Center of Liver, Digestive and Metabolic Diseases Dept of Vascular Medicine Targeting cholesterol excretion Lily Jakulj, MD
Reverse cholesterol transport BS PL CH CH BS, neutral sterols
1. Plasma HDL-c determines RCT BS PL CH CH BS, neutral sterols
2. Obligate role of bile in RCT BS PL CH CH BS, neutral sterols
1. Plasma HDL-c and RCT Biliary and fecal cholesterol excretion not impaired in ABCA1-/- mice 1,2 rhdl increased plasma HDL-c and centripetal flux to the liver, but not FSE in ABCA1-/- mice 3 Upregulation of individual steps in RCT did not affect FSE in normolipidemic mice 4 Human FSE studies conflicting 1. Groen, JCI 2001 2. Xie, JLR 2009 3. Jolley, JLR 1998 4. Alam, JBC 2001
Tissue cholesterol efflux (mg/kg/hr) Impaired in vivo tissue cholesterol efflux in subjects with genetically low HDL-c 6 p = 0.03 4 * * 2 3.5 0.6 4.3 0.5 0 APOA1 or ABCA1 def. n=7 controls n = 6 mean HDL-c: mean HDL-c: 0.33 mmol/l (SD 0.34) 1.42 mmol/l (SD 0.28) Holleboom, Jakulj, Groen, Stroes, unpublished
Fecal excretion (mg/day) FSE is equal in low HDL-carriers and controls 2000 1500 Carriers Controls 1000 500 0 neutral sterols bile acids Holleboom, Jakulj, Groen, Stroes, unpublished
1. Plasma HDL-c determines RCT Plasma HDL-c does not adequately reflect RCT in mice and humans with isolated low HDL-c Despite impaired TCE in carriers of mutations in APOA1 or ABCA1, compensating mechanisms exist Fecal sterol excretion, the obligate endpoint of RCT may depend on alternative (non-hdl?) pathways
2. Obligate role of bile in RCT BS PL CH CH BS, neutral sterols Yu, PNAS 2002
Non-biliary cholesterol excretion Increased FNS loss in dogs with complete biliary diversion and cholesterol free diet (Pertsemlidis, JCI 1973) Genetically modified mice with impaired biliary secretion: C7a hydroxylase -/- (Schwarz, JLR 1998) Abcb4-/- (Kruit, Gastroenterology 2005) Hepatic NPC1L1 +++ (Temel, JCI 2007) Hepatic ACAT2 -/- (Brown, JBC 2008)
NPC1L1 Liver-Tg mice > 90% reduction of biliary cholesterol secretion Normal FNS excretion Normal intestinal cholesterol absorption Similar plasma cholesterol levels as in wildtype Temel, JCI 2007
Intestinal perfusion experiments perfusion inflow 3ml/h + /- TC/PC mixed micelles 90-min perfusate collection 1x/15min Bile cannulation 14 C injection in tail vein Van der Velde, Groen, Gastroenterology 2007
µmol/day.100g body weight Trans-intestinal cholesterol excretion 4 3 2 1 0 proximal medial distal Van der Velde & Groen, Gastroenterology 2007
In vivo stable isotope study Quantification of fractional and absolute contributions to FNS loss in vivo in mice +/ LXR agonist van der Veen, Groen, JBC 2009
LXR-induced increase in FNS loss is largely due to TICE stimulation van der Veen, Groen, JBC 2009
Stimulation of TICE in mouse models Liver HDL LDL Plasma TICE + 1. Lumenal acceptors (PL) 2. High Fat Diet (fatty acids) 3. PPARδ agonist Bile 4. LXR agonist Absorption Diet Feces Shedding/Synthesis 1. Van der Velde, Groen, Gastroenterology 2007 2. Van der Velde, Groen, Am J Physiol Gastrointest Liver Physiol 2008 3. Vrins, Groen, JLR 2009 4. van der Veen, Groen, JBC 2009
Is TICE an anti-atherogenic mechanism? BS PL CH CH BS, neutral sterols
Macrophage-specific RCT assay degoma, JACC 2008
Non-biliary Φ-RCT in liver-npc1l1++ mice BS PL CH CH 3 H-chol BS, neutral sterols 3 H-chol + Temel, Cell Metabolism 2010
Obligate biliary Φ-RCT in Abcb4 -/- mice BS PL CH CH 3 H-chol BS, neutral sterols 3 H-chol - Nijstad, Gastroenterology 2011
Underlying mechanisms? Van der Velde, World J Gastroenterol 2010 Van der Velde, Brufau, Groen, Curr Opin Lipidol 2008
TICE in humans? Fecal sterols of non-dietary origin present in patients with complete biliary obstruction 1 Bile diversion in hofh patients produced a 6-8-fold increase in GI sterol output 2 Human intestinal perfusion studies: TICE estimated as ~44% of total FNS loss 3 1 Cheng, Proc Soc Exp Biol Med 1959 2 Deckelbaum, NEJM 1977 3 Simmonds, JCI 1967
Human TICE studies Proof-of-concept in patients with total biliary obstruction In vivo stable isotope study in subjects with intact enterohepatic cycle
Proof-of-concept: bile-diverted subjects Liver HDL LDL Plasma 50mg 13 C-chol Bile TICE Bile Absorption Diet Feces Shedding/Synthesis 13 C-chol
Human in vivo stable isotope study Male subjects, n 15 Age, years 61.7 ± 3.4 BMI, kg/m 2 25.7 ± 2.5 Total cholesterol, mmol/l 5.59 ± 0.65 LDL-cholesterol, mmol/l 3.74 ± 0.50 HDL-cholesterol, mmol/l 1.32 ± 0.27 Triglycerides, mmol/l 1.01 [ 0.66 2.61] Jakulj, Stroes, Groen, unpublished
Human in vivo flux study i.v. 13 C 2 cholesterol 50 mg oral D 7 cholesterol 50 mg * ** *** Blood (fasting) Blood (post-prandial) Bile - 48-24 0 24 48 72 96 120 144 168 * * * * * * ** *** ** ** oral D 4 - CA and D 4 - CDCA 50 mg feces feces feces feces feces feces feces feces feces - 48 oral D 4 sitostanol 3 mg t.i.d.
FNS excretion (gram/day) TICE ± 30% of FNS loss in humans 1,6 1,4 1,2 1,0 shedding TICE Bile derived 0,8 Non-absorbed 0,6 0,4 0,2 0,0 Jakulj, Stroes, Groen, unpublished
2. Obligate role of bile in RCT Non-biliary cholesterol excretion contributes to plasma cholesterol elimination in mice and men TICE might serve as an attractive target to improve RCT Focus on underlying molecular mechanisms and possibilities to stimulate TICE in humans
Acknowledgements A.K. Groen T.H. van Dijk T. Boer R. Boverhof F. Stellaard G. Brufau E.S.G. Stroes A.G. Holleboom R. Franssen K.P. van Lienden E.A.J. Rauws K.A.C. Booij / D.J. Gouma