Fat and Viral Liver Disease

Francesco NegroFrancesco Negro Viropathology UnitViropathology Unit

University of Geneva Medical CenterUniversity of Geneva Medical Center Geneva, SwitzerlandGeneva, Switzerland

Mainz, September 20, 2008Mainz, September 20, 2008

Steatosis and HBV

Steatosis in HBV infection:prevalence and correlates

n Steatosis Correlates

Thomopoulos 2006* 233 42 (18%) BMI, fasting glucose (MV)

Altlparmak 2005* 164 64 (39%) Age, BMI, cholesterol, triglycerides (UV)

Gordon 2005 17 9 (53%) C-peptide, glucose, waist circumference (MV)

*Patients with and without steatosis were comparable in terms of HBV DNA levels

Steatosis and HCV

Feature PrevalenceCell ballooning 89%Nuclear modifications 88%Eosinophilic cytoplasm 73%Focal sinusoidal activation 92%Steatosis 59%

Histopathological features of chronic non-A, non-B hepatitis

WIESE & HAUPT, Dtsch Z Verdau Stoffwechselkr 1985;45:101-110

"Follow-up analysis showed that there were no changes of these criteria up to the 6th year of disease"

Prevalence of Steatosis in Chronic Hepatitis C

Steatosis occurs in:• ~50% of chronic hepatitis C patients, overall considered

HOURIGAN et al, Hepatology 1999;29:1215LEANDRO et al, Gastroenterology 2006;130:1636

• ~40% of chronic hepatitis C patients without known factors of fatty liver (overweight, alcohol drinking, drugs, dyslipidemia)

RUBBIA-BRANDT et al, J Hepatol 2000;33:106

• ~20% of chronic hepatitis B patientsCZAJA et al, J Hepatol 1998;29:198

THOMOPOULOS et al, Eur J Gastroenterol Hepatol 2006;18:233

1 Occurrence and severity of steatosis is associated with genotype 3

MIHM et al, 1997; RUBBIA-BRANDT et al, 2000; ADINOLFI et al, 2001KUMAR et al, 2002; MONTO et al, 2002; POYNARD et al, 2003

HOFER et al, 2002; WESTIN et al, 2002

2 The score of steatosis correlates with the level of HCV RNA in serum and liver, but only in patients with genotype 3

RUBBIA-BRANDT et al, 2000; ADINOLFI et al, 2001

3 Virological response to α-IFN is associated with the disappearance of the steatosis, which recurs at the time of virological relapse

RUBBIA-BRANDT et al, 2001; KUMAR et al, 2002; POYNARD et al, 2003

Steatosis is a cytopathic effect of HCVSteatosis is a cytopathic effect of HCV

Triglycerides levels in HCV-transfected vs. untransfected Huh-7 cells

0

0.1

0.2

0.3

0.4

0.5

0.6

1b 2a 3a 3h 4h 5a GFP

O. D

.

ABID et al, J Hepatol 2005;42:744-751

Triglycerides levels in HCV-transfected vs. untransfected Huh-7 cells

0

0.1

0.2

0.3

0.4

0.5

0.6

1b 2a 3a 3h 4h 5a GFP

O. D

.

ABID et al, J Hepatol 2005;42:744-751

~3-fold

Liver steatosis in hepatitis C: proposed mechanisms - 1

• Decreased lipoprotein secretion– via inhibition of the MTP activity

PERLEMUTER et al, FASEB J 2002

– via non-specific inhibition of secretory pathways by ROSOKUDA et al, Gastroenterology 2002LERAT et al, Gastroenterology 2002

– via downregulation of MTPMIRANDOLA et al, Gastroenterology 2006

• Decreased degradation– in mitochondria, via downregulation of CPT-1

YAMAGUCHI et al, Dig Dis Sci 2005

HCV core protein transgenic mouseHCV core protein transgenic mouse

MTP

intracytoplasmicTG storage

STEATOSIS

HCV coreprotein

VLDLassembly

impairedVLDL secretion

TG Apo B

PERLEMUTER et al, FASEB J 2002;16:185

Intrahepatic MTP mRNA levels are inversely correlated with steatosis scores

MIRANDOLA et al, Gastroenterology 2006;130:1661-9steatosis score

Intr

ahep

atic

MTP

mRN

A le

vels P = 0.0017

Serum lipid profile changes in HCV

In chronic hepatitis C, Apolipoprotein B levels:– are inversely correlated with steatosis score– revert to normal upon response to therapy

