ARTICLES© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

Crucial role of a long-chain fatty acid elongase, Elovl6, inobesity-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 & Nobuhiro Yamada1
Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that
improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision
is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in
the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes theconversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat
diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia andhyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 andsuppression of hepatic protein kinase C e activity resulting in restoration of Akt phosphorylation. Collectively, these data show that
hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance andstress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes andcardiovascular risks, even in the presence of a continuing state of obesity.

Insulin resistance is associated with obesity and is the major pathogenic indicator of early stages of type 2 diabetes. Epidemiologicalstudies have shown that intake of excess saturated fatty acids is theprincipal lifestyle-related cause of insulin resistance and obesityrelated diseases, including metabolic syndrome1. In general, it has
been thought that dietary saturated fatty acids are detrimental,monounsaturated fatty acids are neutral and polyunsaturated fatty
acids are beneficial, although intracellular events mediated by thesefatty acids have not been fully characterized. During high-fat dietfeeding, the influx of free fatty acids results in the accumulation of
triglycerides, promoting a lipotoxic state, and this induces insulin
resistance in skeletal muscle, adipose tissue and liver2. Intracellularaccumulation of fatty acids activates both oxidative degradation andincorporation of these fatty acids into triglycerides as a protective
adaptation against their cytotoxicity. When these mechanisms are
overwhelmed, fatty acids may activate proinflammatory and stressresponsive signals such as the nuclear factor-kB and c-Jun N-terminal
kinase pathways. These events, in turn, inhibit insulin signaling
through abnormal phosphorylation or degradation of insulin signaling molecules, leading to metabolic deterioration3–6. Recent data alsosuggest that the Janus kinase (JAK)-signal transducer and activator oftranscription (STAT)-3–suppressor of cytokine signaling (SOCS)-3
pathway that mediates leptin signals is involved in insulin resistanceand leptin resistance7–9.
Lipogenesis is a key event in the energy storage system. The
biosynthesis of fatty acids is sequentially catalyzed by the enzymes
acetyl–coenzyme A (CoA) carboxylase, fatty acid synthase (FAS) andstearoyl-CoA desaturase (SCD)-1 (Supplementary Fig. 1a online).
The entire pathway is controlled by the transcription factor sterol
regulatory element–binding protein (SREBP)-1c (refs. 10–12). Saturated fatty acids, as well as high-carbohydrate diets, activate SREBP-1cand enhance lipogenesis10,13. Previously, we have shown that SREBP1c inhibits insulin receptor substrate (IRS)-2 and contributes toinsulin resistance in liver, implicating a link between lipogenesis andinsulin resistance14. The elongation of long-chain fatty acids (ELOVL)
family member 6 (Elovl6, also known as LCE and FACE) has beenshown to be a target of SREBP-1 by microarray analysis of SREBP-1
transgenic mice, and it was predicted to be important for tissue
fatty acid composition15,16. In this study, we have examined theeffects of Elovl6 on obesity, hepatic steatosis and insulin resistanceto better understand the roles of fatty acid composition in theseobesity-related states.

1Department of Internal Medicine (Endocrinology and Metabolism) Graduate School of Comprehensive Human Sciences and 2Center for Tsukuba Advanced Research
Alliance University of Tsukuba, 1-1-1 Tennodai, Tsukuba Ibaraki 305-8575, Japan. 3Department of Metabolic Disease, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8655, Japan. 4Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Correspondence should be addressed to H.S. (shimano-tky@umin.ac.jp).

Received 24 May; accepted 28 August; published online 30 September 2007; doi:10.1038/nm1662

