data suggest that in addition to their rolein the development of hepatic steatosis [2] fatty acids playan important role in disease progression Free fatty acids areelevated in patients with NASH and are positively correlatedwith disease severity [3 4] Suppression of circulating fattyacids improved hepatic insulin sensitivity and reduced liverenzymes in healthy individuals [5] Data such as these haveled to the emerging concept that elevated fatty acids andproducts of fatty acidmetabolism rather than the triglyceridepool per se promote hepatotoxicity and disease progressionin NAFLD A number of recent in vitro and in vivo studieshave demonstrated that the different forms of free fatty acidsexert remarkably different effects Exposure of a variety ofcell types including hepatocytes to long-chain saturatedfatty acids led to increased inflammation ER stress and celldeath [11 15 25] It has been proposed that a key eventdetermining saturated fatty acid-induced toxicity involves itsreduced ability to form triglyceride [15 26] In this paperwe hypothesized that the differential effects of saturated fattyacids on PTEN expression mediated saturated fatty acid-induced toxicity However the results do not support thishypothesis

The present series of experiments were performed in theabsence of insulin and as a result have likely minimized theimpact of PTEN expression on saturated fatty acid-mediatedcytotoxicity PTEN is an important regulator of insulinsignaling and recent studies have demonstrated that theability of PTEN to regulate hepatic lipid synthesis and storageis mediated via these effects and in concert with Akt2 [27]Thus we cannot rule out the possibility that PTEN may con-tribute to saturated fatty acid-mediated cytotoxicity underconditions in which insulin is present The present series ofexperiments do suggest that saturated fatty acid-mediatedchanges in PTEN expression do not restrict expansion ofthe triglyceride pool nor do they account for the subsequentinduction of ER stress and apoptosis

In the present study the role of PTEN in saturated fattyacid-induced cytotoxicitywas examined inH4IIE andHepG2liver cells Palmitate and stearate increased the expressionof PTEN whereas the unsaturated fatty acids oleate andlinoleate reduced PTEN expression in both cell typesSiRNA-mediated knockdown of PTEN did not increase livercell triglyceride stores or reduce palmitate- or stearate-mediated ER stress or cell deaths

Disclosure

No conflict of interests financial or otherwise is declared bythe authors

Authorsrsquo Contribution

C L Gentile andM J Pagliassotti conceived of and designedthe research D Wang and Y Wei performed experimentsD Wang Y Wei and M J Pagliassotti analyzed the dataM Frye C L Gentile and M J Pagliassotti interpreted theresults of the experiments and performed statistical analyses

D Wang Y Wei and M J Pagliassotti wrote the paper MFrye and C L Gentile edited the paper all authors approvedthe final version of the paper

Acknowledgments

This work was supported by National Institutes of HealthGrants DK072017 (M J Pagliassotti) K01-OD010971 (MFrye) and K01-DK087777 (C L Gentile)

References

[1] D H Akbar and A H Kawther ldquoNon-alcoholic fatty liverdisease and metabolic syndrome what we know and what wedonrsquot knowrdquo Medical Science Monitor vol 12 no 1 pp RA23ndashRA26 2006

[2] K L Donnelly C I Smith S J Schwarzenberg J Jessurun MD Boldt and E J Parks ldquoSources of fatty acids stored in liverand secreted via lipoproteins in patients with nonalcoholic fattyliver diseaserdquo Journal of Clinical Investigation vol 115 no 5 pp1343ndash1351 2005

[3] V Nehra P Angulo A L Buchman and K D Lindor ldquoNutri-tional and metabolic considerations in the etiology of nonal-coholic steatohepatitisrdquo Digestive Diseases and Sciences vol 46no 11 pp 2347ndash2352 2001

[4] F Diraison P H Moulin and M Beylot ldquoContribution ofhepatic de novo lipogenesis and reesterification of plasma nonesterified fatty acids to plasma triglyceride synthesis duringnon-alcoholic fatty liver diseaserdquo Diabetes and Metabolism vol29 no 5 pp 478ndash485 2003

[5] S Rigazio H R Lehto H Tuunanen et al ldquoThe lowering ofhepatic fatty acid uptake improves liver function and insulinsensitivity without affecting hepatic fat content in humansrdquoAmerican Journal of Physiology-Endocrinology and Metabolismvol 295 no 2 pp E413ndashE419 2008

[6] P Puri R A Baillie M M Wiest et al ldquoA lipidomic analysis ofnonalcoholic fatty liver diseaserdquo Hepatology vol 46 no 4 pp1081ndash1090 2007

