εSupplementary Figure 1
Role of Elovl6 in mammalian fatty acid synthesis and hepatic insulin
sensitivity.
(a) Schematic representation of the pathways controlling long chain fatty acidsynthesis.

Elovl6 has a specific activity for the elongation of C12–16 saturated-and monounsaturated- fatty acids.

During fatty acid synthesis, palmitate (16:0),

produced by fatty acid synthase (FAS) in the cytosol, is transferred to
endoplasmic reticulum (ER) membranes, where Elovl6 and stearoyl-CoA
desaturase (SCD) are sequentially involved to produce the main final product,
18:1.

(b) Proposed molecular basis for Elovl6 deficiency-mediated metabolic

effects in mice.

Elovl6 deficiency altered hepatic fatty acid composition; thedecreased levels of 18:0 and 18:1, and increased levels of 16:0 and 16:1. Thechanges of a fatty cid chain length (decrease C18 fatty acids) and ratio of fatty
acids (increase 16:1/16:0 ratio) could reduce SREBP-1 and PPARα in the liver.
The reduction of SREBP-1 leads to increase IRS-2 levels and insulin sensitivity,and decrease fatty acid synthesis by the reduction of lipogenic gene expression.
The reduction of lipogenesis could lead to decreased hepatic diacylglycerol
contents, which leads to decrease PKCε activity and increase insulin sensitivity.
The reduction of de novo lipogenesis could also reduce PPARα activity andexpression.

The reduction of PPARα leads to decrease lipid oxidation by

reduction of gene expression of fatty acid oxidation enzymes.

Elovl6 deficiency

suppressed both synthesis and degradation of fatty acids, resulting in slightly
increased triglycerides in the liver.

Taken together, Elovl6 deficiency improves

hepatic insulin sensitivity without amelioration of obesity and hepatosteatosis.

g
30

20

10

0

30
25
20
15
10
5
0
12:0
14:0
16:0
16:1n-7
18:0
18:1n-9
18:2n-6
18:3n-6
18:3n-3
20:0
20:1n-9
20:2n-6
20:3n-9
20:3n-6
20:4n-6
20:5n-3
22:0
22:1n-9
22:4n-6
22:5n-3
24:0
22:6n-3
24:1n-9

0

12:0
14:0
16:0
16:1n-7
18:0
18:1n-9
18:2n-6
18:3n-6
18:3n-3
20:0
20:1n-9
20:2n-6
20:3n-9
20:3n-6
20:4n-6
20:5n-3
22:0
22:1n-9
22:4n-6
22:5n-3
24:0
22:6n-3
24:1n-9

LTR

40

30

SA GEO pA10

ND

PGK puro

E
Probe
E

Exon 1,2 neo

*

20

* *

40

*
*

E

SD
LTR

AAAAn

B

1.5
1
0.5
0
2
20 0:0:1
20 n-9:3
20 n-9:3n6
22
2 2 :0:1
22 n-9:5n3
24
2 4 :0:1
n9

Exon 2
A

12:0
14:0
16:0
16:1n-7
18:0
18:1n-9
18:2n-6
18:3n-6
18:3n-3
20:0
20:1n-9
20:2n-6
20:3n-9
20:3n-6
20:4n-6
20:5n-3
22:0
22:1n-9
22:4n-6
22:5n-3
24:0
22:6n-3
24:1n-9f
Mol % of total fatty acidse

Mol % of total fatty acidsa

Mol % of total fatty acids

Supplementary Figure 2
Exon 3LTR2d*b WT (10.5 kb)
cpuro Exon 3,4,5

*
ND

ND

*

*

+/+

Mut ( 7.4 kb)

E

+/–
–/–

+/+ +/––/–AAAAn

WT (A x B)

Mut (LTR2 x B)

Number of genotype

MatingNumber of litters
+/+
+/–
–/–

+/– M x +/– F
85
158
303
50

–/– M x +/– F
52
NA
273
127

*
Wild-type
Elovl6–/–***
Wild-type
Elovl6–/–*

Wild-type
Elovl6–/–*

Supplementary Figure 2
Generation of Elovl6–/– mice.
(a) Schematic representation of the Elovl6 locus after insertion of the retroviralgene trap.

