Supporting Information in the Materials and Methods
Materials
Anti-PTP1B, anti-p-Tyr (4G10), and anti-p-IRS1 (Ser307) antibodies were purchased from
Millipore (Billerica, MA). Anti-PTP1B antibody used for the in vivo assay was purchased from
Abcam (Cambridge, MA, UK). Antibodies directed against hemagglutinin (HA), IRS1, IRS2,
IR and HNF4 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Akt
antibody, anti-GSK3 antibody, and phospho-specific antibodies directed against p-Akt
(Thr308), p-GSK3 (Ser9), and p-Thr (42H4) were supplied from Cell Signaling Technology
(Danvers, MA). TNFα was provided from R&D systems (Minneapolis, MN). Horseradish
peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgGs were purchased from Zymed
Laboratories (South San Francisco, CA). Bovine serum albumin, and anti-β-actin and antiphosphorylated serine antibodies were supplied from Sigma–Aldrich (St. Louis, MO).
Isoliquiritigenin (IsoLQ) was purchased from Sigma-Aldrich (St. Louis, MO) for in vitro assay.
ILQ used for in vivo assay and liquiritigenin (LQ) were synthesized, as previously stated, and
their chemical structures were confirmed by spectroscopic analyses.1,2
Animal treatments
Animal studies were conducted in accordance with the guidelines of the institutional animal
use and care committee at Seoul National University. The detailed protocol was presented in the
previous study.1 Male 6-week-old C57BL/6 mice were housed with a 12 h light-dark cycle and
fed either a normal diet (ND) or a high-fat diet (HFD) (fat 60% w/w; Dyets, Inc., Bethlehem,
PA) for 11 weeks. Subsequently, mice were orally administered with IsoLQ or LQ (10 or 30
mg/kg/day) dissolved in 40% polyethyleneglycol 400 (Sigma) 5 times per week during last 5
weeks. Control animals received vehicle only, as described before.1 Glucose tolerance test was
conducted 2 weeks before sacrifice. Mice were fasted overnight and then dosed orally with
glucose (2 g/kg). Tail vein blood glucose level was measured at 0, 30, 60, and 120 min after
1
administration. Liver homogenates prepared from the sacrificed animals were subjected to
immunoblottings. These samples were also used in the previous study.1 To assess the effects of
IsoLQ on blood glucose and insulin levels in mice fed a HFD for 9 weeks, a separate set of
experiments were performed. At the end of treatment, mice were fasted overnight (~12 h), and
blood samples were drawn by retro-orbital puncture. Fasting blood glucose was measured using
AccuChek Active (Roche, Germany), whereas serum insulin levels were determined using Ultra
Sensitive Mouse Insulin ELISA kit (Crystal Chem, Downers Grove, IL). HOMA-IR was
calculated as the product of fasting glucose (mmol/l) and insulin (U/ml) divided by 22.5.3 For
another set of experiments, male Lepob/ob mice were purchased from Jackson Laboratory (Bar
Harbor, ME). Lepob/ob mice were gavaged with IsoLQ (30 mg/kg, 5 times per week) for 3 weeks.
The mice were fasted for 6 h after vehicle or IsoLQ treatment at 8 AM, and then sacrificed for
blood glucose and insulin.
Immunoblot analysis
Immunoblot analysis was performed according to the previously published procedures. 4 The
cells were centrifuged at 3,000g for 3 min and allowed to swell after the addition of lysis buffer
in the ice for 30 min. The lysates were centrifuged at 10,000g for 10 min to obtain supernatants.
Proteins of interest in lysates were resolved using 6%, 7.5% or 9% gels and were transferred to
nitrocellulose membrane. The bands were developed using ECL chemiluminescence system
(GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).