[7] S E Choi I R Jung Y J Lee et al ldquoStimulation of lipogenesis aswell as fatty acid oxidation protects against palmitate-inducedINS-1 120573-cell deathrdquo Endocrinology vol 152 no 3 pp 816ndash8272011

[8] J E de VriesMM Vork T HM Roemen et al ldquoSaturated butnot mono-unsaturated fatty acids induce apoptotic cell deathin neonatal rat ventricular myocytesrdquo Journal of Lipid Researchvol 38 no 7 pp 1384ndash1394 1997

[9] L L Listenberger D S Ory and J E Schaffer ldquoPalmitate-induced apoptosis can occur through a ceramide-independentpathwayrdquo Journal of Biological Chemistry vol 276 no 18 pp14890ndash14895 2001

[10] K Maedler G A Spinas D Dyntar W Moritz N Kaiser andM Y Donath ldquoDistinct effects of saturated and monounsatu-rated fatty acids on 120573-cell turnover and functionrdquoDiabetes vol50 no 1 pp 69ndash76 2001

[11] Y Wei D Wang F Topczewski and M J Pagliassotti ldquoSat-urated fatty acids induce endoplasmic reticulum stress andapoptosis independently of ceramide in liver cellsrdquo AmericanJournal of Physiology-Endocrinology and Metabolism vol 291no 2 pp E275ndashE281 2006

[12] H Malhi S F Bronk N W Werneburg and G J Gores ldquoFreefatty acids induce JNK-dependent hepatocyte lipoapoptosisrdquo


Journal of Biological Chemistry vol 281 no 17 pp 12093ndash121012006

[13] P Puri FMirshahi O Cheung et al ldquoActivation and dysregula-tion of the unfolded protein response in nonalcoholic fatty liverdiseaserdquo Gastroenterology vol 134 no 2 pp 568ndash576 2008

[14] A E Feldstein A Canbay P Angulo et al ldquoHepatocyte apop-tosis and Fas expression are prominent features of humannonalcoholic steatohepatitisrdquo Gastroenterology vol 125 no 2pp 437ndash443 2003

[15] L L Listenberger X Han S E Lewis et al ldquoTriglyceride accu-mulation protects against fatty acid-induced lipotoxicityrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 100 no 6 pp 3077ndash3082 2003

[16] Z Z Li M Berk T M McIntyre and A E Feldstein ldquoHepaticlipid partitioning and liver damage in nonalcoholic fatty liverdisease role of stearoyl-Coa desaturaserdquo Journal of BiologicalChemistry vol 284 no 9 pp 5637ndash5644 2009

[17] Y Wei D Wang and M J Pagliassotti ldquoSaturated fatty acid-mediated endoplasmic reticulum stress and apoptosis are aug-mented by trans-10 cis-12-conjugated linoleic acid in liver cellsrdquoMolecular and Cellular Biochemistry vol 303 no 1-2 pp 105ndash113 2007

[18] M P Myers J P Stolarov C Eng et al ldquoP-TEN the tumor sup-pressor from human chromosome 10q23 is a dual-specificityphosphataserdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 17 pp 9052ndash90571997

[19] B Stiles Y Wang A Stahl et al ldquoLive-specific deletion ofnegative regulator Pten results in fatty liver and insulin hyper-sensitivityrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 101 no 7 pp 2082ndash2087 2004

[20] M Vinciguerra C Veyrat-Durebex M A Moukil L Rubbia-Brandt F Rohner-Jeanrenaud and M Foti ldquoPTEN down-regulation by unsaturated fatty acids triggers hepatic steatosisvia an NF-kappaBp65mTOR-dependent mechanismrdquo Gastro-enterology vol 134 no 1 pp 268ndash280 2008

[21] M Vinciguerra F Carrozzino M Peyrou et al ldquoUnsaturatedfatty acids promote hepatoma proliferation and progressionthrough downregulation of the tumor suppressor PTENrdquo Jour-nal of Hepatology vol 50 no 6 pp 1132ndash1141 2009

[22] E G Bligh and W J Dyer ldquoA rapid method of total lipidextraction and purificationrdquo Canadian Journal of Biochemistryand Physiology vol 37 no 8 pp 911ndash917 1959

