Cholesterol and Sphingolipids in Non-Alcoholic fatty liver disease.

Transcription

1 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

2 NAFLD FATTY LIVER DISEASE Steatosis Steatohepatitis NASH Cirrhosis/HC ASH ALD

3 Inflammation Hepatocellular death Oxidative stress Fibrosis

4 ?

5 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

6 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

7 Cholesterol loading sensitizes hepatocytes to TNF Mari et al, Cell Metab, 2006

8 Mitochondrial free cholesterol loading increases membrane order and depletes mgsh Mari et al, Cell Metab 2006

9 Impact of free cholesterol in human NASH?

10 Enhanced free cholesterol in human NASH. Caballero, J Hepatol, 2009

11 StARD1, a mitochondrial cholesterol carrier, is overexpressed in human NASH. # #. FC 11+/-3 13+/-4 52+/-6 (µg/mg protein) Caballero, J Hepatol, 2009

12 TNF/Fas 2-OGC GSH StARD1 CL GSH CLOOH GSH 2-OGC Susceptibility to TNF/Fas ASH/NASH

13 2-OGC GSH? StARD1? CL GSH CLOOH TNF/Fas GSH 2-OGC Susceptibility to TNF/Fas ASH/NASH

14 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

15 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

16 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

17 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 Mari et al, J. Clin. Invest 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

18 EXPERIMENTAL DESIGN 1 HFD (60% cal) MCD OLESTEROL FILIPIN ASMase -/- mice 2 HFD (60% cal) + Amitriptyline SPHINGOMYELIN ASMase +/+ mice (WT mice)

19 ASMase is required for HFD or MCD-induced macrosteatosis Fucho et al, 2013

20 HFD MCD ASMase Steatosis ASMase is required for HFD and MCD induced steatosis

21 ASMase? HFD ER stress steatosis

22 ASMase deficiency prevents HFD-induced ER stress ATF4 PDI Edem Fucho et al, 2013

23 But ASMase KO mice is sensitive to tunicamycin-induced ER stress and steatosis Fernandez et al, 2013 Fucho et al, 2013

24 HFD ASMase ER stress ASMase is required for HFD induced ER stress Does ASMase directly induce ER stress?

25 ASMase induces ER stress and StARD1 upregulation via disruption of ER Ca 2+ homeostasis Fernandez et al, 2013

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27 If cholesterol is not in mitochondria, where does cholesterol accumulate in ASMase null mice?

28 ASMase null mice exhibit lysosomal cholesterol accumulation Fucho et al, 2013

29 What is the impact of increased lysosomal cholesterol? Lysosomal membrane permeabilization (LMP) Lipotoxicity Cell death

30 ASMase null mice are resistant to LMP and palmitic acid-induced lipotoxicity Fucho et al, 2013

31 1. ASMase regulates lysosomal cholesterol homeostasis. 2. The deficiency in ASMase determines increased lysosomal cholesterol content which, in turn, decreases LMP.

32 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

33 Amitriptyline inhibits HFD-induced obesity and glucose intolerance Fucho et al, 2013

34 Amitriptyline inhibits HFD-induced steatosis, liver damage, fibrosis and inflammation Fucho et al, 2013

35 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.

36 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

37 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