qRT-PCR assays of mRNAs
Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) and was reversetranscribed. The resulting complementary DNA was amplified by quantitative real-time PCR
(qRT-PCR). qRT-PCR was performed using a Light CyclerDNA master SYBR green-I kit
(Roche, Mannheim, Germany). The following primer sequences were used: mouse PTPN1 5’-
2
CTGACACCTGCCTCTTACTG-3’ (sense) and 5’-CACTTGACTGGGCTCTGC-3’ (antisense);
human
PTPN1,
5’-GGGGTGTCGTCATGCTCAA-3’
GCCATGTGGTATAGTGGAAATGT-3’
(antisense);
(sense)
mouse
and
G6Pase
5’5’-
TCCTGGGACAGACACACAAG-3’ (sense) and 5’-CAACTTTAATATACGCTATTGG-3’
(antisense); and rat G6Pase 5’-GCAGGTAAAATCCAAGTGCGAA-3’ (sense) and 5’GCAGGTAAAATCCAAGTGCGAA-3’ (antisense).
qRT-PCR assays of microRNAs
Total RNA was extracted using Trizol (Invitrogen). The complementary DNAs were
generated using the miScript Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany)
conducted with a Light Cycle 1.5 (Roche, Mannheim, Germany). According to the
manufacturer’s instructions, 1 g total RNA, 1 l miScript Reverser Transcriptase Mix, and 4 l
miScript RT buffer were mixed and incubated for 1 h at 37℃, then at 95℃ for 5 min. qRT-PCR
was performed using the miScript SYBR Green PCR kit (Qiagen). The 20 l PCR mixture
included 2 l reverse transcription product, 10 l 2×QuantiTect SYBR Green PCR Master Mix,
2 l 10×miScript Universal Primer, 2 l primer, and 4 l Rnase-free water. The reaction
mixtures were incubated at 95℃ for 15 min, followed by 40 amplification cycles of 94℃ for
15 sec, 55℃ for 30 sec, and 70℃ for 30 sec. The transcripts of U6 small RNA were quantified
using the Hs_RNU6B_2 miScript Primer Assay (Qiagen, Hilden, Germany) to normalize
microRNA levels. The following primer sequences were used: human pri-miR-122, 5’ACCCTTTCCCTTTTCAGCAT-3’
(sense)
and
5’-GGGAGATGAGGGGAGAGAAG-3’
(antisense); and human miR-122, 5’-TGGAGTGTGACAATGGTGTTTG-3’.
Cell culture
Primary rat hepatocytes were isolated from Sprague–Dawley rats weighing ~400 g, under
3
the guidelines of the institutional animal use and care committee.5 Briefly, under anesthesia with
Zoletil, livers were perfused with Ca2+-free Hank’s buffered salt solution (Invitrogen) for 10 min,
followed by continuous perfusion with a 0.1% w/v collagenase (Sigma, Type IV). The whole
liver was removed, and then minced in the phosphate-buffered saline. Cell suspension was
filtered through the gauze, and purified with Percoll. Hepatocytes were harvested into collagencoated plates (5105 cells/well) in Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum (FBS), 50 units/ml penicillin, and 50 µg/ml streptomycin. HepG2
(human hepatoma), H4IIE (rat hepatoma), C2C12 (murine myoblast), and 3T3-L1 (mouse
preadipocyte) cell lines were purchased from American Type Culture Collection (Rockville,
MD). The cells with less than 20 passage numbers were used. The cells were maintained in the
DMEM containing 10% FBS, 50 units/ml penicillin, and 50 µg/ml streptomycin. To
differentiate C2C12 cells to myotubes, the cells were plated in 12-well plates, and the medium
was changed to DMEM supplemented with 1% FBS after the cells reached 100% confluence.
The medium was renewed every 2 days, as previously described.4 To differentiate 3T3-L1
preadipocytes to mature adipocytes, 2 day-post confluent preadipocytes were treated with 0.5
mM isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1 µg/ml insulin for 2 days. They
were then incubated in the medium containing insulin for 2 additional days and thereafter
exposed to the complete medium without insulin for 2 to 4 days.
Transfection of miR mimic or its inhibitors
Synthetic microRNA duplexes were synthesized, as previously described.6 The following
sequences were used: miR-122, 5’-UGGAGUGUGACAAUGGUGUUUGU-3’ (guide) and 5’AAACACCAUUGUCACACUCCAUA-3’ (passenger). miRIDIAN miRNA inhibitor control or
hsa-miR-122 was supplied by Dharmacon (Dharmacon, Chicago, IL). Cells were transiently
transfected with 100 nM of control mimic or miR-122 mimic, or 20 nM of miR-122 inhibitors
4
or respective negative control RNAs using FuGENE® HD Reagent (Roche, Indianapolis, IN)
according to the manufacturer’s protocol.