[23] A van Schadewijk E F A vanrsquot Wout J Stolk and P SHiemstra ldquoA quantitativemethod for detection of splicedX-boxbinding protein-1 (XBP1) mRNA as a measure of endoplasmicreticulum (ER) stressrdquo Cell Stress Chaperones vol 17 no 2 pp275ndash279 2012

[24] J E Schaffer ldquoLipotoxicity when tissues overeatrdquoCurrentOpin-ion in Lipidology vol 14 no 3 pp 281ndash287 2003

[25] D Wang Y Wei and M J Pagliassotti ldquoSaturated fatty acidspromote endoplasmic reticulum stress and liver injury in ratswith hepatic steatosisrdquo Endocrinology vol 147 no 2 pp 943ndash951 2006

[26] M D Mantzaris E V Tsianos and D Galaris ldquoInterruptionof triacylglycerol synthesis in the endoplasmic reticulum is theinitiating event for saturated fatty acid-induced lipotoxicity inliver cellsrdquo FEBS Journal vol 278 no 3 pp 519ndash530 2011

[27] L He X Hou G Kanel et al ldquoThe critical role of AKT2 inhepatic steatosis induced by PTEN lossrdquo American Journal ofPathology vol 176 no 5 pp 2302ndash2308 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of hepatic steatosis and regulation of hepatic triglyceridestores [20] Liver specific deletion of PTEN resulted in hepaticsteatosis in mice [19] and PTEN expression was reduced inthe liver of rats with steatosis and high plasma levels of fattyacids as well as in human livers characterized by steatosis[20] In addition unsaturated fatty acids which are readilyincorporated into hepatocyte triglycerides reduce the expres-sion of PTEN in HepG2 cells [20 21] Therefore the aimof this study was to examine the role of PTEN in saturatedfatty acid-mediated ER stress and apoptosis in liver cells Wehypothesized that saturated fatty acid-mediated ER stress andapoptosis involved increased expression of PTEN

2 Materials and Methods

21 Cell Culture The rat hepatoma liver cell line H4IIEand the human hepatoma cell line HepG2 (American TypeCulture Collection Manassas VA USA) were cultured inDMEM containing 8mM glucose and supplemented with10 fetal bovine serum penicillin and streptomycin sulfateExperiments were performed in the absence (LG) or presenceof fatty acids that were complexed to bovine serum albuminat a 2 1 molar ratio [11] and were 16 h in duration Eachexperiment was performed in triplicate and a total of 6independent experiments were performed for each cell lineand treatment

22 Transfection PTEN SiRNAor scrambled SiRNA (50 nMCell Signaling Waverly MA USA) was transfected intoH4IIE or HepG2 liver cells using TransIT-siQuest transfec-tion reagent (Mirus Bio Madison WI USA) Experimentswere performed 24 h following transfection

23 Triglyceride Analysis Total lipids were extracted usingthe methods of Bligh and Dyer [22] Triglyceride concentra-tion was determined using a kit (Zen-Bio Research TrianglePark NC USA)

24 RNA Isolation and Analysis Total RNA was extractedwith Trizol reagent using the manufacturerrsquos protocol (Invit-rogen Carlsbad CA USA) Real Time PCR was per-formed following reverse transcription using 05 120583g ofDNAase-treated RNA Superscript II RnaseH and randomhexamers PCR reactions were performed using transcribedcDNA and IQ-SYBR green master mix (Bio Rad HeculaCA USA) as described in detail previously [11] Analysis ofspliced XBP1 was based on previously described methods[23] using the following primers Forward 51015840GTCTGC-TGAGTCCGCAGCAGG31015840 and Reverse 51015840GATTAGCAG-ACTCTGGGGAAG31015840

25 Immunoblot Analysis Cell processing and immunoblotanalysis were performed as described previously [11] Primaryantibodies included total and phosphorylated eukaryoticinitiation factor2120572 (eIF2120572 Cell Signaling) and glucose-reg-ulated protein-78 (GRP78 Santa Cruz Biotechnology SantaCruz CA USA) Proteins were detected using horseradishperoxidase-conjugated secondary antibodies and density was

determined using a UVP Bioimaging system (Upland CAUSA)

26 Analysis of Akt Total and phosphorylated (serine 473)Akt was determined using the STAR (Signal TransductionAssay Reaction) ELISA kit (Millipore) per themanufacturerrsquosinstructions This kit is based on a solid phase sandwichenzyme-linked immunosorbent assay in which 96-well platesare coated with a monoclonal Akt antibody and followingincubation with cell lysates Akt is detected using a specificrabbit anti-Akt1 antibody or phosphorylated Akt is detectedusing a specific rabbit anti-phospho-Akt (Ser473) antibody

