This study investigated BAR130, a novel dual agonist of LXRα and GPBAR1 receptors. In vitro experiments showed that BAR130 activated both receptors in a concentration-dependent manner. When administered to mice, BAR130 did not increase liver lipids or inflammation markers. It increased expression of the GPBAR1 target gene GLP-1 in the intestine but did not induce expression of lipid metabolism genes in the liver. BAR130 represents a promising approach to target metabolic disorders by activating LXRα and GPBAR1 without causing liver lipid accumulation.

1. BAR130, a hyodeoxycholic derivative as the first example of dual LXRα/GPBAR1 agonist
Carino Adriana*, Cipriani Sabrina #, Marchianò Silvia*, Biagioli Michele* , Zampella Angela † , De Martino Simona † and Fiorucci Stefano*
*Dipartimento di Scienze Chirurgiche e Biomediche, Università degli studi di Perugia Nuova Facoltà di Medicina e Chirurgia, Sant’Andrea delle Fratte, Perugia, Italy
#Dipartimento di Medicina, Università degli studi di Perugia Nuova Facoltà di Medicina e Chirurgia, Sant’Andrea delle Fratte, Perugia, Italy
†Dipartimento di Farmacia, Università di Napoli Federico II, Napoli, Italy
Background.The liver X receptors (LXRs), activated by oxysterol and by secondary bile acid hyodeoxycholic acid (HDCA), heve been found essential in the regulation of lipid homeostasis in mammals. Unfortunately, LXRα activates lipogenic
enzymes causing accumulation of lipid in the liver. In the present study, we report the identification of a side chain modified HDCA (BAR130) that represents the first example of a potent dual LXRα/GPBAR1 agonist.
Aim of the study. To investigate in vitro and in vivo effects of BAR130, a dual GPBAR1/LXRα ligand.
Material and Methods. HepG2 and Glutag cells (a mouse counterpart of intestinal L cells) were exposed to GW3965 or TLCA (10µM), HDCA (10µM) along with increasing concentrations of BAR130 (1, 5, 10, 25, 50 µM); total RNA was isolated for RT-PCR.
C57BL6 mice were administered with BAR130 (30 mg/Kg daily by gavage) or vehicle (distilled water) for 2 weeks. Mice were sacrificed and blood, liver and terminal ileum collected for plasma AST, cholesterol and tryglicerides measurement, liver hystopathology
analysis (H/E) and for evaluation of steatosis marker genes expression (RT-PCR).
Results. BAR130 transactivates LXRα and GPBAR1 with an EC50 value of 3.2±0.03 and 4.9±0.02 µM, respectively. When administered in vitro to HepG2 cells, BAR130 increases the expression of LXRα target genes, ABCA1 and SREBP1C, in a concentration-
dependent manner with an EC50 of 8.3 µM and 5.8 µM respectively. BAR130 increases the expression of pro-glucagon mRNA (a GPBAR1 target gene) in GLUTAG cells with an EC50 of 6.5 µM. Treating mice with BAR130 did not increased AST, cholesterol
and tryglicerides plasma levels. Further on, rt-PCR analysis of liver samples demonstrated that the BAR130 did not induce the expression of lipidogenic genes including FAS, SREBP1C and CD36. The result was confirmed by Liver histology (H/E). BAR130
increased the expression of GPBAR1 target gene GLP1 in the ileum and reduced markers of inflammation in macrophages.
Conclusions. This study describes the first dual LXRα and GPBAR1 ligand. BAR130 represents a promising approach to target metabolic disorders.
Figure3: Effects of BAR130 on hepatic lipid metabolism and on terminal ileum after administration on intact mice. C57BL6
mice were treated with BAR130 (30 mg/Kg daily per os) for two weeks. Results are the mean ± SE of 3-5 mice per group; *p<0.05
versus control mice. Serum levels of AST, total cholesterol and triglycerides; Hematoxilin and Eosin liver staining; Relative hepatic
mRNA expression of genes involved in fatty acids metabolism (FASN, SREBP1C, CD36) and genes for nuclear receptors (PPARα,
PPARγ, LXR α); Relative mRNA expression of GLP-1, FGF21, FXR and LXRα genes in terminal ileum. (Figure 3, A-C)
Figure 1: Concentration-response curves for BAR130. HepG2 cells were transiently transfected
with the reporter vector p(UAS)5XTKLuc, a vector containing the ligand binding domain of LXRα
cloned upstream of the GAL4-DNA binding domain (i.e. pSG5-LXRαLBD-GAL4DBD) and with the
reporter vector pGL4.70 (Promega), encoding the human Renilla gene. To evaluate GPBAR1
mediated transactivation, HEK-293T cells were transfected with pGL4.29 (Promega), a reporter vector
containing a cAMP response element (CRE) that drives the transcription of the luciferase reporter
gene luc2P, with pCMVSPORT6-human GPBAR1, and with pGL4.70 Renilla. 24 hours post-
transfection cells were stimulated with increasing concentrations of BAR130 (1, 5, 10, 25 and 50 µM).