Transient transfection and reporter gene assays
The plasmid encoding for a dominant-negative mutant form of JNK1 (DN-JNK1) was a gift
from Dr. Dhanasekaran.7 The plasmid encoding HA-tagged JNK1 (HA-JNK1) was provided
from Dr. Gutkind.8 V5-tagged human JNK2 (V5-JNK2) was kindly provided from Dr. H.S.
Choi (Chosun University, Gwangju, Korea).9 The plasmid encoding PTP1B (pJ3H-PTP1B) was
supplied from Addgene (Addgene, Cambridge, MA). Cells were transfected with the plasmids
by using FuGENE HD (Roche, Indianapolis, IN). Briefly, cells were plated at a density of 7105
cells in 6-well plates. The cells were incubated with the plasmids of interest and Fugene reagent
for 6 h for transfection, and continuously incubated for additional 24 h in minimal essential
medium containing 1% FBS. Luciferase activity assays were performed following
manufacturer’s protocols. Briefly, HepG2 cells were seeded in 6-well plates, co-transfected with
miR-122 mimic (or its inhibitor) and 3’UTR vector. Medium was changed 12 h after
transfection, and the cells were harvested 36 h thereafter. Firefly and Renilla luciferase activities
were measured sequentially using the dual luciferase assay kit (GeneCopoeia) using POLAR
Star Omega plate reader (BMG LabTech). The activities were normalized with those of Renilla
luciferase and expressed as relative luciferase activity units. Data represent 3 independent
experiments. In pMiR-122a luciferase reporter assays, they were transiently transfected with
pMiR122a-Luc. Luciferase activity was measured by adding luciferase assay reagent (Promega,
Madison, WI).
Immunoprecipitation and immunoblot assays
To assess phosphorylated forms of IR, IRS1, IRS2 and HNF4, total cell lysates (500
µg/ml each) were incubated with anti-IR, anti-IRS1, anti-IRS2, or anti-HNF4 antibody
5
overnight at 4°C. The antigen-antibody complex was immunoprecipitated after incubation for 2
h at 4°C with protein G-agarose. Immune complexes were solubilized in 2×Laemmli buffer.
Protein samples were resolved and immunoblotted with anti-phospho-specific antibody.
Chromatin immunoprecipitation assays
Cells were transfected with HA-JNK1 for 48 h, and then formaldehyde was added to the
cells to a final concentration of 1% for cross-linking of chromatin. The chromatin
immunoprecipitation assay was performed according to the chromatin immunoprecipitation
(ChIP) assay kit protocol (Upstate Biotechnology, Lake Placid, NY). PCR was performed using
the specific primer flanking the HRE region of the miR-122 gene promoter (sense, 5’TGACCAAAGGTGGTGCTGAC-3’, and antisense, 5’-ACAGCTTCTGTGGCTTTAGGC-3’,
160 bp).
Glucose production
Glucose production assay was performed as previously described.4 HepG2 cells in 12-well
plate were serum-starved and incubated with IsoLQ, LQ or insulin for 6 h. The cells were
further incubated in 0.4 ml/well of phenol red-free, glucose-free DMEM containing 2 mM
pyruvate and 20 mM lactate in the presence of each agent. After 3 h, medium was collected and
subjected to glucose measurement by the Amplex® Red glucose/glucose oxidase assay kit
(Invitrogen). The glucose level was normalized based on protein contents.
Glucose uptake
Glucose uptake was analyzed according to methods described previously.4 After serum
deprivation, differentiated myotubes and adipocytes were incubated with IsoLQ in the presence
or absence of 20 ng/ml TNF for 6 h. After the change of the culture medium with PBS, cells
were treated with 10 nM insulin for 10 min, and glucose uptake was determined by 2-deoxy-D-
6
[1-3H]glucose incorporation into the cells for 20 min. The reaction was terminated by adding
ice-cold PBS. After washing 3 times, the cells were dissolved in 0.5 N NaOH containing 0.1%
SDS. Radioactivity was measured by a scintillation counter. Specific uptake was assessed by
subtracting nonspecific uptake, which was measured by incubating cells with 20 M
cytochalasin B.
