Download Hepatic Carboxylesterase 1 Is Essential for Both Normal and

Hepatic Carboxylesterase 1 Is Essential for Both Normal and Farnesoid X
Receptor-Controlled Lipid Homeostasis
Jiesi Xu,* Yuanyuan Li,* Wei-Dong Chen,* Yang Xu,* Liya Yin, Xuemei Ge, Kavita
Jadhav, Luciano Adorini, and Yanqiao Zhang
*Co-equally contribute to this work
Experimental Procedures
Mice, Diets and Ligands. C57BL/6 mice, ob/ob mice, db/db mice, and Fxrmice were
purchased from the Jackson Laboratories (Bar Harbor, Maine, USA). All mice were fed
a standard chow diet. Specific FXR agonists GW4064 (1) (30 mg/kg, twice a day) and
OCA (INT-747) (2) (30 mg/kg/d) have been described previously and were administered
by gavage. Unless otherwise stated, male mice were used and all mice were fasted for
5-6 h prior to euthanization. All the animal studies have been approved by the
Institutional Animal Care and Use Committee at Northeast Ohio Medical University.
Adenovirus. Ad-Ces1-GFP was constructed by cloning mouse Ces1 cDNA into pAdshuttle-IRES-hrGFP vector (Stratagene, CA) as described previously (3, 4). To generate
adenovirus expressing small hairpin RNA against Ces1 (Ad-shCes1), oligonucleotides
were designed using BLOCK-iT™ RNAi Designer (Invitrogen, CA), annealed, and
ligated to pEnter/U6 vector (Invitrogen, CA). Adenovirus was then generated following
the instructions provided by Invitrogen. Three different shRNA oligonucleotides against
1
murine Ces1 were designed. The sequences that produced the most inhibitory effect on
endogenous
Ces1
expression
are:
5’-
GCTGATTCCAGCAGCTATTGACGAATCAATAGCTGCTGGAATCAGC-3’ (top strand)
and
5’-GCTGATTCCAGCAGCTATTGATTCGTCAATAGCTGCTGGAATCAGC-3’
(bottom strand). All the adenoviruses were grown in 293A cells and purified by cesium
chloride gradient centrifugation. About 1-2x109 plaque formation units (pfu) of
adenoviruses were transfused into each mouse intravenously. Unless otherwise stated,
7 days post infection, mice were fasted for 5-6 h and then euthanized.
Chromatin Immunoprecipitation (ChIP) Assay. ChIP assays were performed as
described previously (5).
Glucose Tolerance Test. Mice were fasted overnight, followed by injected
intraperitoneally (i.p.) with D-glucose (2g/kg). Glucose tolerance test was performed as
described previously (3).
Real-Time PCR.
RNA was isolated using TRIzol Reagent (Invitrogen, CA). mRNA
levels were determined by quantitative reverse-transcription polymerase chain reaction
(qRT-PCR) on a 7500 real-time PCR machine from Applied Biosystems (Foster City,
CA) by using SYBR Green Supermix (Roche, Indianapolis, IN). Results were calculated
using Ct values and normalized to 36B4 mRNA level.
2
Western Blot Assay. Western blot assays were performed using whole liver lysates
(3) or nuclear lysates of the liver samples (6) as described previously. CES1 antibody
was purchased from Abcam (Cambridge, MA, USA). -actin antibody was from Novus
Biologicals (CO). p-AKT(ser473) and AKT were from Millipore (Billerica, MA). SREBP-1
antibody was from Novus Biologicals (Littleton, CO). SREBP-2 antibody was from
Cayman Chemicals (Ann Arbor, MI). Histone antibody was from Cell Signaling (Beverly,
MA).
Electrophoretic Mobility Shift Assay (EMSA).
Oligonucleotides containing the
putative FXR response element (FXRE; DR-5; -218 bp) were annealed, and EMSA and
competition studies were performed as previously described (5, 7). The wild-type
oligonucleotide sequence used for EMSA was ATG TAA GAT GTT CCT TGG TTA GTT
TAT GGA CCT CTG TTA TCT GAG AGC TGT CCA ATG G (top strand). The mutant
oligonucleotide sequence was ATG TAA GAT GTT CCT TAA TTA GTT TAT GCA AAT
CTG TTA TCT GAG AGC TGT CCA ATG G (top strand) (mutation sites are underlined).
