1. Nucler receptors and lipid
metabolism focus on FXR
Prof. Stefano Fiorucci
Seminario 21 maggio 2010

2. RINGRAZIAMENTI
Dr.ssa Barbara Renga
Dr.ssa Sabrina Cipriani
Dr. Andrea Mencarelli
Dr. Giuseppe Palladino
Dr. Marco Migliorati

4. Androgen
Estrogen
Mineralcorticoids
Glucocorticoids
Neutral pathway Acidic pathway
PR
VDR
FXR
PXR
LXR

5. Acetyl-CoA Acetoacetyl-CoA
CA
HMG-CoA Primary BAs
synthase
CDCA CA
HMG-CoA
HMG-CoA reductase
Acidic pathway
CDCA
Secondary
BAs
CYP27A1 CYP7B1 LCA DCA
CYP8B1
CA
Cholesterol
CYP8B1 CYP27A1
Cunjugated BAs
CYP7A1 NH3+CH2COO- NH3+(CH2)2SO3O-
+ glycine + taurine
CYP27A1 CDCA
Neutral pathway
(human) (mouse)

6. BILE ACIDS RECEPTORS
G protein coupled receptors
- TGR5
- Glucagon receptor
- FPR (formyl peptide receptor)
- Muscarinic receptor
NUCLEAR RECEPTORS:
- Farnesoid-x-receptor (FXR)
- Pregnane-x-receptor (PXR)
- Constitutive androstane receptor (CAR)
- Vitamin D receptor (VDR)
TYROSIN KINASE RECEPTORS:
- Epidermal growth factor receptor
- Insulin receptor
Cipriani et al., TIPS 2009

7. Structure/function organization of nuclear receptors
Olefsky, J. M. J. Biol. Chem. 2001;276:36863-36864

8. Insulin Drug
PGC1α CAR
CAR PXR
HNF-4 PXR
FoxA2
Insig-1
PPAR
CPT1A SCD1 SREBP1-c
FAE CD36
HMGCS2 apoA1
β-oxidation Lipid
Lipogenesis accumulation
Ketogenesis
HDL-cholesterol
Up-take
Repress hepatic energy metabolism

9. Drug Insulin GLucagon
Drug
CAR PGC1α
PXR FoxO1 CREB
CAR
PXR
IRS
CRE HNF-4
PBREM/XREM
Drug metabolism Gluconeogenesis
CYP3A G6Pase
CYP2B PEPCK1

10. Figure 1 A/B C D E
hinge DIMERIZATION
A NH2 DBD LBD COOH
AF1 AF2
FXRα1-2 FXRα3-4
(MYTG)
B 1 2 3 4 5 6 7 8 9 10 11
hinge DIMERIZATION
FXRα1 NH2 AF1 DBD LBD AF2 COOH
MYTG
FXRα2 NH2 AF1 DBD LBD AF2 COOH
FXRα3 NH2 AF1 DBD LBD AF2 COOH
MYTG
FXRα4 NH2 AF1 DBD LBD AF2 COOH
Fiorucci S, et al. Trends in Mol Med 2007
C CoActivator
PGC1-α
DRIP205
CoRepressor PRMT1 SRC-1
CARM1 LX
LL X X LL
LX Histones
AF2 AF2 AF2 AF2 Acetylation/Methylation
LB LB LB LB
D D Histones D D
De-Acetylation
AF1 AF1 AF1 AF1 Ac AcAc AcAc Ac
Bile acids
DBD DBD DBD DBD
FXR RXR FXR RXR
9-cis RA
AGGTCA n TGACCT
Ac AcAc AcAc Ac