27 Cell Viability Cell death was evaluated using the CellDeath Detection ELISA kit (Roche Diagnostics PenzbergGermany) This assay is based on the quantitative sandwich-enzyme-immunoassay principle using mouse monoclonalantibodies directed against DNA and histones Cell sur-vival was evaluated using 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assays (Promega IncMadison WI USA) based on the supplierrsquos protocolCaspase-3 activity was determined with the ColorimetricCaspase-3 Activation Assay which uses a caspase specificpeptide conjugated to the color reporter p-nitroaniline (RampDSystems Minneapolis MN USA)

28 Statistics Statistical comparisons were made based ona factorial design using 2-way ANOVA with Bonferronirsquospost hoc test or 1-way ANOVA with the least significantdifference testThe level of significancewas119875 lt 005 Data arereported as means plusmn standard deviations for 6 independentexperiments in each cell type

3 Results

31 Individual Fatty Acids Differentially Influence PTEN Ex-pression The unsaturated fatty acids oleate and linoleatereduced PTEN expression whereas the saturated fatty acidspalmitate and stearate increased PTEN expression in bothH4IIE and HepG2 liver cells (Figure 1(a))

32 SiRNA-Mediated Knockdown of PTEN Does Not IncreaseTriglyceride Stores or Mitigate Saturated Fatty Acid-InducedER Stress and Cell Death Previous studies demonstratedthat liver specific PTEN deletion increased hepatic steatosistherefore we hypothesized that increased expression of PTENmay account for saturated fatty acid-mediated cytotoxicityby restricting the incorporation of these fatty acids intothe triglyceride pool SiRNA for PTEN resulted in a 75reduction in the expression of PTEN (Figure 1(b)) and pre-vented the saturated fatty acid-induced increase in PTEN(data not shown) However PTEN knockdown did notincrease triglyceride concentrations in response to oleatelinoleate palmitate or stearate in H4IIE or HepG2 liver cells(Figure 1(c)) PTEN knockdown did not reduce markers ofER stress (Figure 2) or cell death (Figure 3) in response topalmitate or stearate


H4IIE HepG2

PTEN

tubu

linPTEN

Tubulin

PTEN

Tubulin3

25

2

15

1

05

0

PTEN

tubu

lin

3

25

2

15

1

05

0LG O250 L250 P250 S250 LG O250 L250 P250 S250

LG O250 L250 P250 S250 LG O250 L250 P250 S250

lowast+

lowast+

lowast+

lowast+

lowastlowast

lowastlowast

(a)

PTEN

Tubulin

PTEN

Tubulin

SiRNA SiRNA



PTEN

tubu

lin

3

25

2

15

1

05

0

PTEN

tubu

lin

3

25

2

15

1

05

0

LG LG O25

0

L250

P250

S250

LG LG O25

0

L250

P250

S250



lowastlowastlowast

lowastlowastlowast

(b)

SiRNA

3

25

2

15

1

05

0

3

25

2

15

1

05

0

ControlSiRNAControl

LG O250 L250 P250 S250 LG O250 L250 P250 S250

lowastlowast



Trig

lyce

ride (

fold

chan

ge)

Trig

lyce

ride (

fold

chan

ge)

(c)



LG O250 P250 S250 LG O250 P250 S250


lowast

lowastlowast

lowast

Splic

ed X

BP1

mRN

A (a

u)

Splic

ed X

BP1

mRN

A (a

u)

0

1

2

3

4

5

0

1

2

3

4

5

H4IIE HepG2

(a)


LG O250 P250 S250LG O250 P250 S250

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

0

02

04

06

08

1

12

0

02

04

06

08

1

12

p-eI

F2120572

eIF2120572

p-eI

F2120572

eIF2120572

p-eIF2120572

eIF2120572p-eIF2120572

eIF2120572

(b)

SiRNASiRNA

SiRNA SiRNA


LG O250 P250 S250 LG O250 P250 S250

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

GRP

78tu

bulin

GRP

78tu

bulin

GRP78

TubulinGRP78

Tubulin

LG O P S LG O P S

0

05

2

1

15

25

0

02

04

06

08

1

12

14

Control Control

(c)





lowast

HepG2H4IIE

LG O250 L250 P250 S250 LG O250 L250 P250 S250

300

250

200

150

100

50

0

300

250

200

150

100

50

0

lowast

lowastlowast

lowastlowast

lowast lowast

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

(a)