Mutagenesis and Transient Transfection Assays.
The mutant pGL3 promoter-
luciferase construct was generated using a QuickChange Site-directed Mutagenesis kit
from Agilent (Santa Clara, CA). HepG2 cells were plated in a 24-well plate and cultured
in DMEM containing 10% FBS. Transient transfections were performed in triplicate as
described (5). Briefly, pGL3-Ces1 luciferase reporter constructs were transfected into
HepG2 cells together with plasmids expressing FXR or RXR, followed by treatment with
either vehicle or GW4064 (1 M). After 36 h, luciferase activities were determined and
3
normalized to -galactosidase activity. To determine the effect of over-expression of
CES1 on PPAR activity, a CES1-expression plasmid was co-transfected with the
3xPPRE-luc plasmid, and luciferase activity was determined as described above.
Lipid and Lipoprotein Analysis. Approximately 100 mg liver was homogenized in
methanol and lipids were extracted in chloroform/methanol (2:1 v/v) as described (8).
Hepatic triglyceride and cholesterol levels were then quantified using Infinity reagents
from Thermo Scientific (Waltham, MA). Hepatic fatty acid profile was quantified using
gas chromatography (GC)-mass spectrometry at the Mouse Metabolic Phenotyping
Center (MMPC) of Case Western Reserve University (Cleveland, OH). Hepatic total free
fatty acids and free cholesterol were quantified using kits from BioVision (Milpitas, CA).
Plasma lipid and glucose levels were also determined using Infinity reagents. Plasma
lipoprotein profile was analyzed by FPLC as described (4). Briefly, after 100 l plasma
was injected, lipoproteins were run at 0.5 ml/min in a buffer containing 0.15 M NaCl,
0.01 M Na2HPO4, 0.1 mM EDTA, pH 7.5, and separated on a Superose 6 10/300 GL
column (GE Healthcare) by using BioLogic DuoFlow QuadTec 10 System (Bio-Rad,
CA). 500 l of sample per fraction was collected.
VLDL Secretion.
C57BL/6J mice were injected intravenously with specific
adenoviruses. On day 6, these mice were fasted overnight, followed by intravenous
injection of Tyloxapol (500 mg/kg). Blood was taken at indicated time points and plasma
TG levels determined. VLDL secretion rate was determined as described (4).
4
Hepatic Lipogenesis. Mice were fasted for 4 h and then injected intraperitoneally with
2H O
2
(20-30 l/g). After 4 h, liver and plasma were snap-frozen in liquid nitrogen. The
newly synthesized palmitate, triglycerides, and cholesterol were measured by mass
spectrometry at MMPC of Case Western Reserve University.
TGH Activity Assay. Cells were lysed in lysis buffer containing 50mM Tris, pH 7.4,
0.25M Sucrose, and 1mM EDTA, 1 mM dithiothreitol, 50 mM NaF and protease inhibitor
cocktail (Sigma). After spin at 14,000 x g, the supernatant was used for TGH activity
assay. For animals, liver was harvested after a 6-h fast. Liver was homogenized in lysis
buffer and then spun at 800 x g to remove cell debris. The supernatant was then
centrifuged at 100,000 × g for 1 h. The cytosolic portion and microsome portion were
used for TGH assay separately.
In brief, 100 l liver extracts (100 μg of protein) were incubated at 37 oC with 100
l substrates containing 0.15 mM cold triolein, 0.32 M [3H]triolein, 10 M egg yolk
lecithin, 100 M sodium taurocholate, 1 mM dithiothreitol, and 50 mM potassium
phosphate
(pH
7.4).
After
1
h,
the
reaction
was
stopped
by
3.75
ml
methanol:chloroform:heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate/0.1M
boric acid. After centrifuge at 800 x g, 1 ml top phase is used for counting radioactivity
using a liquid scintillation counter.
