Hepatitis C virus-induced metabolic reprogramming and pathological consequences

Birke Bartosch

Team 15Mechanisms of chronic hepatitis B and C pathogenesis and novel antiviral strategies

Cancer Research Center LyonINSERM 1052

Hepatitis C virus-induced metabolic reprogramming and

pathological consequences

Hepatitis CRiga – February 11th, 2013

10th Annual Conference of the New Visby Network on

Hepatocarcinogenesis – a multistep process

Risk FactorsHCV, HBV

AlcoholAflatoxins

Genetic diseasesDiabetes, Obesity

Metabolic syndrome

Normal liver Chronic Hepatitis Cirrhosis Carcinoma

Chronic inflammation

Fibrosis

Genetic + Epigenetic Alterations

Transformationp53, RB, TGFβ,

beta catenin

15 to 40 years 3 to 5% per year

Indirect neoplastic transformation

Direct neoplastic transformation

Hepatitis C Virus Pathogenesis

Liver cancer

Cirrhosis

Metabolic Syndrome

Obesity, Diabetes

Cell cycle control ↓Cellular proliferation ↑

Metabolicreprogramming

Mitochondrialdysfunction ↑

Fibrosis↑Genomic instability ↑

DNA damage ↑Steatosis ↑

Insulin resistance ↑

ClinicalPathology of HCV

HCV

NAFLDNon-alcoholic liver disease

NASHNon-alcoholic steatohepatitis

ALDAlcoholic liver disease

Inflammation ↑

Bartosch et al. J Hepatology, 2009

Lipogenesis

Glucose

Glycolysis

ApoB

TriglyceridesLipid droplet

Specific infectivity Immune escape

HCV infection and hepatic metabolism

vLDL

Cirrhosis HCC

HCV-induced metabolic changes:

Role in viral replication?

Contribution to pathology?

vLDL

Steatosis

Oxidative stressInflammationFibrosis

Insulin Resistance

Lipo/viro particle

assembly

Methodology

In vitro:Infection of hepatoma cells Huh-7.5 with HCVcc of genotype 2a (JFH1 strain)

• RTqPCR of enzymes implicated in liver metabolism• Metabolic flux analysis in infected and uninfected control cells• Infection assays in cell lines with altered metabolism

In vivo: Biopsies from HCV patients at different stages of disease

• RTqPCR of enzymes implicated in liver metabolism

glucose

GLUT

PEP

lactate pyruvate

Gly

coly

sis

acetyl CoA

TCA cycle

mitochondria

fatty acids triglycerides

LipogenesisACC, FAS, SCD

TG

lipid droplet

vLDL synthesis

ER

MTP, ApoB

vLDL

cytosol

vLD

L as

sem

bly

HC

V as

sem

bly

low density HCV virion

Liver carbohydrate metabolism

malate

OAA

Blood

Liver parenchyma

glucose

GLUT

PEP

lactate pyruvate

Gly

coly

sis

acetyl CoA

TCA cycle

mitochondria



TG

lipid droplet

vLDL synthesis

ER

MTP, ApoB

vLDL

cytosol

vLD

L as

sem

bly

HC

V as

sem

bly


Liver carbohydrate metabolism

malate

OAA

Insulin /IR

IRS 1/2

PI3/Akt

FoxO1

nucleus

HGP / vLDL synthesis genes:PEPCK, G6Pase / MTP, APOB

Lipogenesis:FAS, ACC

Glycolysis, lipogenesis:PK / FAS, ACC, SCD

Glucose metabolism:PK, HK1/2, LDHA, MCT4, GLUT1/3

Glucose/glutamine metabolism:PKM2, HK2, SLC745, GLS

SREBP-1c

ChREBP

HIF-1a

c-MYC

Blood

Liver parenchyma

Cell seeding+/- HCV Infection

-1 0

8-hoursupernatantharvest

3 5

Glucose / Lactate measurementsMethod:


Glucose utilization Lactate production

Day 3Post-infection

Contro

l med

ium

Contro

l med

iumNo g

lutam

ineNo g

lucos

e

Reduced glucose utilization by HCV infected cells

Cell seeding+/- HCV Infection

-1 0


3 5

Glucose / Lactate measurementsMethod:


Reduced glucose utilization by HCV infected cells

Day 5Post-infection


Lactate production In the absence of glucose

Contro

l med

iumNo g

lutam

ineNo g

lucos

e

Expression of metabolic enzymes in HCV infection

RT qPCR of proliferative HCV infected versus uninfected Huh7.5 cells

glucose

GLUT

G6P Pentose Phosphate Pathway

PEP

pyruvateLPK

Gly

coly

sis

acetyl CoA

TCA cycle

mitochondria

acetyl-CoAmalate

OAA

lactate

MCT

lactate

glutamine

SLC1A5 SLC7A5

glutamineGLS

pyruvate

ME

PC

G6PDH



TG

lipid droplet

vLDL synthesis

ER

MTP, ApoB

vLDL

cytosol

vLD

L as

sem

bly

HC

V a

ssem

bly


PFKL

HCV-induced metabolic reprogramming

PKM2

Xu5P

-> Nucleoside pool-> RedoxBalance

lactate

Evidence for a glutamine-based glucose-independent metabolism in HCV infected Huh7.5 cells

Cell seeding

-5 -4

Cell seeding

-1 5/14

Method:+/- HCV Infection

Day

Cell count

0

Cell countCell count

1

Change medium+/- glc or gln

Cell growth analysis after glucose or glutamine deprivation


Cell seeding

-5 -4

Cell seeding

-1 5/14


Day

Cell count

0


1



N = 4

Time (day) Time (day)

Cel

l Cou

nt

Cel

l Cou

nt-HCV +HCV

glucose

GLUT


PEP

pyruvateLPK

Gly

coly

sis

acetyl CoA

TCA cycle

mitochondria

acetyl-CoA

a-KG

malate

OAA

lactate

MCT

lactate

glutamine

SLC1A5 SLC7A5

glutamineGLS

pyruvate

ME

PC

G6PDH



TG

lipid droplet

vLDL synthesis

ER

MTP, ApoB

vLDL

cytosol

vLD

L as

sem

bly

HC

V a

ssem

bly


PFKL

HCV-induced metabolic reprogramming

PKM2

Xu5P

-> Nucleoside pool-> RedoxBalance


Cell seeding

-5 -4

Cell seeding

-1 5/14


Day

Cell count

0


1



Glutamine is an essential source to the TCA cycle in HCV infected cells

N = 4

Time (day) Time (day)

Cel

l Cou

nt

Cel

l Cou

nt

-HCV +HCV

glucose

GLUT

G6P

PEP

pyruvateLPK

Gly

coly

sis

acetyl CoA

TCA cycle

mitochondria

acetyl-CoA

a-KG

malate

OAA

lactate

MCT

lactate

glutamine

SLC1A5 SLC7A5

glutamineGLS

pyruvate

ME

PC

G6PDH



TG

lipid droplet

vLDL synthesis

ER

MTP, ApoB

vLDL

cytosol

vLD

L as

sem

bly

HC

V a

ssem

bly


PFKL

PKM2

Xu5P

c Myca regulator of cell cycle and cell metabolism

New concepts in cancer cell metabolism

c-MYC expression is increased in HCV infected cells and biopsies

Cell seeding(Huh7.5)

+/- HCV Infection(JFH1) MOI1

-1 0

RNAextraction

1

RNAextraction

2

RNAextraction

5

RT-qPCR analysisMethod:

In vitro time course infection (Huh7.5 cells/JFH1 virus) n=4

Time (day)

c-MYC expression is increased in HCV infected cells and biopsies



-1 0

RNAextraction

1

RNAextraction

2

RNAextraction

5


In vitro time course infection (Huh7.5 cells/JFH1 virus) n=4

Time (day)

