Supplementary Materials and Methods

!Isolation of Membrane Proteins and Mass Spectrometry

For proteomic analysis, 10-week-old C57 control and db/db mice were used. Isolation of membrane proteins

from mouse liver and mass spectrometry analysis were performed as previously described.1 In brief, mouse

liver lysates were diluted with 100 mM sodium carbonate (pH 11.5) at 0°C for 30 min. The suspensions were

centrifuged at 50,000 rpm for 1 hr at 4°C. The membrane pellets were rinsed with distilled water and

dissolved in SDS for PAGE. For mass spectrometry, 20 µg of protein samples were separated by 12% SDS-

PAGE. Trypsin-digested peptides were analyzed using an ESI-7 Tesla Fourier Transform Ion Cyclotron

Resonance (FT-ICR) mass spectrometer (Thermo Scientific, MA). For protein identification, MS/MS spectra

were searched using MASCOT (version 2.2, Matrix Science, UK).

!Cyp4a shRNA-lentivirus production

For generating recombinant lentivirus, 12 individual pGIPZ-shRNA clones against Cyp4a (6 shRNA clones

for Cyp4a10, 2 shRNA clones for Cyp4a12a, 4 shRNA clones for Cyp4a12b, 5 shRNA clones for Cyp4a14)

were purchased from Invitrogen (Carlsbad, CA). HEK293T cells were maintained in 10% fetal bovine serum

(FBS) and 1% penicillin/streptomycin in Dulbecco’s modified Eagle medium (DMEM) at 37°C and 5% CO2.

24 h before transfection, 6 × 106 HEK293T cells were seeded into 100 mm culture dish. The following day,

the trans-lentiviral packaging mix encoding viral proteins Gag-Pol, Rev, and VSV-G and the purchased

lentiviral transgene plasmids were co-transfected into each well for lentivirus production using calcium

phosphate. 14 h after transfection, the DNA-reagent mixture was removed and replaced with 5% FBS in

fresh DMEM. At 48 h post-transfection, lentiviral supernatants were harvested and filtrated with 0.45 µm

filter. One volume of cold PEG-it Virus Precipitation Solution (System Biosciences, Mountain View, CA)

was added to every 4 volumes of lentiviral particle-containing supernatant. The supernatant/PEG-it mixture

was centrifuged at 1,500 x g for 30 min at 4°C and the remaining virus pellet was resuspended in 10 µl cold

DMEM. The resulting lentiviral particles were stored at - 80°C until to be used.

!Animals and Chemical and shRNA-lentivirus Treatment

Male C57BL/6J and C57BL/KsJ-db/db mice were purchased from Japan SLC. HET0016 (5 mg/kg/day,

Cayman Chemical, MI) or clofibrate (400 mg/kg/day, Sigma-Aldrich, MO) were injected intraperitoneally.

Control littermate mice for HET0016 were treated with an equivalent volume of DMSO, and controls for �1

clofibrate were treated with an equivalent volume of corn oil. For chemical treatment, 8-week-old mice were

assigned to four groups according to genotype and treatment: (1) C57+DMSO; (2) C57+HET0016; (3) db/db

+DMSO; (4) db/db+HET0016. Mice in these four groups were treated with indicated reagents for 2 weeks.

For analyzing genetic function of CYP4A, lentiviral particles carrying Cyp4a shRNAs were delivered into 8-

week-old db/db mice liver using the hydrodynamic tail vein injection and the mice were grown for 2 weeks.

For the dietary-induced diabetes model, 8-week-old C57 control mice were randomly assigned to three

groups fed normal diet (ND) (AIN-93G) or high-fat diet (HFD; 45% energy-derived calories) (FeedLab,

Korea) and treated with the following reagents for 12 weeks: (1) ND+DMSO; (2) HFD+DMSO; (3) HFD

+HET0016. The body weight of each mouse was recorded before the start of the ND/HFD regimen, every

other day throughout treatment, and at the time of sacrifice.

