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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
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NAFLD
NASH Cirrhosis/HC
ALD
Steatosis
ASH
Steatohepatitis
FATTY LIVER DISEASE
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Inflammation
Hepatocellular death
Oxidative stress
Fibrosis
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?
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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
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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
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Cholesterol loading sensitizes hepatocytes to TNF
Mari et al, Cell Metab, 2006
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Mari et al, Cell Metab 2006
Mitochondrial free cholesterol loading increases membrane order and
depletes mGSH
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Impact of free cholesterol in
human NASH?
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Caballero, J Hepatol, 2009
Enhanced free cholesterol in human NASH.
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Caballero, J Hepatol, 2009
StARD1, a mitochondrial cholesterol carrier, is overexpressed in human NASH.
#
.
#
FC
(µg/mg protein)11+/-3 13+/-4 52+/-6
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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
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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
?
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Mechanisms involved in ALD/NAFLD
Alcohol Obesity
ALD NAFLD
ER stress Mitochondrial
cholesterol loading
Methionine metab Insulin resistance
AcetaldehydeType 2 diabetes
Homocysteine
Disease progression
?
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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
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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
?
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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
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EXPERIMENTAL DESIGN
ASMase-/- mice
HFD (60% cal)
MCD
ASMase+/+ mice
(WT mice)
HFD (60% cal)
+
Amitriptyline
1
2
CHOLESTEROL
FILIPIN
SPHINGOMYELIN
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ASMase is required for HFD or MCD-induced macrosteatosis
Fucho et al, 2013
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HFD MCD
ASMase
Steatosis
ASMase is required for HFD and MCD induced steatosis
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HFD ER stress steatosis
ASMase ?
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ASMase deficiency prevents HFD-induced ER stress
Fucho et al, 2013
ATF4 PDI Edem
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But ASMase KO mice is sensitive to tunicamycin-induced ER stress and steatosis
Fernandez et al, 2013
Fucho et al, 2013
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HFD
ASMase
ER stress
ASMase is required for HFD induced ER stress
Does ASMase directly induce ER stress?
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ASMase induces ER stress and StARD1 upregulation
via disruption of ER Ca2+ homeostasis
Fernandez et al, 2013
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If cholesterol is not in mitochondria,
where does cholesterol accumulate in
ASMase null mice?
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Fucho et al, 2013
ASMase null mice exhibit lysosomal cholesterol accumulation
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What is the impact of
increased lysosomal cholesterol?
Lysosomal membrane permeabilization (LMP)
Lipotoxicity
Cell death
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Fucho et al, 2013
ASMase null mice are resistant to LMP and palmitic acid-induced lipotoxicity
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1. ASMase regulates lysosomal cholesterol homeostasis.
2. The deficiency in ASMase determines increased
lysosomal cholesterol content which, in turn, decreases
LMP.
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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
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Amitriptyline inhibits HFD-induced obesity and glucose intolerance
Fucho et al, 2013
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Amitriptyline inhibits HFD-induced steatosis, liver damage, fibrosis and inflammation
Fucho et al, 2013
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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.
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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
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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