LG O250 L250 P250 S250LG O250 L250 P250 S250

120

100

80

60

40

20

0

120

100

80

60

40

20

0

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)lowast

lowastlowast

lowast

lowast lowastlowast

lowast

(b)



LG O250 L250 P250 S250 LG O250 L250 P250 S250

08

06

04

02

0

08

06

04

02

0

lowast

lowastlowast

lowast


Casp

ase-

3 (U

middotmg

prot

einminus

1)

Casp

ase-

3 (U

middotmg

prot

einminus

1)

(c)




Tota

l Akt

(OD

at4

50

nm)

Tota

l Akt

(OD

at4

50

nm)

0

05

1

15

2

0

05

1

15

2H4IIE HepG2

(a)

Phos

pho-

Akt

(OD

at450

nm)


05

1

15

2

Phos

pho-

Akt

(OD

at450

nm)

0

05

1

15

2






lowast

lowast

lowastlowast

lowast

lowast

lowast

lowastlowast

(b)



4 Discussion












Disclosure





Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H4IIE HepG2

PTEN

tubu

linPTEN

Tubulin

PTEN

Tubulin3

25

2

15

1

05

0

PTEN

tubu

lin

3

25

2

15

1

05

0LG O250 L250 P250 S250 LG O250 L250 P250 S250

LG O250 L250 P250 S250 LG O250 L250 P250 S250

lowast+

lowast+

lowast+

lowast+

lowastlowast

lowastlowast

(a)

PTEN

Tubulin

PTEN

Tubulin

SiRNA SiRNA



PTEN

tubu

lin

3

25

2

15

1

05

0

PTEN

tubu

lin

3

25

2

15

1

05

0

LG LG O25

0

L250

P250

S250

LG LG O25

0

L250

P250

S250



lowastlowastlowast

lowastlowastlowast

(b)

SiRNA

3

25

2

15

1

05

0

3

25

2

15

1

05

0

ControlSiRNAControl

LG O250 L250 P250 S250 LG O250 L250 P250 S250

lowastlowast



Trig

lyce

ride (

fold

chan

ge)

Trig

lyce

ride (

fold

chan

ge)

(c)



LG O250 P250 S250 LG O250 P250 S250


lowast

lowastlowast

lowast

Splic

ed X

BP1

mRN

A (a

u)

Splic

ed X

BP1

mRN

A (a

u)

0

1

2

3

4

5

0

1

2

3

4

5

H4IIE HepG2

(a)


LG O250 P250 S250LG O250 P250 S250

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

0

02

04

06

08

1

12

0

02

04

06

08

1

12

p-eI

F2120572

eIF2120572

p-eI

F2120572

eIF2120572

p-eIF2120572

eIF2120572p-eIF2120572

eIF2120572

(b)

SiRNASiRNA

SiRNA SiRNA


LG O250 P250 S250 LG O250 P250 S250

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

GRP

78tu

bulin

GRP

78tu

bulin

GRP78

TubulinGRP78

Tubulin

LG O P S LG O P S

0

05

2

1

15

25

0

02

04

06

08

1

12

14

Control Control

(c)





lowast

HepG2H4IIE

LG O250 L250 P250 S250 LG O250 L250 P250 S250

300

250

200

150

100

50

0

300

250

200

150

100

50

0

lowast

lowastlowast

lowastlowast

lowast lowast

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

(a)

LG O250 L250 P250 S250LG O250 L250 P250 S250

120

100

80

60

40

20

0

120

100

80

60

40

20

0

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)lowast

lowastlowast

lowast

lowast lowastlowast

lowast

(b)



LG O250 L250 P250 S250 LG O250 L250 P250 S250

08

06

04

02

0

08

06

04

02

0

lowast

lowastlowast

lowast


Casp

ase-

3 (U

middotmg

prot

einminus

1)

Casp

ase-

3 (U

middotmg

prot

einminus

1)

(c)




Tota

l Akt

(OD

at4

50

nm)

Tota

l Akt

(OD

at4

50

nm)

0

05

1

15

2

0

05

1

15

2H4IIE HepG2

(a)

Phos

pho-

Akt

(OD

at450

nm)


05

1

15

2

Phos

pho-

Akt

(OD

at450

nm)

0

05

1

15

2






lowast

lowast

lowastlowast

lowast

lowast

lowast

lowastlowast

(b)