!Cell Culture and Chemical Treatment

HepG2 cells were cultured in low glucose DMEM (Gibco, CA) supplemented with heat-inactivated 10%

FBS (Gibco) and antibiotics. For the HET0016 study, cells were pre-treated with 4 µM HET0016 for 6 h

before replacing the culture media with media containing 5 µg/ml tunicamycin (Sigma-Aldrich), 1 µM

thapsigargin (Sigma-Aldrich), or 50 mM glucose (i.e., high glucose). For siRNA inhibition studies, cells

were transfected with pre-made siRNAs against human Cyp4a11 (1038727, Bioneer, Korea) and Cyp4a22

(171405, Bioneer), at a final concentration of 50 nM each, using Lipofectamine RNAiMax reagent

(Invitrogen, Carlsbad, CA). After 48 h of Cyp4a siRNA transfection, cells were treated with 5 µg/ml

tunicamycin, 1 µM thapsigargin, or 50 mM glucose (high glucose) for 24 h. Control cells were treated with

DMSO.

!Histopathology Analysis of Mice

At the end of treatment, glucose tolerance test (GTT) and insulin tolerance test (ITT) were carried out in

mice fasted for 6 h by intraperitoneal injection of 1 g/kg glucose or 0.75 U/kg insulin (Humulin N, Eli Lilly

and Company, IN) dissolved in PBS, respectively. Tail-blood glucose concentrations were determined using

a One Touch Ultra glucometer (LifeScan, CA) before (0 min) and 15, 30, 60, 90, and 120 min after glucose

or insulin injection. Tissue isolation for histological examination was performed as previously described.1

Isolated liver tissue was fixed in 10% neutral-buffered formalin solution (Sigma-Aldrich), and paraffin

sections were stained with hematoxylin-eosin.

!�2

Measurement of Metabolites

Blood was collected from the orbital sinus at the time of sacrifice. Serum insulin concentration was

determined using a commercial mouse insulin ELISA kit (Shibayagi, Japan). Serum adiponectin was

monitored using a mouse adiponectin EIA kit (SPI-Bio, France). Serum levels of aspartate aminotransferase

(AST), alanine aminotransferase (ALT), free fatty acid (NEFA), low-density lipoprotein (LDL), high-density

lipoprotein (HDL), total cholesterol, and triglyceride were measured using the Modulor DDP Chemistry

Analyzer (Roche). Lipid peroxidation was measured by quantifying a natural bi-product of lipid

peroxidation, malondialdehyde (MDA), in liver homogenates using the OxiSelectTMTBARS Assay Kit (Cell

Biolabs, CA). The hepatic triglyceride level was determined using the Triglyceride Quantification Kit

(Sigma-Aldrich).

!Preparation of Microsomes from Mouse Liver

Hepatic microsomes were prepared from fresh mouse liver as previously described2 with minor

modifications. Isolated liver was thoroughly perfused with ice-cold 1.15% (w/v) KCl solution. Next, the liver

was homogenized with four volumes of homogenizing buffer (0.1 M Tris-HCl, pH 7.4; 0.1 M KCl; 1 mM

EDTA, pH 7.5; 25 µM butylated hydroxytoluene). The homogenate was centrifuged at a low centrifugal

force (1,000 × g; 15 min; 4℃) to remove undisrupted cells, nuclei, and mitochondria. From the supernatant,

the microsomes were precipitated at a higher centrifugal force (100,000 × g; 60 min; 4℃). The firmly

packed pellet was resuspended in 3 ml ice-cold pyrophosphate buffer (0.1 M potassium pyrophosphate; 1

mM EDTA, pH 7.5; 20 µM butylated hydroxytoluene) using homogenizer, and then centrifuged again

(100,000 × g; 60 min; 4℃). The final washed microsome pellet was suspended in 2 ml ice-cold microsome

buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA, pH 7.5; 20% (v/v) glycerol).

!Western Blots and Real-time RT-PCR

Western blots were performed according to standard protocol as previously described.3 Blots were visualized

using a luminescent image analyzer ImageQuant LAS-4000 mini (GE Healthcare). Primary antibodies are

listed in Supplementary Table 1. RNA extraction and cDNA synthesis were performed as described

previously.1 Real-time PCR was performed with the LightCycler®480 DNA SYBR Green I Master (Roche),

and the PCR products were detected by on the LightCycler®480 Real-Time PCR System (Roche). Primer

sets are described in Supplementary Table 2.