Fatty Acid Oxidation. AML12 cells were cultured in DMEM containing 10% FBS in 12well dishes and infected with either Ad-GFP or Ad-Ces1. After 48 h, the media were
removed and washed with 1XPBS. The cells were then cultured in DMEM containing
5
0.5% fatty acid-free BSA, 0.5 Ci [3H]palmitate and 500 M cold palmitate. Fatty acid
oxidation was performed as described (9). Briefly, after incubation for 3 h, the
supernatant was collected. 0.1 ml supernatant was added to a round-bottomed
Eppendorf tube containing 0.9 ml of charcoal slurry. The samples were left at room
temperature for 30 min with intermittent shaking (at least once every 5 minutes), and
then centrifuged at 13,000 rpm for 15 minutes. 0.2 ml of the supernatant was carefully
taken out and added to a scintillation vial containing 2.8 ml of scintillation liquid and the
radioactivity was determined on a liquid scintillation counter.
Chromatin Immunoprecipitation (ChIP) Assay.
described previously (5).
6
ChIP assays were performed as
References
1.
Maloney PR, Parks DJ, Haffner CD, Fivush AM, Chandra G, Plunket KD, Creech
KL, et al. Identification of a chemical tool for the orphan nuclear receptor FXR. Journal
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Pellicciari R, Fiorucci S, Camaioni E, Clerici C, Costantino G, Maloney PR,
Morelli A, et al. 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective
FXR agonist endowed with anticholestatic activity. J Med Chem 2002;45:3569-3572.
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Zhang Y, Lee FY, Barrera G, Lee H, Vales C, Gonzalez FJ, Willson TM, et al.
Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in
diabetic mice. Proc Natl Acad Sci U S A 2006;103:1006-1011.
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Yin L, Ma H, Ge X, Edwards PA, Zhang Y. Hepatic hepatocyte nuclear factor
4alpha is essential for maintaining triglyceride and cholesterol homeostasis. Arterioscler
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5.
Ge X, Yin L, Ma H, Li T, Chiang JY, Zhang Y. Aldo-keto reductase 1B7 is a target
gene of FXR and regulates lipid and glucose homeostasis. J Lipid Res 2011;52:15611568.
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Zhang Y, Yin L, Hillgartner FB. SREBP-1 integrates the actions of thyroid
hormone, insulin, cAMP, and medium-chain fatty acids on ACCalpha transcription in
hepatocytes. J Lipid Res 2003;44:356-368.
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Lee H, Zhang Y, Lee FY, Nelson SF, Gonzalez FJ, Edwards PA. FXR regulates
organic solute transporters alpha and beta in the adrenal gland, kidney, and intestine. J
Lipid Res 2006;47:201-214.
8.
Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J
Biochem Physiol 1959;37:911-917.
9.
Rune A, Osler ME, Fritz T, Zierath JR. Regulation of skeletal muscle sucrose,
non-fermenting 1/AMP-activated protein kinase-related kinase (SNARK) by metabolic
stress and diabetes. Diabetologia 2009;52:2182-2189.
7
Figure legends
Figure S1. Hepatic CES1 is regulated by fasting/refeeding. C57BL/6 mice were
fasted for 8 or 24 h, or fasted for 24 h and then refed for 24 h. Hepatic mRNA levels of
glucose-6-phosphotase (G6pase) and Ces1 were determined by qRT-PCR. * p<0.05, **
p<0.01
Figure S2. Hepatic expression of CES1 does not affect plasma triglyceride or
cholesterol levels. (A-C) C57BL/6 mice were i.v. injected with adenovirus expressing
GFP or Ces1 (n=8 mice per group). After 7 days, plasma total cholesterol (TC) and
triglyceride levels were determined (A). Hepatic mRNA levels were quantified by qRTPCR (B, C). SR-BI, scavenger receptor group B, type I. Abcg5, ATP-binding cassette
transporter G5. Elovl6, very long chain fatty acid elogase 6. Adrp (perilipin 2), adipose
differentiation-related protein. Acox1, acyl-CoA oxidase 1. Mcad, median-chain acylCoA dehydrogenase. Vlcad, very long-chain acyl-CoA dehydrogenase. Fabp5, fatty acid
binding protein 5. Acl, ATP citrate lyase. Cyp4a14, cytochrome P450, family 4,
subfamily a14. Scd1, Stearoyl-CoA desaturase-1.
Figure S3. Over-expression of CES1 tends to increase plasma -hydroxybutyrate
levels. C57BL/6 mice were i.v. injected with adenovirus expressing GFP or Ces1. After
7 days, plasma b-hydroxybutyrate was quantified (n=8 per group).