HCV type 3a lowers serum cholesterol levels:HCV 1 HCV 3 HCV 4 P

188 ± 36 147 ± 42 172 ± 35 <0.01

Hypocholesterolemia in genotype 3a:– returns to normal in sustained virological responders– is not shared by other HCV genotypes

HOFER et al, Am J Gastroenterol 2002;97:2880

SERFATY et al, J Hepatol 2001;34:428

Liver steatosis in hepatitis C: proposed mechanisms - 2

• Increased ex novo synthesis of fatty acids– via activation of RxRα

TSUTSUMI et al, Hepatology 2002

– via enhanced LXRa/RxRα binding to LXR-RE in a PA28γ-dependent manner

MORIISHI et al, Proc Natl Acad Sci USA 2007

– via upregulation of sterol responsive element binding protein (SREBP)-1c

SU et al, Proc Natl Acad Sci USA 2002WARIS et al, J Virol 2007

JACKEL-CRAM et al, J Hepatol 2007

HCV induces lipogenic enzymes in Huh-7

WARIS et al, J Virol 2007;81:8122-8130

What is the HCV sequence responsible for TG accumulation?

1 501b MSTNPKPQRK TKRNTNRRPQ DVKFPGGGQI VGGVYLLPRR GPRLGVRAPR2a MSTNPKPQRK TKRNTNRRPQ DVKFPGGGQI VGGVYLLPRR GPRLGVRATR3a MSTLPKPQRK TKRNTIRRPQ DVKFPGGGQI VGGVYVLPRR GPRLGVCATR3h MSTLPKPQRK TKRNTIRRPQ NVKFPGGGQI VGGVYVLPRR GPTLGVRAAR4h MSTNPKPQRK TKRNTNRRPM DVKFPGGGQI VGGVYLLPRR GPRLGVRATR5a MSTNPKPQRK TKRNTSRRPQ DVKFPGGGQI VGGVYLLPRR GPRMGVRATR

51 1001b KTSERSQPRG RRQPIPKARR PEGRTWAQPG YPWPLYGNEG MGWAGWLLSP2a KTSERSQPRG RRQPIPKDPR STGRSWGRPG YPWPLYGNEG LGWAGWLLSP3a KTSERSQPRG RRQPIPKARR SEGRSWAQPG YPWPLYGNEG CGWAGWLLSP3h KTSERSQPRG RRQPIPKARR NEGRTWAQPG YPWSLYGNEG CGWAGWLLSP4h KTSERSQPRG RRQPIPKARP SEGRSWAQPG YPWPLYGNEG CGWAGWLLSP5a KTSERSQPRG RRQPIPKARQ STGRSWGQPG YPWPLYANEG LGWAGWLLSP

101 1501b RGSRPSWGPT DPRRRSRNLG KVIDTLTCGF ADLMGYIPLV GAPLGGAARA2a RGSRPSWGPT DPRHRSRNLG KVIDTLTCGF ADLMGYIPVV GAPVGGVARA3a RGSRPSWGPN DPRRRSRNLG KVIDTLTCGF ADLMGYIPLV GAPVGGVARA3h RGSRPHWGPN DPRRRSRNLG KIIDTLTCGF ADLMGYIPLV GAPVGGVARA4h RGSRPSWGPN DPRRRSRNLG KVIDTLTCGF ADLMGYIPLV GAPVGGVARA5a RGSRPNWGPN DPRRRSRNLG KVIDTLTCGF ADLMGYIPLV GGPVGGVARA