Confirmation in liver biopsies

**



-1 0

RNAextraction

1

RNAextraction

2

RNAextraction

5


Glutamine transporters Glutaminase

Currently beeing confirmed in biopsie samplesIn vitro time course infection

(Huh7.5 cells/JFH1 virus) n=4

Time (day) Time (day) Time (day)

c Myc target genes are induced by HCV

Establishment of a Huh7.5 cell line silenced for MYC

Strong decrease of HCV RNA level in shMYC cell line despite a reduction of only 60% in MYC mRNA levels (*)

MYC seems to be essential for HCV replication

24h

48h

shCTR

Lsh

MYC

MYC mRNA level

c Myc required for HCV replication

c-myc mediates HCV-induced glutamine dependence but not decrease of glucose utilization


c-mycinduced

HCVinfected

glucose

GLUT

G6P R5P Xu5P

Pentose Phosphate Pathway

PEP

pyruvateLPK

Gly

coly

sis

acetyl CoA

TCA cycle

mitochondria

acetyl-CoA

a-KG

malate

OAA

lactate

MCT

lactate

glutamine

SLC1A5 SLC7A5

glutamineGLS

pyruvate

ME

PC

G6PDH PGD TALDO TKT

fatty acids


PFKL

PKM2

Glycolytic reprogramming in HCV infection

NADP+ NADPH

Biopsies

Huh7.5 cells

.02-

NAD + NADH, H +

mitochondria

Cellular defence against oxidative stress

.02

-

H2O2

MnSOD

.02

-

CuZnSOD

mGPX

H2O + 02 H2O + 02

CAT

cGPX

GSH

GSSG

GSH

GSSG

GR

NADPH

NADP+

lactate glucose

GLUTMCT

G6P R5P Xu5P

Pentose Phosphate Pathway

PEP

pyruvatelactate

Gly

coly

sis

G6PDH PGD TALDO TKT

TCA cycle

a-KG

NAD + NADH, H +

H2O2

glutamine

glutamine

Huh7.5seeding

Oxidative stressMeasurement

(H2O2 using DCFDA / O2.- using DHE)

HCVccinfection

Day 1p.i.

Day 2p.i.

Day 3p.i. Time (days)

Reactive oxygen species (ROS) production during HCVcc infection

Huh7.5seeding



HCVccinfection

Day 1p.i.

Day 2p.i.



• Glutathione turnover during infectionn= 3

• O2.- production 3 days p.i.

MOI

n= 1

• H2O2 production 3 days p.i.

MOI

** **

**

n= 3

Huh7.5seeding



HCVccinfection

Day 1p.i.

Day 2p.i.



• Glutathione turnover during infectionn= 3

• O2.- production 3 days p.i.

MOI

n= 1

• H2O2 production 3 days p.i.

MOI

** **

**

n= 3

Increase in O2.- but not in H202 levels:

Overall no increase in oxidative stress

HCV induces glutathione peroxidases 1 and 4 mRNA

seeding RNA extractionHCVccinfection

Day 1p.i.


Enzyme expression structureGPx1 strong expression tetramerGPx2 weak expression tetramerGPx3 Plasmatic GPx tetramer

GPx4strong expression and specific of membranes lipid

peroxides monomer

• qPCR analysis at day 3 p.i. n= 12

HCV induces glutathione peroxidases 1 and 4 activity

seeding RNA extractionHCVccinfection

Day 1p.i.


• Western-blot Analysis at day 3 p.i. • Activity test at day 3 p.i.

Densitometric analysis of Western-blots

GPx1

β-actin

NS3- +

HCV infection- +

β-actin

NS3

GPx4

**

n= 3 n= 4

Substrate : tBOOH

Measure the decrease at 340 nm

tBOOH or CHP

Substrate : CHP

Effects of GPx1 & GPx4 silencing on HCV replication, secretion & infectivity

seedingcells/cm2

HCVcc infection& siRNA

transfection

Time (days)

Extraction of :-Intra RNA-extra RNA-supernatant

Day 1p.i.