4 Discussion












Disclosure





Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LG O250 P250 S250 LG O250 P250 S250


lowast

lowastlowast

lowast

Splic

ed X

BP1

mRN

A (a

u)

Splic

ed X

BP1

mRN

A (a

u)

0

1

2

3

4

5

0

1

2

3

4

5

H4IIE HepG2

(a)


LG O250 P250 S250LG O250 P250 S250

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

0

02

04

06

08

1

12

0

02

04

06

08

1

12

p-eI

F2120572

eIF2120572

p-eI

F2120572

eIF2120572

p-eIF2120572

eIF2120572p-eIF2120572

eIF2120572

(b)

SiRNASiRNA

SiRNA SiRNA


LG O250 P250 S250 LG O250 P250 S250

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

GRP

78tu

bulin

GRP

78tu

bulin

GRP78

TubulinGRP78

Tubulin

LG O P S LG O P S

0

05

2

1

15

25

0

02

04

06

08

1

12

14

Control Control

(c)





lowast

HepG2H4IIE

LG O250 L250 P250 S250 LG O250 L250 P250 S250

300

250

200

150

100

50

0

300

250

200

150

100

50

0

lowast

lowastlowast

lowastlowast

lowast lowast

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

(a)

LG O250 L250 P250 S250LG O250 L250 P250 S250

120

100

80

60

40

20

0

120

100

80

60

40

20

0

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)lowast

lowastlowast

lowast

lowast lowastlowast

lowast

(b)



LG O250 L250 P250 S250 LG O250 L250 P250 S250

08

06

04

02

0

08

06

04

02

0

lowast

lowastlowast

lowast


Casp

ase-

3 (U

middotmg

prot

einminus

1)

Casp

ase-

3 (U

middotmg

prot

einminus

1)

(c)




Tota

l Akt

(OD

at4

50

nm)

Tota

l Akt

(OD

at4

50

nm)

0

05

1

15

2

0

05

1

15

2H4IIE HepG2

(a)

Phos

pho-

Akt

(OD

at450

nm)


05

1

15

2

Phos

pho-

Akt

(OD

at450

nm)

0

05

1

15

2






lowast

lowast

lowastlowast

lowast

lowast

lowast

lowastlowast

(b)



4 Discussion












Disclosure





Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

HepG2H4IIE

LG O250 L250 P250 S250 LG O250 L250 P250 S250

300

250

200

150

100

50

0

300

250

200

150

100

50

0

lowast

lowastlowast

lowastlowast

lowast lowast

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

ELIS

A-ba

sed

cell

deat

h (m

Umiddot103

cells

minus1)

(a)

LG O250 L250 P250 S250LG O250 L250 P250 S250

120

100

80

60

40

20

0

120

100

80

60

40

20

0

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)

MTT

assa

y (

of L

G sc

ram

bled

SiR

NA

)lowast

lowastlowast

lowast

lowast lowastlowast

lowast

(b)



LG O250 L250 P250 S250 LG O250 L250 P250 S250

08

06

04

02

0

08

06

04

02

0

lowast

lowastlowast

lowast


Casp

ase-

3 (U

middotmg

prot

einminus

1)

Casp

ase-

3 (U

middotmg

prot

einminus

1)

(c)




Tota

l Akt

(OD

at4

50

nm)

Tota

l Akt

(OD

at4

50

nm)

0

05

1

15

2

0

05

1

15

2H4IIE HepG2

(a)

Phos

pho-

Akt

(OD

at450

nm)


05

1

15

2

Phos

pho-

Akt

(OD

at450

nm)

0

05

1

15

2






lowast

lowast

lowastlowast

lowast

lowast

lowast

lowastlowast

(b)



4 Discussion












Disclosure





Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Tota

l Akt

(OD

at4

50

nm)

Tota

l Akt

(OD

at4

50

nm)

0

05

1

15

2

0

05

1

15

2H4IIE HepG2

(a)

Phos

pho-

Akt

(OD

at450

nm)


05

1

15

2

Phos

pho-

Akt

(OD

at450

nm)

0

05

1

15

2






lowast

lowast

lowastlowast

lowast

lowast

lowast

lowastlowast

(b)



4 Discussion












Disclosure





Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Disclosure





Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Saturated Fatty Acid-Induced Cytotoxicity in Liver … · 2019. 7. 31. · fatty...

Documents

Transcript of Research Article Saturated Fatty Acid-Induced Cytotoxicity in Liver … · 2019. 7. 31. · fatty...