Expression of the trapped gene is disrupted because the inserted

sequence causes the endogenous transcript to be divided into two separated
transcripts, neither of which can produce the endogenous protein.

LTR, longterminal repeat; SA, splice acceptor sequence; neo, neomycin resistancecassette; pA, polyadenylation sequence; PGK, phosphoglycerate kinase-1
promoter; puro, puromycin resistance cassette; SD, splice donor sequence; E,
EcoRI.

Primers used for genotyping by PCR are primer A, primer B, and primer

LTR2.

The location of the probe used for Southern blot analysis is denoted by

the horizontal filled rectangle labeled “probe”. (b) Southern blot analysis of tailDNA from wild-type (+/+), heterozygous (+/–), and homozygous (–/–) mice.
Genomic DNA was digested with EcoRI and detected using probe to yield the
expected fragments of 10.5 kb (wild-type allele) and 7.4 kb (mutated allele).
PCR analysis of genomic DNA prepared from mouse tail.
survival chart.
from tail.

(c)(d) Elovl6–/– mouse

Genotype was determined by PCR analysis of DNA preparedM, male; F, female; NA, not applicable.

(e–g) Fatty acid composition(mol % of total) of plasma (e), white adipose tissue (f), and skeletal muscle (g) inwild-type and Elovl6–/– mice.

Total lipids were extracted from livers of mice fed

a normal chow diet, and lipid fractions were quantified by gas chromatography.
Values are means ± SEM for n = 3–5 mice. *, p < 0.05 as compared with theirrespective wild-type.

ND, not detectable.

Supplementary Figure 3

Wild-type
Elovl6–/–100

Wild-type
Elovl6–/–

500
400
300
200
100
0

0

15 30 60 120

2.5
2
1.5
1
0.5
0

Wild-type
Elovl6–/–

600
500
400
300
200
100
0

15 30 60 120
Time (min)10*Fasted

6
5
4
3
2
1
0

300
250
200
150
100
50
0

Fasted

100
80
60

*

40
20

** * ** ** **15 30 60 120
Time (min)

**

*
*
**

0 15 30 60 90 120

Time (min)Wild-type
Elovl6–/–Fed

Wild-type
Elovl6–/–0Fed
Wild-type
Elovl6–/–

Wild-type
Elovl6–/–

Wild-type
Elovl6–/–

0

2

d

*
0 15 30 60 120

fInsulin (ng/ml)

Glucose (mg/dl)

g**Time (min)Glucose (mg/dl)

Body weight (g)

50
40
30
20
10
0

Wild-type
Elovl6–/–3Fed

Wild-type
Elovl6–/–Time (min)eFasted4

Insulin (ng/ml)

20*

Insulin (ng/ml)

30

250
200
150
100
50
0

Glucose (%basal)*

40

Insulin (ng/ml)

Glucose (mg/dl)

c

b

10
8
6
4
2
0

*

*
FastedhGlucose (%basal)

Body weight (g)Wild-type
Elovl6–/–

Glucose (mg/dl)aFedWild-type
Elovl6–/–

110
90
70
50
30

* **
0

*

*

15 30 60 90 120

Time (min)Supplementary Figure 3
Effects of the aging and body weight differences on the insulin sensitivityof Elovl6–/– mice.
(a–d) Protection from age-associated insulin resistance in chow-fed Elovl6–/–
mice.

(a) Body weight of 6–8 month old wild-type and Elovl6–/– mice fed a chowdiet.

(b) Plasma glucose (left) and insulin (right) concentrations in 6–8 month

old wild-type and Elovl6–/– mice on fasted or fed state.

(c) Plasma glucose (left)

and insulin (right) levels during an intraperitoneal glucose tolerance test. (d)Insulin tolerance tests in 6–8 month old wild-type and Elovl6–/– mice (0.5 U/kg ofbody weight).

(e–h) Protection from insulin resistance in HF-HS–fed Elovl6–/–

mice were not caused by the body weight difference.

Six-week-old wild-type

and Elovl6–/– mice were fed HF-HS diet for 8-12 weeks, and bodyweight-matched wild-type and Elovl6–/– mice were used for each experiment.
(e) Body weight of HF-HS–fed wild-type and Elovl6–/– mice used in thisexperiments.
state.