!�3

CYP4A Enzymatic Activity Assay

The products of lauric acid by liver microsomal extract of control and db/db mice were determined by gas

chromatography/mass spectrometry (GC/MS). Metabolites were generated by the incubating 100 µM lauric

acid and 0.2 mg liver microsomal extract from control and db/db mice in 0.5 ml of 100 mM potassium

phosphate buffer (pH 7.4) for 30 min at 37℃. After incubation, metabolites were extracted using CH2Cl3,

and the organic solvent was removed under a stream of nitrogen. The residue was dissolved in N,O-

bis(trimethylsilyl)-trifluoroacetamide (BSTFA: 50 µl) containing 1% (v/v) trimethylchorosilane. The solution

was transferred to a glass vial and incubated at 75℃ for 20 min to yield trimethylsilylated products. GC/MS

analysis was carried out on a Shimadzu QP2010 (column length, 30 m; internal diameter, 0.25 mm; film

thickness, 0.1 um) with electron-impact ionization.4 The GC oven temperature was programmed for 1 min at

70℃ followed by a rise to 170℃ at 25℃/min, to 200 °C at 5℃/min, and to 280℃ at 20℃/min. The oven

was finally held at 280℃ for 5 min. The MS source and interface were maintained at 250℃ and 280℃,

respectively, and a solvent delay of 4 min was used. The products were identified by their characteristic mass

fragmentation patterns.5 The distribution of products was determined based on the relative peak areas of the

gas chromatogram.

!Hyperinsulinemic-euglycemic clamp

A hyperinsulinemic-euglycemic clamp study was performed as previously described.6 Briefly, a catheter

(silicone tubing, Helix Medical, CA, USA) was inserted into the right internal jugular vein of mice four

days before clamp. After overnight fasting, a hyperinsulinemic-euglycemic clamp was conducted with a

primed (900 pmol/kg body weight), and continuous infusion of human regular insulin (Novolin, Novo

Nordisk, Denmark) at a rate of 15 pmol/kg/min, and 20% glucose was infused to maintain constant

glucose levels. Blood samples were collected from the tail vessels, and the plasma glucose levels were

measured using a GM9 Micro-Stat Analyzer (Analox Instruments Ltd, London, UK). The basal hepatic

glucose production (HGP) and insulin-stimulated rates of the whole body glucose uptake were estimated

with a continuous infusion of [3-3H] glucose (PerkinElmer Life and Analytical Sciences, Boston, MA,

USA) for 2 h before the clamps (0.05 µCi/min) and throughout the clamps (0.1 µCi/min), respectively.

Plasma levels of [3-3H]glucose and 3H2O were determined after deproteinization of plasma samples at

80, 90, 100, 110, and 120 min of clamps. Whole body glucose uptake rate was calculated as the ratio of

the [3H] glucose infusion rate [disintegrations per minute (dpm/min)] to the specific activity of plasma

�4

glucose (dpm/mg). Insulin-stimulated HGP during the clamps was determined by subtracting the glucose

infusion rate from the whole-body glucose uptake rate.

!!Supplementary References

1. Kim GH, Park EC, Yun SH, et al. Proteomic and bioinformatic analysis of membrane proteome in type

2 diabetic mouse liver. Proteomics 2013.

2. von Jagow R, Kampffmeyer H, Kiese M. The preparation of microsomes. Naunyn Schmiedebergs

Arch Exp Pathol Pharmakol 1965;251:73-87.

3. Park EC, Cho GS, Kim GH, et al. The involvement of Eph-Ephrin signaling in tissue separation and

convergence during Xenopus gastrulation movements. Dev Biol 2011;350:441-50.

4. Gustafsson MC, Roitel O, Marshall KR, et al. Expression, purification, and characterization of

Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of

P450 BM3 from Bacillus megaterium. Biochemistry 2004;43:5474-87.

5. Lentz O, Li, Q. S., Schwaneberg, U., Lutz-Wahl, S., Fischer, P., Schmid, R. D. Modification of the

fatty acid specificity of cytochrome P450 BM-3 from Bacillus megaterium by directed evolution: a

validated assay. J Mol Catal B: Enzym 2001;15:123-133.

6. Sung HK, Kim YW, Choi SJ, et al. COMP-angiopoietin-1 enhances skeletal muscle blood flow and

insulin sensitivity in mice. Am J Physiol Endocrinol Metab 2009;297:E402-9.