1b LAHGVRVLED GVN YATG NLP GCSFSIFLLA LLSCLTIPAS A151 191

2a LAHGVRVLED GIN YATG NLP GCSFSIFLLA LLSCISVPVS A3a LAHGVRALED GIN FATG NLP GCSFSIFLLA LFSCLVHPAA S3h LAHGVRAVED GIN YATG NLP GCSFSIFLLA ILSCLTVPAS G4h LAHGVRALED GIN YATG NLP GCAFSIFLLA LLSCLTVPAS A5a LAHGVRALED GVN YATG NLP GCSFSIFILA LLSCLTVPAS A

The HCV-3a triglyceride accumulation is due to a Y164F mutation within the core D2 domain

HOURIOUX et al, Gut 2007;56:1302-1308

Core 1a

Core 1a Y164F

F164Y is responsible for FAS activation by HCV 3a core

JACKEL-CRAM et al, J Hepatol 2007;46:999-1008

HCV-induced steatosis:1) Why?

2) Does it matter?

HCV core and NS5A proteins colocalize with lipid droplets in hepatocytes

• LD are physiological structures of fat storage in hepatocytes• Colocalization is an essential step for virion assembly• The domain 2 (D2), especially A147V, has a critical role in

the core-LD interaction and allows efficient virion assemblySHAVINSKAYA et al, J Biol Chem 2006;282:37158-37169

• Dysruption of the domain 2 (D2) of the core and of domain III of NS5A abrogate colocalization to LD and assembly of infectious particles

BOULANT et al, J Biol Chem 2006;281:22236-22247APPEL et al, PLoS Pathog 2008;4:e1000035

HCV core and NS5A colocalize with LD and direct virion assembly

APPEL et al, PLoS Pathog 2008;4:e1000035

HCV core colocalization with LD occurs independently of its steatogenic effects

PIODI et al, Hepatology 2008;48:16-27

What is better for HCV:1,000,000 small lipid droplets

or 1 big steatosis droplet?

1,000,000lipid droplets

(each Ø 0.1 μm)4,187 μm3 125,600 μm2

One steatosis droplet

(Ø 10 μm)4,187 μm3 1,256 μm2

The HCV MAID StudyPredictors of fibrosis (n = 3,068)

All pts.

HCV genotype BMI1 2 3 4 <25 25-30 >30

n 3068 1694 563 669 142 1481 1287 300

Activity 5.33 4.35 4.52 11.1 4.11 6.49 4.90 NSMale gender 1.92 1.79 NS NS NS 1.91 1.91 NSSteatosis 1.66 1.72 NS NS NS 1.61 NS NSAge 1.04 1.04 NS 1.05 NS 1.02 1.05 1.08Genotype 2 0.688 - - - - NS NS NSDiabetes NS 4.52 NS NS NS NS NS NSAlcohol abuse NS NS NS NS NS NS 1.69 NS

LEANDRO et al, Gastroenterology 2006

Steatosis at baseline and SVR

POYNARD et al, Hepatology 2003;38:75-85

0102030405060708090

3 non-3 1, 4, 5, 6

SteatosisNo steatosis

P=0.33

P<0.001

P<0.001

(n = 134) (n = 746)(n = 900)

Steatosis follows insulin resistance,not the other way around

FARTOUX et al, Gut 2005;54:1003-1008

HCV-induced steatosisSummary - 1

• Steatosis in hepatitis C is significantly associated with genotype 3a

• In patients with HCV 3a, TG accumulation correlates with viral load and pattern of response to therapy, and seems to be due to impaired VLDL secretion + ex novo lipogenesis

• The sequence responsible for TG accumulation may be the Y/F polymorphism at residue 164

HCV-induced steatosisSummary - 2

• HCV induced steatosis:– is NOT associated with increased liver fibrosis– is NOT associated with reduced response to IFN-α– is NOT associated with increased insulin resistance– is unlikely to benefit HCV replication or assembly– it may only have diagnostic significance

In chronic viral liver disease,is steatosis

ALWAYS due to the virus?