Day 2p.i.

Day 3p.i.

***

• extracellular viral RNA

n= 3n.s.

n= 3n.s.

• Specific infectivity

n=4

n=3n.s.

• intracellular viral RNA

n= 4

n= 3

n.s.

• Efficiency of siRNA

GPx4, but not GPx1, is a pro-viral factor for HCV replication & infectivity

***

GPx1 silencing:

GPx4 silencing:

HCV replication is sensitive to lipid peroxidation

Huh7.5 seeding HCVcc infection& CHP incubation

Time (days)

Extraction of :-Intra RNA-extra RNA-supernatant

• intracellular viral RNA• Cumene hydroperoxide (CHP) incubation induces lipid peroxidation

Linden A. et al, Toxicol In Vitro 2008

• Measurement of hydroxyalkenals and MDA in infected vs uninfected cells

• Collaboration with Institut Multidiscplinaire de Biochimie des Lipides• Measurement by GC-MS

• α-tocopherol blocks CHP mediated lipid peroxidation

Huang H. et al, Proc Natl Acad Sci USA 2007

n= 3

Lipid peroxidation inhibits

HCV replication

Role of GPx4 ?

Day 1p.i.

Day 2p.i.

Day 3p.i.

NS5A induces GPx4 transcription

Huh7.5 seeding +/- Protein induction with Doxycycline

Time (days)

RNA & proteinextraction

- + - + - +Core

β-actin β-actin β-actin

NS3/4A NS5A

Doxycycline

- + Doxycycline(5 µg / mL)

7.5 TA GFP

7.5 TA Core 7.5 TA NS3/4A 7.5 TA NS5A

• qPCR analysis 3 post induction: • Western-blot analysis 3 post induction to check protein expression: n= 2

Day 1 Day 2 Day 3

Conclusions

.02

- .02

-

TCA cycle

NAD + NADH, H +

IVIIIIIImitochondrion

cytosol

extracellular

ER

E1E2


vLDL

Core

HCV genome

NS5B

HCV replication and morphogenesis

glucose


PEP

Gly

coly

sis

glutamine

pyruvate

Conclusions

.02

-

H2O2

.02

-

H2O + 02 H2O + 02

TCA cycle

NAD + NADH, H +

H2O2

IVIIIIIImitochondrion

cytosol

extracellular

ER

E1E2


vLDL

Core

HCV genome

NS5B

HCV replication and morphogenesis

GSH

GSSG

GSH

GSSG

GR

NADPH

NADP+

glucose


PEP

Gly

coly

sis

glutamine

pyruvate

Phospholipid peroxidation

Constant turn overGPx4

GPx4

NS5A

Perspectives

• Validation in clinical studies• NMR flux and GC-MC analysis• What about NASH, chronic HBV, HCC?

in vitroclinical cohorts

• « metabolic » cancer drugs for treatment of liver diseases

• Understand the role of alterd carbohydrate metabolism and glutaminolysis not only in liver disease but also in cancer

AcknowledgementsCancer Research Center LyonTeam 15 : U1052Fabien Zoulim

Pierre LevyCharlène BraultDavid DurantelRomain ParentMaud MicheletAurélie SalleKennel Audrey

Hepato-GastroenterologyHCL LyonFabien Zoulim

U823, Grenoble, FrancePatrice MarcheChristian Villiers

U886, Lyon, FranceMichel Ovize

U1060, Lyon FranceMichel GuichardantHubert Vidal

Pathologisches Institut, Köln, GermanyMargarethe OdenthalHans-Michael SteffenHannah EischeidUlrike Koize

CRMN, Lyon, FranceBénédicte Elena-HerrmannGilles RautureauElodie Jobard

Engelhard Institute, Moskow, RussiaAlexander Ivanov

Documents

Hepatitis C virus-induced metabolic reprogramming and pathological consequences