(f) Plasma glucose (left) and insulin (right) levels in fasted or fed(g) Plasma glucose (left) and insulin (right) levels during an

intraperitoneal glucose tolerance test.

(h) Insulin tolerance tests in bodyweight-matched wild-type and Elovl6–/– mice fed HF-HS diet (1.0 U/kg of bodyweight).

n = 8–10 per group in a–d and n = 10–12 per group in e–h.

0.05; **, p < 0.01 as compared with their respective wild-type.

*, p <

Supplementary Figure 4

a

Chow

– ++ – ++ – ++ – ++Chow HF-HS+/+ –/– +/+ –/–pAkt(Ser473)

pJNK

Akt

WAT Akt phosphorylation(multiple of basal chow +/+)

Genotype

Wild-type chow
Elovl6–/– chow
Wild-type HF-HS
Elovl6–/– HF-HS(Thr183/Tyr185)

+/+

–/–

+/+

–/–
Wild-type
Elovl6–/–

12
10

JNK
pIKKa(Ser180)/IKKb(Ser181)

8

IKKb

6

pSTAT-3

4(Tyr705)

20

JNK phosphorylationration(multiple of chow-fed WT)

Insulinb

HF-HS

STAT-3
–

+
Chow

c

–

+

Insulin

HF-HS

d

+/
+
–/
–
+/
+
–/
–

Chow HF-HS

Fads2

Chow
Insulin

Fads1

Foxo1

Elovl2Foxa2

Elovl5LaminA/C

Socs3

Genotype

Cish

6
5
4
3
2
1
0

–

+

+/+

–

HF-HS
+

–/–

–

+

+/+

–

+

–/–

Supplementary Figure 4
Effects of HF-HS diet on insulin signaling and inflammation in the white
adipose tissue (WAT) and livers of wild-type and Elovl6–/– mice.
(a) Immunoblot analysis (top) and densitometric quantification (bottom) of
Ser473-phosphoylated Akt (pAkt) and total Akt in response to a bolus injection of
insulin in WAT.

The blot is representative of three independent experiments.

Data represent means ± SEM (n = 6 per group).

(b–d) Examination of protein

and mRNA levels in the livers of wild-type and Elovl6–/– mice fed a standard
chow or HF-HS diet.

(b) Protein levels of inflammatory and cytokine signaling

molecules were examined by immunoblotting in livers of wild-type and Elovl6–/–
mice fed a chow or HF-HS diet for 12 weeks (left).

Densitometric quantificationof the JNK was also shown (n = 8–11 per group, right).

(c) Northern blot

analysis of various mRNA levels in the liver of wild-type and Elovl6–/– mice fed astandard chow or HF-HS diet for 12 weeks. Fads2, Δ6 desaturase; Fads1, Δ5
desturase; Socs3, suppressor of cytokine signaling 3; Cish, cytokine inducible
SH2-containing protein.

(d) Immunoblot analysis of nuclear abundance of

Foxo1 and Foxa2 in the livers of wild-type and Elovl6–/– mice fed on a standard
chow or HF-HS diet.

Mice in 24 h fasted state were injected intraperitoneallywith PBS or human regular insulin (1.0 U/kg of body weight). After 60 min,livers were rapidly excised and nuclear extracts were prepared.

Supplementary Figure 5bChow HF-HS

Chow HF-HS

Pparg

+/
+
–/
–
+/
+
–/
–

Ucp1

Elovl6

Ucp2

Slc2a4

Ucp3

Fasn

Pparg

Cfd
Scd1

Slc2a4

Adfp

Ppara

Insr

Insr

Lep

Ppargc1a

Irs1

Irs1

Adipoq

Cpt1b

Irs2

Irs2

Retn

Acox1

Rplp0

Irs3

Rplp0

d

Chow HF-HS

+/

+/
+
–/
–
+/
+
–/
–

+
–/
–
+/
+
–/
–

Chow HF-HS

Acox1

Elovl6

Ucp2
Srebf1

Ucp3

Ppara

Irs1

Ppard

Irs2

Ppargc1a

Slc2a42-deoxyglucose uptale

Scd1

Insulin –Insulin +

+/+

+/+

–/–
+

Palmitate1
0.8

Insulin –Insulin +

0.6
0.4
0.2
0

GFP Elovl6 GFP Elovl6 Adenovirus
Elovl6–/–

Wild-type+

Liver TC (mg/g)