8
Figure S4. Effects of shCes1 over-expression on CES1 mRNA level. AML12 cells
were infected with Ad-Ces1, followed by infection with either Ad-shLacZ (control) or AdshCes1 for 48 h (n=2 per group). Hepatic mRNA levels were quantified by qRT-PCR.
The third Ad-shCes1 (Ad-shCes1-c) had the most inhibitory effect on Ces1 expression
and was selected for further studies.
Figure S5. Effects of hepatic CES1 knockdown on gene expression.
(A, B)
C57BL/6 mice were i.v. injected with either Ad-shLacZ or Ad-shCes1. After 7 days,
hepatic mRNA levels were quantified by qRT-PCR (n=8 per group). Lrp, LDLR-related
protein. Soat1, acyl-Coenzyme A: cholesterol acyltransferase 1. Lcat, Lecithincholesterol acyltransferase. Cyp7a1, cholesterol 7 alpha-hydroxylase. Bsep, bile salt
export protein. L-FABP, liver-type fatty acid-binding protein. Angptl3, angiopoietin-like 3.
HL, hepatic lipase. Hsl, hormone sensitive lipase.
Figure S6. Effects of knock down of hepatic CES1 on hepatic levels of free
cholesterol and FFA and VLDL secretion. C57BL/6 mice were i.v. injected with either
Ad-shLacZ or Ad-shCes1 (n=8 mice per group). After 7 days, hepatic free cholesterol
(A) and free fatty acid (B) levels were quantified using kits from BioVision. Hepatic
protein levels were analyzed by Western blot assays (C) and VLDL secretion
determined (n=8 mice per group).
Figure S7. Effects of the FXR agonist OCA (INT-747) on lipid and glucose
homeostasis in C57BL/6 mice. C57BL/6 mice were injected i.v. with either Ad-shLacZ
9
or Ad-shCes1. On the next day, these mice were gavaged with either vehicle or OCA
(n=7-9 mice per group). After 7 days, mice were fasted for 5 h. Plasma glucose levels
(A) and hepatic cholesterol levels (D) were measured. Plasma cholesterol (B) and
triglyceride (C) lipoprotein profiles were determined by FPLC.
Figure S8. Effects of the FXR agonist OCA on lipid homeostasis in ob/ob mice.
Ob/ob mice were injected i.v. with either Ad-shLacZ or Ad-shCes1. On the next day,
these mice were gavaged with either vehicle or OCA (n=6 per group). After 7 days, mice
were fasted for 5 h. Plasma total cholesterol (TC) (A), plasma triglycerides (TG) (B),
hepatic TC (C) and hepatic TG (D) levels were determined. Hepatic mRNA levels of
Ces1 (E) and Shp (F) were quantified by qRT-PCR. * p<0.05, ** p<0.01. NS, nonspecific.
10
Figure S1
Relative mRNA
4
3
**
**
2
1
0
Fed
Fasted, 8 h
Fasted, 24 h
Refed, 24 h
**
G6pase
Ces1
11
C
2
B
Ad-GFP
Ad-Ces1
TG
14
12
10
8
6
4
2
0
Relative mRNA
120
100
80
60
40
20
0
Relative mRNA
A
Plasma lipids (mg/dL)
Figure S2
**
Ad-GFP
Ad-Ces1
TC
Ad-GFP
Ad-Ces1
1
*
0
12
*
-hydroxybutyrate
(M)
Figure S3
800
p=0.07
600
400
200
0
Ad-GFP
Ad-Ces1
13
Figure S4
Relative mRNA
1.5
1
*
*
0.5
0
**
shLacZ
shCes1-a shCes1-b shCes1-c
14
Figure S5
Relative mRNA
A
Relative mRNA
B3
2
Ad-shLacZ
Ad-shCes1
1
0
Ad-shLacZ
Ad-shCes1
2
1
0
15
Figure S6
B
11
*
10
9
8
Ad-shLacZ
5
4
3
2
1
0
Ad-shCes1
D
C
6
FFA (nmol/mg)
12
Free cholesterol
(g/mg)
A
shLacZ shCes1
Mtp
ApoB100
ApoB48
-actin
16
Ad-shLacZ
Ad-shCes1
Figure S7
A
B
C
D
17
Figure S8
A
B
C
D
E
F
18