Steatosis in chronic hepatitis BSteatosis (n=42)* No steatosis (n=191) p

Male gender 28 (66.7%) 136 (71.2%) NSAge 46.2 ± 14.5 44.4 ± 16.7 NSHBV DNA (cp x 106) 55.5 ± 132.6 58.6 ± 127 NSActivity ≥ 7 17 (40.5%) 66 (34.6%) NSFibrosis ≥ 3 17 (40.5%) 77 (40.3%) NSBMI ≥ 25 30 (71.4%) 83 (43.5%) 0.001Fasting glucose** 112.4 ± 30.1 101.6 ± 26.5 0.041Cholesterol** 210.7 ± 51.9 204.5 ± 47.2 NSTriglycerides** 123.1 ± 70 129.6 ± 73.1 NS

THOMOPOULOS et al, Eur J Gastroenterol Hepatol 2006;18:233-7

* Moderate to heavy alcohol drinkers were excluded; ** mg/dl

In most chronic hepatitis C patients who have non-3a genotype infection and who do not drink alcohol:

Severity of steatosis correlates with the body mass index (and not with HCV RNA level), hence the steatosis is « metabolic »

ADINOLFI et al, 2001

The steatosis occurrence and severity is not (or only partially) modified by successful antiviral treatment

KUMAR et al, 2002; POYNARD et al, 2003

HCV-unrelated steatosis

Metabolic steatosis is due to insulin resistance

BROWNING & HORTON, J Clin Invest 2004;114:147

Risk factors for steatosis in 44 non-3a, alcohol abstinent chronic hepatitis C patients

BMI >25

HOMA >2

no risk factors

BMI >25 / HOMA >2

MUZZI et al, J Hepatol 2005;42:41-46

30%20%

18%32%

The HCV MAID StudyPredictors of fibrosis (n = 3,068)

All pts.

HCV genotype BMI1 2 3 4 <25 25-30 >30

n 3068 1694 563 669 142 1481 1287 300

Activity 5.33 4.35 4.52 11.1 4.11 6.49 4.90 NSMale gender 1.92 1.79 NS NS NS 1.91 1.91 NSSteatosis 1.66 1.72 NS NS NS 1.61 NS NSAge 1.04 1.04 NS 1.05 NS 1.02 1.05 1.08Genotype 2 0.688 - - - - NS NS NSDiabetes NS 4.52 NS NS NS NS NS NSAlcohol abuse NS NS NS NS NS NS 1.69 NS

LEANDRO et al, Gastroenterology 2006

By MV, the HOMA score (but not steatosis) is a factor independently associated with fibrosis score (P<0.001) and with its progression rate (P=0.03)

HUI et al, Gastroenterology 2003

Insulin resistance and/or diabetes are associated with severity of fibrosis

RATZIU et al, 2003; HUI et al, 2003; FARTOUX et al, 2005MUZZI et al, 2005; LEANDRO et al, 2006

Fibrogenesis in Chronic Hepatitis C:Fibrogenesis in Chronic Hepatitis C:Steatosis or Insulin Resistance?Steatosis or Insulin Resistance?

Steatosis at baseline and SVR

POYNARD et al, Hepatology 2003;38:75-85

0102030405060708090

3 non-3 1, 4, 5, 6

SteatosisNo steatosis

P=0.33

P<0.001

P<0.001

(n = 134) (n = 746)(n = 900)

Insulin resistance decreases SVR in chronic hepatitis C (genotype 1)

ROMERO-GOMEZ et al, Gastroenterology 2005;128:636-641

0

20

40

60

80

100

<2 2 - 4 >4HOMA-IR score

χ2 = 9.752, p<0.001

% S

VR

POUSTCHI et al, J Hepatol 2008;48:28-34

Insulin resistance decreases SVR in chronic hepatitis C (genotypes 2 and 3)

Increasing insulin sensitivity in chronic viral liver disease

• Increase physical activity• Reduce body weight• Insulin sensitizers

–Metformin–Thiazolidindiones

Fat and viral liver diseaseClinical management summary

• HCV-induced steatosis seems an innocent bystander, and disappears with successful antiviral therapy

• The management of steatosis unrelated to HBV/HCV is the management of its cause:– In case of alcohol abuse, treat alcohol abuse– In case of metabolic syndrome, insist on

lifestyle changes