*

30
20
10

1
8:

0
8:

+1

+1

6:

0

0
+1

Liver TG (mg/g)

40

Genotype
Palmitate

Wild-type
Elovl6–/–

O

0

8:
1

8:

+1

0

0
6:

8:
0
+1
8:
1

+1

:0

+1
6

O

0

1

+1

50

* * *

2

+1

100

3

O

150

4

CH

Insulin (ng/ml)

200

Wild-type
Elovl6–/–CH

Wild-type
Elovl6–/–

CH

Glucose (mg/dl)eGenotype

1.2

Rplp0

Cpt1b

–/–
–

Insr

(multiple of basal +/+)

Fasn

** **

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Wild-type
Elovl6–/–

6
5
4
3
2
1
0

*

O

c

CH

Acox1

2-deoxyglucose uptale

Cpt1b(multiple of basal +/+)

Ucp2

8:
0
+1
8:
1

Scd1

0

Adrb3

Fasn

6:

Srebf1

Srebf1+1

Elovl6

Chow HF-HS

+1

+/
+
–/
–
+/
+
–/
–

+/
+
–/
–
+/
+
–/
–

Chow HF-HS

+/
+
–/
–
+/
+
–/
–

a

Supplementary Figure 5
Effects of Elovl6 deficiency on mRNA levels in the white adipose tissue(WAT),

brown

adiposetissue(BAT)

andskeletalmuscle,and2-deoxyglucose uptake in the skeletal muscle cells, and effects of dietary
fatty acid supplementation on plasma and liver metabolic parameters inwild-type and Elovl6–/– mice.
(a–c) Northern blot analysis of various mRNA levels in WAT (a), BAT (b) andskeletal muscle (c) from wild-type and Elovl6–/– mice fed on a standard chow dietor HF-HS diet for 12 weeks.

Cpt1b, carnitine palmitoyltransferase 1b, muscle;Insr, insulin receptor; Pparg, peroxisome proliferator-activated receptor gamma;
Slc2a4, glucose transporter 4; Adrb3, b3-adorenergic receptor; Cfd, adipsin;
Adfp, adipose differentiation-related protein; Lep, leptin; Adipoq, adiponectin;
Retn, resistin; Ppargc1a, peroxisome proliferator-activated receptor gamma
coactivator-1a; Ppard, peroxisome proliferator-activated receptor delta.

(d)Basal and insulin-stimulated 2-deoxyglucose uptake was assayed in myotubes
prepared from wild-type and Elovl6–/– mice following incubation in the absenceor presence of palmitate (0.5 mM) for 16 h (top), and infection with adenovirus
expressing GFP or Elovl6 (MOI 100) with palmitate (0.5 mM) for 16 h (bottom),followed by incubation in the absence or presence of insulin (100 nM) for 15 min.
Values are represented as means ± SEM (n = 6 per group).
compared with their respective wild-type controls.

**, p < 0.01 as(e) Effects of tripalmitin(16:0), tristearin (18:0), or triolein (18:1) supplementation on plasma glucose,
plasma insulin, liver triglyceride and liver total cholesterol levels in wild-type andElovl6–/– mice.

Mice were fed high carbohydrate fat-free diet (CHO), tripalmitin,tristearin, or triolein-supplemented diets (20% by weight) for 2 weeks.
per group.

*, p < 0.05 as compared with their respective wild-type.

n = 8–10

Crucial role of long chain fatty acid elongase (Elovl6)
in obesity-induced insulin resistanceTakashi Matsuzaka1,2 , Hitoshi Shimano1,2,*, Naoya Yahagi2,3, Toyonori Kato1, Ayaka
Atsumi1, Takashi Yamamoto1, Noriyuki Inoue1, Mayumi Ishikawa1, Sumiyo Okada1,
Naomi Ishigaki1, Hitoshi Iwasaki1, Yuko Iwasaki1, Tadayoshi Karasawa1, Shin
Kumadaki1, Toshiyuki Matsui1, Motohiro Sekiya3, Ken Ohashi3, Alyssa H Hasty4,
Yoshimi Nakagawa1,2, Akimitsu Takahashi1, Hiroaki Suzuki1, Sigeru Yatoh1, Hirohito
Sone1, Hideo Toyoshima1, Jun-ichi Osuga3, and Nobuhiro Yamada1

Supplementary Methods

Materials.

