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 thaasd Seoul, Nov 7 2013
NAFLD FATTY LIVER DISEASE Steatosis Steatohepatitis NASH Cirrhosis/HC ASH ALD
Inflammation Hepatocellular death Oxidative stress Fibrosis
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Mechanisms involved in ALD/NAFLD Alcohol Methionine metab Homocysteine Acetaldehyde Obesity Insulin resistance Type 2 diabetes ALD NAFLD ER stress Mitochondrial dysfunction NASH: mitochondrial disease Disease progression
Mitochondrial Cholesterol in Steatohepatitis * Lombardi diet feeding (choline deficient) 2-14 days * * Ob/ob mice Ob/ob mice Triglyceride/FFA loading Cholesterol * loading TNF 2% cholesterol feeding (HC) NPC1 knockout mice TNF FFA GSH TNFR1/R2 endosomes GSH TNFR1/R2 ApoB + TG Susceptibility to TNF/Fas? ER Oil-Red ASH/NASH Filipin
Cholesterol loading sensitizes hepatocytes to TNF Mari et al, Cell Metab, 2006
Mitochondrial free cholesterol loading increases membrane order and depletes mgsh Mari et al, Cell Metab 2006
Impact of free cholesterol in human NASH?
Enhanced free cholesterol in human NASH. Caballero, J Hepatol, 2009
StARD1, a mitochondrial cholesterol carrier, is overexpressed in human NASH. # #. FC 11+/-3 13+/-4 52+/-6 (µg/mg protein) Caballero, J Hepatol, 2009
TNF/Fas 2-OGC GSH StARD1 CL GSH CLOOH GSH 2-OGC Susceptibility to TNF/Fas ASH/NASH
2-OGC GSH? StARD1? CL GSH CLOOH TNF/Fas GSH 2-OGC Susceptibility to TNF/Fas ASH/NASH
Mechanisms involved in ALD/NAFLD Alcohol Methionine metab Homocysteine Acetaldehyde Obesity Insulin resistance Type 2 diabetes ALD NAFLD ER stress? Mitochondrial cholesterol loading Disease progression
Tunicamycin-induced ER stress induces StARD1 expression which is prevented by TUDCA PMH + Tm StARD1 SREBP-2 Thus, StARD1 is a novel target gene regulated by ER stress Fernandez et al, 2012
Mechanisms involved in ALD/NAFLD Alcohol Methionine metab Homocysteine Acetaldehyde Obesity Insulin resistance Type 2 diabetes ALD NAFLD ER stress Lipogenesis Insulin resistance? Mitochondrial dysfunction Oxidative stress Cell death mchol, mgsh Disease progression
Ceramide generation in cells Serine+ Palmitoyl-CoA SPT 3-ketoesphinganine Sphinganine ER (de novo synthesis) Sphingosine Dihydroceramide CERAMIDE Pro-apoptotic Pro-fibrotic Pro-NAFLD ASMase NSMase ASMase Endosomes/lysosomes Sphingomyelin 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
EXPERIMENTAL DESIGN 1 HFD (60% cal) MCD OLESTEROL FILIPIN ASMase -/- mice 2 HFD (60% cal) + Amitriptyline SPHINGOMYELIN ASMase +/+ mice (WT mice)
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
ASMase? HFD ER stress steatosis
ASMase deficiency prevents HFD-induced ER stress ATF4 PDI Edem Fucho et al, 2013
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 Ca 2+ homeostasis Fernandez et al, 2013
If cholesterol is not in mitochondria, where does cholesterol accumulate in ASMase null mice?
ASMase null mice exhibit lysosomal cholesterol accumulation Fucho et al, 2013
What is the impact of increased lysosomal cholesterol? Lysosomal membrane permeabilization (LMP) Lipotoxicity Cell death
ASMase null mice are resistant to LMP and palmitic acid-induced lipotoxicity Fucho et al, 2013
1. ASMase regulates lysosomal cholesterol homeostasis. 2. The deficiency in ASMase determines increased lysosomal cholesterol content which, in turn, decreases LMP.
EXPERIMENTAL DESIGN 1 HFD (60% cal) MCD ASMase -/- mice 2 HFD (60% cal) + Amitriptyline (an ASMase inhibitor) ASMase +/+ mice (WT mice) HFD 0 wk WT mice+hfd HFD+Ami 8 wk 16 wk
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 Methionine metab Homocysteine Acetaldehyde Obesity Insulin resistance Type 2 diabetes ALD NAFLD ER stress Lipogenesis Insulin resistance ASMase Mitochondrial dysfunction Oxidative stress Cell death amitriptylline Lysosomal LMP Lipotoxicity Cell death Disease progression
Thank you!! 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 Grant Support: NIAAA, NIH, SAF, FIS, CiberEHD