�5

A

B

0

2

4

6

8

10

Cyp4

a10

Cyp4

a12

Cyp4

a14

C57db/db

Rela

tive

mRN

A le

vel

CYP4A

C57 db/db

β-actin

CYP2E1

POR

Micr

osom

e

C Dnm

ol p

rodu

ct!

/min

/mg

prot

ein

0

50

100

150

200

C57

db/db

E

0

1

2

3

4

5

6

Cyp1

7a1

Cyp2

b10

Cyp2

b13

Cyp2

b19

Cyp2

b9Cy

p4a1

2aCy

p2a2

1Cy

p3a2

5Cy

p2c3

8Cy

p4a1

0Cy

p4a1

4Cy

p2t4

Cyp2

a22

Cyp4

a12b

Cyp3

a13

Cyp2

b23

Cyp4

f15

Cyp2

a5Cy

p2c3

9Cy

p2c3

7Cy

p2j5

Cyp2

c50

Cyp2

c67

Cyp2

c29

Cyp2

a12

Cyp3

a44

Cyp2

c54

Cyp3

a41a

Cyp2

c40

POR

Cyp2

d34

Cyp2

c68

Cyp2

d26

Cyp3

a11

Cyp2

d9Cy

p2e1

Cyp2

d22

Cyp2

d10

Cyp2

f2Cy

p2a4

Cyp2

d12

Cyp4

v3Cy

p2c4

4Cy

p1a2

Cyp8

b1Cy

p4f1

4Cy

p51

Cyp2

d11

Cyp3

a16

Cyp2

c70

Cyp7

b1Cy

p27a

1Cy

p2j6

Cyp4

a30b

Cyp4

f13

db/db

C57Re

lativ

e ex

pres

sion

(db/db

/ C5

7)

C57

C57

C57

C57

db/db

db/db

db/db

db/db

Cyp4a

Actin

C57

4 5 6 7 8 9 10 4 5 6 7 8 9 10

db/dbAge

(week)

Supplementary Figure 1

Supplementary Figure 1. Expression of CYP4A. (A) The relative expression profiles (db/db / C57BL/6J) of CYP450 proteins of

10-week-old C57BL/6J and db/db mice livers. Proteins expressed exclusively in either C57BL/6J or db/db liver are indicated by

the corresponding mouse strain name. The red line indicates equivalent expression levels (i.e., a 1-fold difference) between

normal and diabetic mouse livers (B) Real-time RT-PCR of mouse Cyp4a mRNAs in the liver. (C) Western blot assay for CYP4A,

CYP2E1, and POR in microsome fractions from liver. (D) CYP4A enzymatic activity assay. (E) Expression of CYP4A during the

development of diabetes.

0

10

20

30

40

50

8 10

Body

wei

ght (

g)

9Age (week)

C57!db/db!db/db + HET0016 1 mg/kg/day!db/db + HET0016 2.5 mg/kg/day!db/db + HET0016 5 mg/kg/day

0

5

10

15

8 10

Body

wei

ght g

ain

(g)

9Age (week)

C57!db/db!db/db + HET0016 1 mg/kg/day!db/db + HET0016 2.5 mg/kg/day!db/db + HET0016 5 mg/kg/day

Supplementary Figure 2

A

B

Supplementary Figure 2. Effect of HET0016 on body weight of db/db mice. Eight-week-old db/db mice were injected with

either HET0016 (1, 2.5, or 5 mg/kg/day) or DMSO intraperitoneally for 2 weeks. (A) Body wight changes. (B) Body weight gain.

HET0016 at the dose of 5 mg/kg/day significantly suppressed the body wight gain in db/db mice.