GW3965 (10 µM) or TLCA (10 μM) were used as a positive control.
For calculation of efficacy data, maximal transactivation of LRE or CRE caused by the compound (10
μM) was compared to maximal transactivation caused by GW3965 (10 µM) or TLCA (10 μM) and by
HDCA (10 μM). (Figure 1, A-C).
Figure 2: In vitro pharmacological evaluation of BAR130 activity.
Quantitative Real-Time PCR analysis of mRNA expression on LXRα
and GPBAR1 target genes. ABCA1 and SREBP1c expression in
HepG2 cells primed with increasing concentration of BAR130 (1, 5, 10
and 25 μM). GW3965 and HDCA were used as positive controls. Pro-
glucagon expression in Glutag cells stimulated with increasing dose of
BAR130 (1, 10, 25 and 50 μM). TLCA and HDCA were used as a
positive control. As shown BAR130 was able to induce the expression
of ABCA1 and SREBP1c genes in HepG2 cells in dose-dependent
manner with an EC50 of 8.3 µM and 5.8 µM respectively, and the
expression of pro-glucagon mRNA in Glutag cells; however, the
induction is dose-dependent only until the 10 µM concentration with an
EC50 of 6.5 µM. Values are normalized to GAPDH and are expressed
relative to those of not treated cells (NT) which are arbitrarily settled to
1. The relative mRNA expression is expressed as 2(-ΔΔCt). *p < 0.05
vs NT (Figure 2, A-C).
0
25
50
75
100
125
10-7 10-6 10-5 10-4
EC50 :3.2  0.03 M
0
130 (M)
%MaximalResponse
0
25
50
75
100
125
10-7 10-6 10-5 10-4
EC50: 4.990.2 M
0
130 (M)
%MaximalResponse
Compound GPBAR1* LXRa**
Efficacy Efficacy
(% vs TLCA) (% vs HDCA) (% vs GW3965) (% vs HDCA)
HDCA 26 15,5
BAR130 55,1 211,9 109,3 703,3
HepG2
NT GWHDCA 1 5 10 25
0
10
20
30
130
(M)
*
*
*
*
EC50:8.3M
ABCA1mRNArel.expression
A. B.
C.
HepG2
NT GW HDCA 1 5 10 25
0
5
10
15
20
25
130
(M)
*
* *
*
*
* EC50 : 5.8 M
SREBP1cmRNArel.expression
GLUTAG
NT TLCA HDCA 1 10 25 50
0
1
2
3
4
130
(M)
*
*
*
*
 EC50 : 6.5 M
Pro-Glucagon
mRNArel.expression
A.
B.
C.
CTRL BAR130
0
50
100
150
200
ASTU/L
CTRL BAR130
0
20
40
60
80
100
TotalCholesterolmg/dL
CTRL BAR130
0
50
100
150
Triglyceridesmg/dL
0
1
2
3
FASNrelativemRNAexpression
(x10^-1)
0.0
0.5
1.0
1.5
2.0
SREBP1crelativemRNAexpression
(x10^-1)
0
1
2
3
4
5
CD36relativemRNAexpression
(x10^-3)
0
1
2
3
4
5
PPARrelativemRNAexpression
(x10^-2)
0
1
2
3
4
5
PPARrelativemRNAexpression
(x10^-3)
0
1
2
3
4
LXRrelativemRNAexpression
(x10^-2)
0
2
4
6
8
10
*
GLP-1relativemRNAexpression
(x10^-2)
0
5
10
15
FGF21relativemRNAexpression
(x10^-5)
0.0
0.5
1.0
1.5
2.0
FXRrelativemRNAexpression
(x10^-2)
0.0
0.5
1.0
1.5
2.0
LXRrelativemRNAexpression
(x10^-2)
Liver histopathology (H&E) 10x
BAR130 (30 mg/kg)Control
A.
B.
C.