11. Fexaramine
GW4064
6E-CDCA
Cipriani S, et al. 2009, Current medicinal Chemistry

13. Cholesterol biliary excretion
LRH-1
SR-B1
↑ABCG5
↑ ABCG8
Glucose metabolism
Bile acid biosynthesis
HNF4 PEPCK
CYP7A1 F 1,6-biPase
CYP8B1 FGF15 or 19,
Foxo1
↑ hFGF-19 JNK G6Pase
↑ mFGF-15 (small intestine) TRB3,Akt? ↑PDK4
↑ SHP
Bile acid conjugation Cellular protection
↑ BACS (IR1) ↑ A-Crystallin (IR1)
↑ BAT (IR1)
Inflammation, coagulation
Bile acid & xenobiotics transport
↑ BSEP (IR1) ↑Fibrinogen
↑ I-BABP (IR1) (intestine) ↑ h kininogen (IR-1)
hIBAT/ASBT (intestine) RXR/RAR(h) ↑ ICAM (FXRE in RARE)
↑ hMDR3, rMDR2 (IR1) FTF/LRH1(rb)
↑ MRP2 (ER8)
RXR/RAR Bile
NTCP BSEP Canaliculus
FXR
Hepatocyte
OATP B1
↑ OATP B3 (IR1)
↑ OATP8 (IR1) MRP2
Bile acid detoxification MDR2
↑ Cyp3A4 (IR1,DR3,IR8) Bile acids NTCP
↑ SULT2A1 (IR0)
↑ UGT 2B4 (1/2 FXRE) MRP3
FXRα
sinusoidal
capillaries
Triglyceride metabolism
↓ApoAI (1/2 FXRE) SHP MDR2
↓ ApoB
↑ ApoCII (IR1)
↓ ApoCIII (HNF4 RE)
↑ ApoA V (IR8)
↑ ApoE CYP7A1
↓ h hepatic lipase CYP8B1
↑ PLTP (IR1) OSTα
↑ hPPARα (imp. DR5)
OSTβ
LXR
SREBP-1c BAs
synthesis
↑ VLDL-R (BA, not FXR)
↑ Syndecan (DR1)
↑ Complement C3 (IR1)
intestine
MTP
Genes in red are inhibited by BAs

14. ileum mesenteric blood
Apical Basolateral
GLP1
Enterocyte
IP3
Gα
Phospholipase Ca2+
tgr5 Gβ Gγ Cβ2
(+) FXR FGF15
Asbt bile
Ostα/β
acids
Ntcp
Hepatocyte
FGF15
FGF4R
Cyp7a1
bile
Bsep acids cholesterol
bile canaliculus portal blood

15. Regulation of bile acids
detoxification by NRs
NR Canalicular Alternative Phase I & 2
Transporters pathway detoxification
FXR SEP, MRP2 OSTΑ/Β CYP3A4, UGT2BS
MRP 3& 4 SULT2A1
PXR MRP2 MRP3 & 4, CYP3A4, UGT2BS
OATP1A4 SULT2A1
CAR MRP2 MRP3 & 4 CYP3A4, UGT2BS
SULT2A1, CYP2B
Biological effects: Induction of canalicular and alternative basolateral bile acid
excretion and induction of phase I and II bile acid detoxification systems

16. CYP7A1
HNF4
CYP8B1
RXR CYP27A1
LXR
LRH1
Ntcp Asbt
HNF1 SHP RXR RAR
SHP HNF4
RXR RAR
FXR
BSEP/Bsep
OATP8
IBABP Sult1a1
Mrp2 Ostα/β
CYP3A4 UGT2B4
MRp2/Mrp2
Sult2a1 CYP7A1
RXR Mrp3 indirectly
PXR UGT1A1
OATP2 MDR1
CYP3A4
MRp2/Mrp2
UGT1A1
Mrp3
CAR RXR Mrp4 CYP2B
Sult2a1 CYP3A4
CYP7A1 SHP
LXRα RXR Abcg5/8 CYP3A4
Mrp3
Fig. 4
VDR RXR CYP3A4
Sult2a1

17. Fiorucci et al. Prog. Lipid Research 2010
Kidney
Systemic circulation
Hepatocytes OSTα/β Mrp3
OSTα/β Mrp4
Mrp4
Mrp2
Asbt
NTCP BSEP SHP CYP7A1
CYP8B1
Mdr1 FXR
BA FXR
BA
Mrp2 BA
JNK
mMdr2
OATPs
hMDR3
FGF-R4 Asbt Mrp2
FGF-15
OSTα/β Asbt
BA
BA FXR
Mdr1 Portal vein FGF15 IBABP
Mrp3
Cholangiocyte
Mrp3 OSTα/β
Bile duct
Small intestine