Statistical analysis
One-way analysis of variance procedures were used to assess significant differences among
treatment groups. For each significant treatment effect, the Newman-Keuls test was utilized to
compare multiple group means. The criterion for statistical significance was set at p<0.05 or
p<0.01.
7
References
1.
Kim YM, Kim TH, Kim YW, Yang YM, Ryu DH, Hwang SJ, et al. Inhibition of liver X
receptor-α-dependent hepatic steatosis by isoliquiritigenin, a licorice antioxidant flavonoid,
as mediated by JNK1 inhibition. Free Radic Biol Med 2010;49:1722-1734.
2.
Kang HE, Jung HY, Cho YK, Kim SH, Sohn SI, Baek SR, et al. Pharmacokinetics of
liquiritigenin in mice, rats, rabbits, and dogs, and animal scale-up. J Pharm Sci
2009;98:4327-4342.
3.
Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis
model assessment: insulin resistance and beta-cell function from fasting plasma glucose
and insulin concentrations in man. Diabetologia 1985;28:412-419.
4.
Bae EJ, Yang YM, Kim JW, Kim SG. Identification of a novel class of dithiolethiones that
prevent hepatic insulin resistance via the adenosine monophosphate-activated protein
kinase-p70 ribosomal S6 kinase-1 pathway. Hepatology 2007;46:730-739.
5.
Hwahng SH, Ki SH, Bae EJ, Kim HE, Kim SG. Role of adenosine monophosphateactivated protein kinase-p70 ribosomal S6 kinase-1 pathway in repression of liver X
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dithiolethiones. Hepatology 2009;49(6):1913-1925.
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Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, et al. miR-122 regulation of lipid
metabolism revealed by in vivo antisense targeting. Cell Metab 2006;3:87-98.
7.
Wadsworth SJ, Gebauer G, van Rossum GD, Dhanasekaran N. Ras dependent signaling by
the GTPase-deficient mutant of Galpha12. J Biol Chem 1997;272:28829-28832.
8.
Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, et al. The small GTP-binding
proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell
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Choi HS, Bode AM, Shim JH, Lee SY, Dong Z. c-Jun N-terminal kinase 1 phosphorylates
Myt1 to prevent UVA-induced skin cancer. Mol Cell Biol 2009;29:2168-2180.
8
10. Cheung O, Puri P, Eicken C, Contos MJ, Mirshahi F, Maher JW, et al. Nonalcoholic
steatohepatitis is associated with altered hepatic microRNA expression. Hepatology
2008;48:1810-1820.
11. Alisi A, Da Sacco L, Bruscalupi G, Piemonte F, Panera N, De Vito R, et al. Mirnome
analysis reveals novel molecular determinants in the pathogenesis of diet-induced
nonalcoholic fatty liver disease. Lab Invest 2011;91:283-293.
12. Li S, Chen X, Zhang H, Liang X, Xiang Y, Yu C, et al. Differential expression of
microRNAs in mouse liver under aberrant energy metabolic status. J Lipid Res
2009;50:1756-1765.
9
Table S1. miRNA levels in the livers of NASH patients.
miRNA candidates
Human NASH[10]
targeting PTP1B
miR-122
down
miR-203
down
miR-135
-
miR-29
-
miR-124
-
miR-506
-
miR-206
-
miR-1
-
NASH, non-alcoholic steatohepatitis
10
Table S2. A summary of reported changes in hepatic miRNA levels in animal models.
miRNA candidates
STZ-induced diabetic
HFD-fed rats[11]
ob/ob mice[12]
mice[12]
targeting PTP1B
miR-122
down
down
down
miR-203
up
-
-
miR-135
up
-
-
miR-29
up
down
-
miR-124
-
-
-
miR-506
-
-
-
miR-206
-
-
-
miR-1
-
-
-
STZ, streptozotocin
11
Fig. S1. The effect of palmitate treatment on miRNA levels in HepG2 cells.
qRT-PCR assays for miRNAs. HepG2 cells were treated with bovine serum albumin (BSA) or
palmitate (PA, 500 M) for 3 h. Data represent the meanSE of 3 separate experiments
(significantly different as compared to BSA, *p<0.05, **p<0.01). N.D., not detected
12