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Macrophage inhibitory cytokine-1 aggravated diet-induced gallstone

formation via increased ABCG5, ABCG8 expressions

Min Hee Kim *, Joo-Won Park , Su-Jeong Kim , Inkeun Park , Woo-Jae Park 1
1
1,2
3
2

1 Department of Biochemistry, College of Medicine, Gachon University, Incheon, South Korea

2 Department of Biochemistry, College of Medicine, Ewha Womans University, Seoul, South Korea

3 Division of Oncology, Department of Internal Medicine, Gachon Univeristy Gil Medical Center, Incheon, South Korea

Abstract MIC-1 increased phosphorylation of AMPK.

Macrophage inhibitory cytokine-1 (MIC-1) is known to be associated with

cachexia and could be overproduced in various types of cancers. MIC-1 reduces

appetite by affecting hypothalamus, leading to reduction of body weight. However,

the effects of MIC-1 on bile acid metabolism and gallstone formation are poorly

understood. In this study, we investigate the mechanism of MIC-1 on bile acid

metabolism and gallstone formation. Male C57BL/6J mice were intraperitoneally

injected with PBS or MIC-1 (200 ug/kg/week) for 6 weeks while feeding of

standard (chow diet) or lithogenic diet. In lithogenic diet fed mice, gallstone

formation in MIC-1 injected mice is increased compared to PBS injected mice. (A) mRNA levels of SHP, FXR, and PXR in the livers of mice injected with

Hepatic cholesterol and bile acid levels are reduced upon MIC-1 injection and MIC-1 were measured by real-time PCR (n=3). (B) Representative western

SREBP-2, a master regulator of cholesterol metabolism, and HMG-CoA blots of proteins in the phosphorylation of ERK, JNK and AMPK. The values

reductase (HMGCR) expressions are reduced. Furthermore, the expressions of are expressed as means ± SEM. Three independent experiments were

Cyp7a1, Cyp27a1, and Cyp7b1, are decreased. To further explore the mechanism, performed.

we examine the expressions of small heterodimer partner (SHP), farnesoid X

receptor (FXR), pregnane X receptor (PXR) and phosphorylation of ERK, JNK.

The expressions of SHP, FXR, PXR are not affected and phosphorylation of ERK AMPK phosphorylation as a result of MIC-1

and JNK is reduced, meaning that these are not the main reason of Cyp7a1 plays a critical role in the downregulation

reduction. We find that AMPK phosphorylation is elevated upon MIC-1 treatment.

AICAR, an AMPK activator, also reduces Cyp7a1 and HMGCR expressions, while of Cyp7a1 and HMG-CoA reductase.

compound C, an AMPK inhibitor, reversed MIC-1-induced Cyp7a1 and HMGCR

reductions. Furthermore, total biliary cholesterol levels in MIC-1 injected mice are Representative western blots of (A)

increased with increased ABCG5/8 expressions. Liver X receptor α, β (LXRα, β), Hep3B cells treated with 5 or 20

LRH1, HNF4α, NR1I3(CAR) expressions, which are upstream of ABCG5/8, are ng/ml MIC-1, (B) Hep3B cells treated

not affected, but MIC-1 treatment increases ABCG5/8 expressions and promoter with 0.1 or 0.5 mM AICAR (AMPK

activities. In conclusion, MIC-1 increases AMPK phosphorylation, which affect activator) and (C) Hep3B cells co-

Cyp7a1 and HMGCR reductions, and ABCG5/8 expressions, which affects treated with 20 ng/ml MIC-1 and 10

gallstone formation. μM compound C (comp C; AMPK

inhibitor). (D) Cyp7a1 reporter

luciferase activity assay after co-

MIC-1 increased diet-induced gallstone treatment of 20 ng/ml MIC-1 and 10

formation. μM compound C (comp C; AMPK

inhibitor). The values are expressed

as means ± SEM. *p<0.05. Three

C57BL/6J mice were fed a lithogenic diet independent experiments were

for 6weeks and administered MIC-1 performed.

(200ug/kg/week) or PBS intraperitoneally.

(A) images of gallstone formation

(leftpanel) and incidence (rightpanel) MIC-1 increases biliary cholesterol secretion

(n=10). Serumcholesterol (B) and serum

bileacid (C) were measured with by overexpression of ABCG5 and ABCG8.

commercial colorimetric kits (n=3). The

values are expressed asmeans ±

standard error of the mean (SEM). (A) mRNA levels and (B)

*p<0.05, **p<0.01. Three independent representative western blots of ABCG5,

experiments were performed. ABCG8, BSEP, and MDR2 in the livers

of mice injected with MIC-1 were

measured (n=3). (C) biliary cholesterol,

(D) biliary bile acid, and (E) biliary
MIC-1 reduced cholesterol and bile acid phospholipid were measured in mice

synthesis. injected with MIC-1. The values are

expressed as means ± SEM. *p<0.05,

(A) Total hepatic **p<0.01, ***p<0.001. Three

cholesterol and (B) total independent experiments were

hepatic bile acid in mice performed.

injected with MIC-1 were

quantified with commercial

kits (n=3). (C) mRNA MIC-1 plays an important role in the

levels of SREBP-2 and

HMG-CoA reductase upregulation of ABCG5 and ABCG8.

(HMGCR) and (D) mRNA

levels of Cyp7a1, Cyp7b1, (A) mRNA levels LXRα, β, LRH1,

Cyp27a1, and Cyp8b1 in HNF4α, NR1I3(CAR) in the

the livers of mice injected livers of mice injected with MIC-1

with MIC-1 were were measured by real-time

measured by real-time PCR (n=3). (B) Representative

PCR (n=3). (E) western blots and reporter

Representative Western activities of ABCG5 and ABCG8

blots of precursor form (p) in Hep3b cells co-treated with 20

of SREBP-2, mature form ng/ml MIC-1 and 10 μM

(m) of SREBP-2, HMGCR, compound C (comp C; AMPK

Cyp7a1 and Cyp27a1.
inhibitor). The values are

Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01. Three independent expressed as means ± SEM.

experiments were performed. The values are expressed as means ± SEM. **p<0.01. Three independent

*p<0.05, **p<0.01, ***p<0.001. Three independent experiments were performed. experiments were performed.