A

β-actin

ATF6(L)

ATF6(S)

IRE1PERK

C57B

L/6J

db/db

B

ERp72

BiP

C57B

L/6J

db/db

XBP1

CHOP

UnsplicedSpliced

p-eIF2α

eIF2α

β-actin

Micr

osom

eTo

tal ly

sate

GAPDH

C10w9w8w7w

db/db

β-actinCHOP

Tota

l lysa

te

Supplementary Figure 3

Supplementary Figure 3. The unfolded protein response (UPR) in the diabetic mice liver. (A) The expression of ER stress

markers and molecular chaperones were determined by Western blotting of the liver tissues from C57BL/6J and db/db mice. ER-

localized proteins, such as ATF6, IRE1, PERK, ERp72, and BiP, were analyzed from microsomal fraction of the liver tissues. (B)

The splicing of XBP1 and the transcription of CHOP were determined by RT-PCR from the liver tissues of C57BL/6J and db/db

mice. (C) The expression of CHOP during the developmental state of type 2 diabetes.

0

1

2

3

Cyp4

a/ac

tin

p-PE

RK/P

ERK

p-eI

F2α/

eIF2α

CHO

P/ac

tin

p-JN

K/JN

K

pTyr

-IR/IR

p-Ak

t/Akt

Casp

ase-

3/ac

tin

Casp

ase-

9/ac

tin

Bax/

actin

Bcl-2

/act

in

db/dbdb/db + clofibrate

ER stress Insulin signaling Apoptosis

Rela

tive

inte

nsity

D

*

**

**

*

***

**

**

***

*

*

A

0.0

0.5

1.0

1.5

Cyp4

a/ac

tin

PERK

/act

in

p-eI

F2α/

eIF2α

CHO

P/ac

tin

p-JN

K/JN

K

0

2

4

6

8

10

pTyr

-IR/IR

p-Ak

t/Akt

0

1

2

3

4

Casp

ase-

3/ac

tin

Casp

ase-

9/ac

tin

Bax/

actin

Bcl-2

/act

in

db/dbdb/db + HET0016

Rela

tive

inte

nsity

B C

**

***

** **

*

** ** **

**

Supplementary Figure 4

Supplementary Figure 4. Effect of CYP4A inducer clofibrate on ER stress, insulin resistance and apoptosis. (A-C) Quantification

of ER stress (A), insulin signaling (B), and apoptosis (C) markers. HET0016 (5 mg/kg/day) or DMSO was administered to 8-week-old

db/db mice by intraperitoneal injection for 2 weeks. (D) Quantification of ER stress, insulin signaling, and apoptosis markers. Clofibrate

(400 mg/kg/day) or corn oil was administered to 8-week-old db/db mice by intraperitoneal injection for 2 weeks. The expression of ER

stress and apoptotic proteins and the activity of in vivo insulin signaling were determined by Western blotting of liver tissues from

indicated mice. Then, the relative intensity was measured. Data are shown as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 for db/db

vs. db/db+HET0016, db/db vs. db/db+clofibrate.

Supplementary Figure 5

0

1

2

3

4

ND HFD

HFD

+ HE

T001

6

Food

inta

ke (g

/day

)

Supplementary Figure 5. Average food intake. C57BL/6J mice were fed with either ND or HFD and injected with either DMSO or

HET0016 (5 mg/kg/day) intraperitoneally for 12 weeks.

p-JNK

JNK

β-actin

PERK

p-eIF2α

eIF2α

CHOP

High

glu

cose!

+ Di

hydr

ochl

orid

e

Low

gluc

ose

High

glu

cose

p-JNK

JNK

β-actin

PERK

p-eIF2α

eIF2α

CHOP

Low

gluc

ose

High

glu

cose

Clofibrate

High glucose!+ HET0016

A B

Supplementary Figure 6

Supplementary Figure 6. Effect of CYP4A inhibitor on ER stress and rescue by CYP4A inducer in HepG2 cells. (A) Effect of

CYP4A inhibitor dihydrochloride on ER stress. HepG2 human hepatoma cells were cultured in low glucose DMEM and pretreated with

dihydrochloride. After 6 h of CYP4A inhibitor pretreatment, the culture media was replaced with high glucose DMEM with or without

CYP4A inhibitor dihydrochloride. (B) Rescue experiment of CYP4A inhibition by using CYP4A inducer clofibrate. HepG2 cells were

cultured in low glucose DMEM and pretreated with CYP4A inhibitor HET0016 for 6 h. The culture media was replaced with high glucose

DMEM with or without indicated reagents, such as CYP4A inhibitor HET0016 and CYP4A inducer clofibrate. Cells were harvested 24

later and the expression of ER stress markers were determined by Western blotting using the indicated antibodies.