18. HDL clearance
Bile acids Syndecan-1
SRBI
pool
+ + LDL-R
+
+ FXR + VLDL-R
+
+ +
SHP
+
+
PPRalpha TG uptake LDL
+ SREBP1c
MTP + +
FA ACC
Β-oxidation FAS TG clearance ApoCII ApoCIII
FA VLDL ANGTPL3
synthesis secretion
LPL
+
VLDL
LPL
TG synthesis/secretion Plasma TG
IDL
glycerol (renmants)
FFA
Lipid storage FA uptake/
+ TG storage
Adopytes
TNFα
differentiation ASP
C3 HDL
SREBP1c FXR
+

19. agonists adipose tissue
FXR
liver Adiponectin
SHP TNFα
FABP
PEPCK
p-IRS-1/2
Glut-4
G6Pase
gluconeogenesis muscle
glycogenosynthesis
p-IRS-1/2
Glut-4
Insulin sensitivity
Glucose uptake
Fig. 13

20. FXR is expressed in adipose tissue and 3T3-L1 pre-adipocytes cell line
e
is su cyte cyte
a s e t dipo ipo iver
ipo rea Ad L
Ad P
FXR 5
d FXR
relative mRNA expression
*
4 *
3
Actin *
2
1
0
NT day 1 day 2 day 4 day 8
3 SHP
e
relative mRNA expression
*
b *
2
1
0
c NT day 1 day 2 day 4 day 8
Rizzo et al. Mol Pharmacol 2005

21. FXR antagonism prevent-adipocytes differentiation
induced by INT- 747
DIM + DIM + INT-747
INT-747 + GUGGULSTERON
DIM a NT
DIM + DIM + INT-747
INT-747 + GUGGULSTERON
10x
40x b c
CEBP PPARγ2 FABP TNF-α SREBP-1c AdipoQ
10.0 d 5 * 40 1.25 ** 4 * 5
NT
*
DIM + INT-747
*
* 4 1.00 4 DIM + INT-747 + GUGGULSTERON
relative mRNA expression
7.5 30 3
3 0.75
** 3
5.0 20
* 2
** 2 ** 0.50 2 **
2.5 10 1
1 0.25 1
**
0.0 0 0 0.00 0 0 Rizzo et al. Mol Pharmacol 2005

22. Retroviral expression of FXR enhances DIM-induced
differentiation in 3T3-L1 cells
vector DI FXR DI
M M
FABP CEBP-α PPAR-γ2 TNF-α
Rizzo et al. Mol Pharmacol 2005

23. INT-747 induces differentiation by PPAR-dependent
and -independent fashion
Rizzo et al. Mol Pharmacol 2005
DIM + DIM + INT-747
INT-747 + 9662
DIM NT
a
DIM + DIM + INT-747
10x
INT-747 + 9662
20x b c
d CEBP-α PPARγ2 FABP SREBP-1c TNF-α AdipoQ
6 5 30 * 6 1.00 5
* * NT
* DIM + INT-747
5 5 *
4 4 DIM + IN-747 + GW 9662
relative mRNA expression
** 0.75
**
4 20 4
3 3
3 ** ** 3 0.50
*
2 2
2 10 2
0.25
1 1
1 1
0 0 0 0 0.00 0

24. H&E Oil Red O CD11b
50000
40000
Area lesion
(pixels)
Control 30000
20000
10000
0
L
e
Kg
g
10
Kg
on
TR
m
g/
g/
si
APO E-/-
al
20
C
m
m
ro
E
si
3
10
g+
o
ro
E
Ap
m
-E
6-
+
10
+6
E
+
E
o
e
E
Ap
6-
o
o
Ap
Ap
IL1b
6E 3 mg/Kg
IL6
MCP1
CD11b
6E 10 mg/Kg
SHP
PPARg
FXRb
Rosiglitazone
FXRa
0.0 2.5 5.0 10 20 30 40 50 60
mRNA
A Mencarelli A., et al, Am J Physiol 2009