We purchased antibodies to IRβ, phospho-IRβ (Tyr972), IRS-1, IRS-2,

and phosphotyrosine 4G10 from Upstate Biotechnology, and antibodies to SREBP-1,
Foxa2, PKCε, and PKCβΙΙ from Santa Cruz Biotechnology.

We obtained antibodies

to Akt, phospho-Akt (Ser473), Foxo1, AMPK, phospo-AMPKα (Ser485), JNK,
Phospho-JNK (Thr183/Tyr185), IKK, phospho-IKK, STAT-3, phospho-STAT-3(Tyr705),

PKCα,

and

PKCδ

from

Cell

Signaling

Technology.

2-Deoxy-D-[1-14C]glucose and [1-3H]mannitol were purchased from PerkinElmer Life
Sciences.

Tripalmitin, tristearin, and triolein were purchased from Wako Pure

Chemicals.

Other chemical compounds were obtained from Sigma.

PCR genotyping.

Routine genotyping was performed on tail DNA by PCR.

We

used a single antisense primer (primer A: 5’- GGATTCCCCACCTATTTCCTTCAG-3’ ) corresponding to intron sequence downstream of the insertion site and two sense
primers, one corresponding to intron sequence upstream of the insertion (primer B: 5’AACCCTTTCCTCCCCAACTTGCTC-3’) and the other within the 3’ end of the
Lexicon insertion (LTR2: 5’- AAATGGCGTTACTTAAGCTAGCTTGC -3’ ). The
primer pair A/B amplifies a fragment of 300 bp, and primer pair LTR2/B amplifies afragment of 173 bp.

Generation of ob/ob-Elovl6–/– Mice.

Mice deficient in both Elovl6 and Lep wereobtained by breeding Elovl6–/– mice to C57BL/6 ob/+ mice (The Jackson Laboratory,
Bar Harbor, Maine).

To generate ob/ob mice lacking the Elovl6 gene, Elovl6–/– males

were first bred to ob/+ females to create compound heterozygous (ob/+-Elovl6+/–). In asecond cross, compound heterozygous were bred with Elovl6–/– mice, and ob/+-Elovl6–/–
offspring were identified. In the third set of crosses, ob/+-Elovl6–/– mice were bred toeach other to produce ob/ob-Elovl6–/– animals.

In parallel, ob/+ mice were bred to eachother to produce ob/ob as well as wild-type animals. The Lep gene genotyping wasdetermined as described previously51.

Assays.

Plasma concentrations of IL-6 and TNF-α were assayed by enzyme-linked

immunosorbent assay kit (PIERCE).

Histology.

Tissue specimens were fixed in 4% paraformaldehyde, and embedded inparaffin. Thin sections were subjected to standard hematoxylin and eosin staining.

Primers for quantitative RT-PCR.

The primer sets were as follows; Elovl6 forward,

5’-CCCGAACTAGGTGACACGAT-3’, Elovl6 reverse, 5’-CCAGCGACCATGTCTTTGTA-3’; Srebf1 forward, 5’- CGGCGCGGAAGCTGT-3’, Srebf1 reverse, 5’-TGCA
ATCCATGGCTCCGT-3’; Ppara forward, 5’-CCTCAGGGTACCACTACGGAGT-3’,
Ppara reverse, 5’- GCCGAATAGTTCGCCGAA-3’, Ppia forward, 5’-CCTGAAGTG
CTCGACATCACA-3’, Ppia reverse, 5’- GCGCTTGTACCCATTGATGA-3’.

Primary cultures from muscle cells and measurement of 2-deoxyglucose uptake.
Primary myotube cultures were prepared by isolating satellite cells from hind-limb
muscles of 8 week-old wild-type and Elovl6–/– mice, and cultured as previously
described52, with some modifications.

Briefly, myoblasts were proliferated for 5-6

days in Dulbecco's minimum essential medium (DMEM) supplemented with 20% fetal
bovine serum (FBS) and antibiotics.