A

0

100

200

300

400

500

600

0 15 30 60 90 120

C57C57 + HET0016

Time after glucose injection (min)

Bloo

d gl

ucos

e (m

g/dl

)

0.0

0.5

1.0

1.5

2.0

2.5

C57

C57

+ HE

T001

6

Insu

lin (n

g/m

l)

B C

C57

C57

+ HE

T001

6

D

TG (μ

g/m

g tis

sue)

0

10

20

30

40

C57

C57

+ HE

T001

6

Supplementary Figure 7

Supplementary Figure 7. Effect of CYP4A inhibitor HET0016 in normal mouse liver. (A) GTT was performed by injecting 1 g/kg

glucose intraperitoneally into C57BL/6J mice treated with 5 mg/kg/day HET0016 or DMSO for 2 weeks. Tail blood glucose levels were

measured at the indicated time points. (B) Fasting serum insulin concentration were measured by enzyme-linked immunosorbent assay.

(C) Photomicrographs following paraffin section and H&E staining of the indicated mouse livers. (D) The hepatic TG level was measured

in lipid extract from the liver tissues. Data are shown as means ± SD.

Supplementary Table 1

Antibody Catalog no. Manufacturer

β-actin sc-47778 Santa Cruz Biotechnology, TXmouse CYP4A sc-98988 Santa Cruz Biotechnology, TXhuman CYP4a11 sc-101385 Santa Cruz Biotechnology, TXG6Pase sc-25840 Santa Cruz Biotechnology, TXPEPCK sc-32879 Santa Cruz Biotechnology, TXPGC-1 sc-13067 Santa Cruz Biotechnology, TXDGAT2 sc66859 Santa Cruz Biotechnology, TXFAS sc20140 Santa Cruz Biotechnology, TXLipin-1 sc-98450 Santa Cruz Biotechnology, TXSCD1 sc-14719 Santa Cruz Biotechnology, TXATF6 MG-273 Imgenex, CAhuman CYP4a22 ab98035 Abcam, UKeIF2α ab5369 Abcam, UKphospho-eIF2α Ser51 ab32157 Abcam, UKIRE1 ab37073 Abcam, UKPERK #3192 Cell Signaling Technology, MAphospho-PERK Thr980 #3179 Cell Signaling Technology, MABiP #3177 Cell Signaling Technology, MACHOP #2895 Cell Signaling Technology, MASAPK/JNK #9252 Cell Signaling Technology, MAphospho-SAPK/JNK Thr813/Tyr185 #9251 Cell Signaling Technology, MAInsulin Receptor β #3025 Cell Signaling Technology, MAphospho-Insulin Receptor β Tyr1150/1151 #3024 Cell Signaling Technology, MAAkt #4691 Cell Signaling Technology, MAphospho-Akt Ser473 #4060 Cell Signaling Technology, MABcl-2 #2876 Cell Signaling Technology, MABax #2772 Cell Signaling Technology, MAcleaved Caspase-3 #9664 Cell Signaling Technology, MAcleaved Caspase-9 #9509 Cell Signaling Technology, MAERp72 kind gift from Dr. O.Y. Kwon, College of Medicine,

Chungnam National University

Supplementary Table 1. Primary antibodies used for Western Blot.

Supplementary Table 2

Gene Primer Sequence

Cyp4a10 forward 5’-AGCCACAAGGGCAGTGTTCAGG-3’

reverse 5’-CCAAGCGGCCATTGGAAGAAAG-3’

Cyp4a12 forward 5’-GCCTTATACGGAAATATGGCA-3’

reverse 5’-TGGAATCCTGGCCAACAATC-3’

Cyp4a14 forward 5’-TGAATTGCTGCCAGATCCCACCAGGATC-3’

reverse 5’-GTTCAGTGGCTGGTCAGA-3’

XBP1 forward 5’-AAACAGAGTAGCAGCGCAGACTGC-3’

reverse 5’-GGATCTCTAAAACTAGAGGCTTGGTG-3’

CHOP forward 5’-CATACACCACCACACCTGAAAG-3’

reverse 5’-CCGTTTCCTAGTTCTTCCTTGC-3’

Supplementary Table 2. Primers used for RT-PCR.