Cholesterol and Sphingolipids in Non-Alcoholic

fatty liver disease .

José C. Fernández-Checa

Liver Unit, Hospital Clinic

CIBEK and IDIBAPS

Instituto Investigaciones Biomédicas Barcelona

Consejo Superior Investigaciones Científicas

Barcelona, Spain

ICDM 2013

& 5 AASD

Seoul, Nov 7 2013

th

NAFLD

NASH Cirrhosis/HC

ALD

Steatosis

ASH

Steatohepatitis

FATTY LIVER DISEASE

Inflammation

Hepatocellular death

Oxidative stress

Fibrosis

?

Mechanisms involved in ALD/NAFLD

Alcohol Obesity

ALD NAFLD

ER stress Mitochondrial

dysfunction

Methionine metab Insulin resistance

NASH: mitochondrial disease

AcetaldehydeType 2 diabetes

Homocysteine

Disease progression

FFA

TGApoB +

TNFR1/R2

TNF

GSH

CH

CH

ER

TNFR1/R2

TNF

GSH

endosomes

Susceptibility to TNF/Fas?

ASH/NASH

Triglyceride/FFA loading Cholesterol loading

Mitochondrial Cholesterol in SteatohepatitisLombardi diet feeding

(choline deficient)

Ob/ob mice

2% cholesterol feeding (HC)

NPC1 knockout mice

Ob/ob mice2-14 days

FFA

TG

CH

*

* *

**

Oil-Red Filipin

Cholesterol loading sensitizes hepatocytes to TNF

Mari et al, Cell Metab, 2006

Mari et al, Cell Metab 2006

Mitochondrial free cholesterol loading increases membrane order and

depletes mGSH

Impact of free cholesterol in

human NASH?

Caballero, J Hepatol, 2009

Enhanced free cholesterol in human NASH.

Caballero, J Hepatol, 2009

StARD1, a mitochondrial cholesterol carrier, is overexpressed in human NASH.

#

.

#

FC

(µg/mg protein)11+/-3 13+/-4 52+/-6

Susceptibility to TNF/Fas

ASH/NASH

CH

CH

CH

CH

CH

CH

CH

GSH

GSH

2-OGC

CLCLOOH

StARD1

CH

CH

CH

CH

CH

CH

GSH

2-OGC

TNF/Fas

Susceptibility to TNF/Fas

ASH/NASH

CH

CH

CH

CH

CH

CH

CH

GSH

GSH

2-OGC

CLCLOOH

StARD1?

CH

CH

CH

CH

CH

CH

GSH

2-OGC

TNF/Fas

?

Mechanisms involved in ALD/NAFLD

Alcohol Obesity

ALD NAFLD

ER stress Mitochondrial

cholesterol loading

Methionine metab Insulin resistance

AcetaldehydeType 2 diabetes

Homocysteine

Disease progression

?

Tunicamycin-induced ER stress induces StARD1 expression which is prevented by

TUDCA

Fernandez et al, 2012

StARD1 SREBP-2

Thus, StARD1 is a novel target gene regulated by ER stress

PMH

+

Tm

Mechanisms involved in ALD/NAFLD

Alcohol Obesity

ALD NAFLD

ER stress Mitochondrial

dysfunction

Methionine metab Insulin resistance

AcetaldehydeType 2 diabetes

Homocysteine

Disease progression

Lipogenesis

Insulin resistanceOxidative stress

Cell death

mChol, mGSH

?

Serine+ Palmitoyl-CoA

SPT

3-ketoesphinganine

Sphinganine

Sphingosine

Dihydroceramide

ER (de novo synthesis)

CERAMIDE SphingomyelinASMase

Endosomes/lysosomes

Pro-apoptotic

Pro-fibrotic

Pro-NAFLD

Garcia-Ruiz et al, J. Clin. Invest. 2003

Mari et al, J. Clin. Invest. 2004

Mari et al, Cell Metab 2006

Mari et al, Gastroenterology 2008

Moles et al, Hepatology 2010

Moles et al, Am J. Pathol 2011

Moles et al, J. Biol. Chem 2012

Fernandez et al, J Hepatol 2013

Fucho et al, Sci Transl Med 2013

Baulies et al, In prep 2013

NSMase

Ceramide generation in cells

ASMase

EXPERIMENTAL DESIGN

ASMase-/- mice

HFD (60% cal)

MCD

ASMase+/+ mice

(WT mice)

HFD (60% cal)

+

Amitriptyline

1

2

CHOLESTEROL

FILIPIN

SPHINGOMYELIN

ASMase is required for HFD or MCD-induced macrosteatosis

Fucho et al, 2013

HFD MCD

ASMase

Steatosis

ASMase is required for HFD and MCD induced steatosis

HFD ER stress steatosis

ASMase ?

ASMase deficiency prevents HFD-induced ER stress

Fucho et al, 2013

ATF4 PDI Edem

But ASMase KO mice is sensitive to tunicamycin-induced ER stress and steatosis

Fernandez et al, 2013

Fucho et al, 2013

HFD

ASMase

ER stress

ASMase is required for HFD induced ER stress

Does ASMase directly induce ER stress?

ASMase induces ER stress and StARD1 upregulation

via disruption of ER Ca2+ homeostasis

Fernandez et al, 2013

If cholesterol is not in mitochondria,

where does cholesterol accumulate in

ASMase null mice?

Fucho et al, 2013

ASMase null mice exhibit lysosomal cholesterol accumulation

What is the impact of

increased lysosomal cholesterol?

Lysosomal membrane permeabilization (LMP)

Lipotoxicity

Cell death

Fucho et al, 2013

ASMase null mice are resistant to LMP and palmitic acid-induced lipotoxicity

1. ASMase regulates lysosomal cholesterol homeostasis.

2. The deficiency in ASMase determines increased

lysosomal cholesterol content which, in turn, decreases

LMP.

ASMase-/- mice

HFD (60% cal)

MCD

ASMase+/+ mice

(WT mice)

HFD (60% cal)

+

Amitriptyline

(an ASMase inhibitor)

1

2

EXPERIMENTAL DESIGN

WT mice+HFD

8 wk 16 wk0 wk

HFD HFD+Ami

Amitriptyline inhibits HFD-induced obesity and glucose intolerance

Fucho et al, 2013

Amitriptyline inhibits HFD-induced steatosis, liver damage, fibrosis and inflammation

Fucho et al, 2013

SUMMARY

1. StARD1 expression and mitochondrial cholesterol loading

are controlled by ER stress

2. ASMase is required for HFD-induced ER stress and steatosis

3. ASMase controls lysosomal cholesterol loading and

lysosomal membrane permeabilization

4. ASMase inhibition by amitriptyline prevents HFD-induced

obesity and glucose intolerance

5. ASMase targeting by amitriptyline rescues HFD-induced

NASH.

ASMase as a novel target for NAFLD

Alcohol Obesity

ALD NAFLD

ER stress Mitochondrial

dysfunction

Methionine metab Insulin resistance

AcetaldehydeType 2 diabetes

Homocysteine

Disease progression

Lysosomal

LMP

Lipogenesis

Insulin resistanceLipotoxicity

Cell deathOxidative stress

Cell death

ASMase amitriptylline

Thank you!!

Grant Support: NIAAA, NIH, SAF, FIS, CiberEHD

Collaborations

Gorka Basañez

Bilbao, Spain

Hide Tsukamoto

Neil Kaplowitz

Los Angeles, CA

B.Karten,

Nova Scotia, CA

S. Zanlungo,

Santiago Chile

C. Trautwein

Aachen, DE