For myotube culture, myoblasts were cultured for

6-7 days in DMEM with 2.5% FBS and antibiotics.

Specific 2-deoxyglucose (2DG)

uptake was determined by subtracing the non-specific uptake of mannitol from the total
uptake of 2DG as described previously53, with some modifications. The cells wereincubated for 15 min in Krebs-Ringer phosphate buffer containing 1 mM 2DG, 0.1

µCi/ml [1-14C]2DG and 0.5 µCi/ml [1-3H]mannitol. After the incubation, the cells
were washed three times with ice-cold PBS and then dissolved in 1 N NaOH. The
sample solution was neutralized with 1 N HCl and counted by a liquid scintillation.

Supplementary References

51.

Yahagi, N. et al. Absence of sterol regulatory element-binding protein-1(SREBP-1) ameliorates fatty livers but not obesity or insulin resistance inLep(ob)/Lep(ob) mice. J Biol Chem 277, 19353-7 (2002).

52.

Megeney, L.A., Kablar, B., Garrett, K., Anderson, J.E. & Rudnicki, M.A. MyoD
is required for myogenic stem cell function in adult skeletal muscle. Genes Dev
10, 1173-83 (1996).

53.

Dobrzyn, A. & Gorski, J. Ceramides and sphingomyelins in skeletal muscles of
the rat: content and composition. Effect of prolonged exercise. Am J Physiol
Endocrinol Metab 282, E277-85 (2002).

Supplementary Table
Phenotypic characteristics of wild-type and Elovl6–/– mice fed a standard
chow or high-fat, high-sucrose (HF-HS) diet.
Diet

Standard chow

HF-HS
–/–

Wild-typeWild-type

Elovl6

Body weight (g)

28.1

0.42

24.6

1.2 *

45.5

1.25

41.0

1.32 *

Liver (% body weight)

3.93

0.13

4.29

0.15

4.09

0.18

4.60

0.21

Epididymal fat (%body weight)

1.53

0.16

1.04

0.05 *

3.44

0.12

3.22

0.18

Brown fat (% body weight)

0.31

0.02

0.25

0.03

0.70

0.03

0.62

0.06

Liver triglyceride (mg/g)

5.2

0.49

6.4

0.59

28.4

4.3

35.5

2.6

Liver cholesterol (mg/g)

1.65

0.10

1.79

0.12

2.70

0.26

3.47

0.37

Plasma FFA (mM)

0.38

0.04

0.49

0.06

0.37

0.02

0.38

0.04

Plasma triglyceride (mg/dl)

123

10.3

114

6.6

105

5.8

112

15.4

Plasma cholesterol (mg/dl)

77.1

2.2

73.9

2.8

129.9

8.4

138.7

14.5

Plasma glucose (mg/dl)

177

6.9

161

6.2

209

7.7

190

9.7

Plasma glucose (fasted,mg/dl)

118

11.1

120

8.2

167

9.2

143

8.9

Plasma insulin (ng/ml)

2.45

0.23

1.10

0.18 *

8.05

0.54

4.15

0.67 *

Plasma insulin (fasted, ng/ml)

0.57

0.11

0.44

0.09

2.56

0.31

1.36

0.36 *

Plasma leptin (ng/ml)

3.45

0.59

1.04

0.46 *

32.0

3.1

12.5

2.9 *

Plasma adiponectin (µg/ml)

45.1

3.6

51.9

5.4

30.3

3.9

29.4

2.7

Plasma TNF-α (pg/ml)

4.11

1.00

5.93

1.92

9.07

3.06

9.81

2.34

Plasma IL-6 (pg/ml)

4.78

0.33

4.24

1.01

4.55

2.48

4.64

1.83

Plasma ALT (IU/l)

11.1

0.8

12.2

1.5

21.8

2.4

13.4

1.9 *

Six-week-old male mice were fed a chow or HF-HS diet for 12 weeks.

Elovl6–/–

Parameter measured

Physiologic andmetabolic parameters were determined throughout standard chow diet or at the end of 12 weeks
of HF-HS diet feeding.

Mice were killed at non-fasted state otherwise noted.

± SEM for n = 15–18 mice.

Values are mean

Asterisks represent significant difference of at least p < 0.05.