NATURE MEDICINEADVANCE ONLINE PUBLICATION

1ARTICLES

-9

:0
24

:1
n

:0

24

-9

22

-9

:3
n
20

:0
20

:1
n
20

0.5

24:1n-9

22:6n-3

24:0

22:5n-3

22:4n-6

22:0

20:5n-3

20:3n-6

0.3

18:1/18:0

16:1/16:0

**

**

0.2

2

**
1

0.1
O
H
C

O
H

ho
w

0

0

C

0

C

0

1

**

3

0.4

ho
w

**

0.1

20:4n-6

20:3n-9

20:2n-6

20:0

20:1n-9

18:3n-3

18:3n-6

**

0.2

O

24:1n-9

22:6n-3

24:0

22:5n-3

22:0

22:1n-9

20:5n-3

20:4n-6*
20:3n-6

20:3n-9

20:2n-6

20:1n-9

20:0

18:3n-3

18:3n-6

18:2n-6

18:0**
18:1n-9

16:0

16:1n-7

14:0

0

*

0.3

4

**

0.5

1.5

H

10

*

*

0.6

**

C

*
*

2

ho
w

20

Wild-type–/–
Elovl60.4
18:0/16:0

*

*

C

e 0.5

*

C

* *

2
20 0:0
:1
n
20 -9
:3
n9
22
22 :0
:1
n9
2
24 4:0:1
n930

16:1/18:1

3
2
10

Elovl6 –/– CHO

40

*

*

*

0

*

*

*

*

*

*

5

18:2n-6

18:0 18:1

10

*

*

15

O

16:1

0

20

H

16:0

0

25

C

**

0.4
0.2

18:0

**

Wild-type CHO*
Mol% of total fatty acids

5

0.6

30

18:1n-9d 50© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

10

Wild-type chow
Elovl6 –/– chow

*

14:0

Rplp0

15

35

ho
w

Elovl6

20

C

–/–

16:0

+/–

Elongase activity(pmol/min per mg protein)

+/+

c 40
Wild-type–/–
Elovl6

16:1n-7b

Mol% of total fatty acidsaFigure 1 Targeting of the Elovl6 gene. (a) Northern blot analysis of Elovl6 expression in the livers of Elovl6+/+, Elovl6+/– and Elovl6–/– mice. Total RNA(15 mg) pooled equally from three mice was subjected to northern blotting, followed by hybridization with the Elovl6 cDNA. A cDNA probe for Rplp0(which encodes acidic ribosomal phosphoprotein P0) was used to confirm equal loading. (b) Assessment of Elovl6 enzymatic activity in liver total membrane
fractions (n ¼ 5 per group) using four separate substrates, 16:0-CoA, 16:1-CoA, 18:0-CoA and 18:1-CoA. (c,d) Fatty acid composition of liver tissue inwild-type and Elovl6–/– mice fed either a normal chow (c) or a CHO diet (d) for 2 weeks (n ¼ 3–5 per group). (e) The ratio of stearate (18:0) to palmitate(16:0), palmitoleate (16:1n-7) to oleate (18:1n-9), 18:1 to 18:0 and 16:1 to 16:0 in livers of wild-type and Elovl6–/– mice. Results are represented asmeans ± s.e.m. *P o 0.05, **P o 0.01 for Elovl6–/– mice as compared to wild-type controls.

RESULTS
Metabolic features of Elovl6-null mice
The Elovl6 gene product belongs to a highly conserved family of
microsomal enzymes involved in the formation of long-chain fatty
acids17. Functional analysis by expression experiments in cultured cells
demonstrated that this enzyme has a role in the elongation of
palmitate (16:0) to stearate (18:0), as well as in the elongationof palmitoleate (16:1n-7) to vaccinate (18:1n-7) (Supplementary
Fig. 1a). To evaluate the importance of Elovl6 in vivo, Elovl6-nullmice were created (Supplementary Fig. 2a–c online). Homozygous
Elovl6 knockout (Elovl6–/–) mice had partial embryonic lethality,
although surviving male and female mutants were fertile (Supplementary Fig. 2d). Gene disruption completely abolished hepatic
Elovl6 expression, resulting in an effective loss of its activity in theliver; this indicates that the hepatic elongase activity for these reactions
is essentially attributable to Elovl6 (Fig. 1a,b). The residual elongase
activity of palmitoleate to vaccinate in Elovl6–/– liver may be attributed
to Elovl5, which has been reported to have a weak activity for thisreaction18. To determine whether the livers of Elovl6–/– mice contained
fewer C18 fatty acids, hepatic fatty acid composition was determined(Fig. 1c,d). Consistent with the absence of elongation of C16 fatty
acids to C18, hepatic concentrations of stearate and oleate (18:1n-9)
were lowered, whereas those of palmitate and palmitoleate wereheightened, as compared to wild-type mice. These trends were more
prominent in the livers of mice fed a fat-free, high-sucrose diet (CHO)
that enhances endogenous hepatic fatty acid synthesis10. This increase
was significant enough to make palmitoleate, which is usually a minor
fatty acid class, the second most abundant fatty acid present in liversof CHO-fed mice (Fig. 1d). The ratios of 18:0/16:0 and 16:1/18:1, themarkers of the elongase activity, were consistently decreased andincreased, respectively, in the livers of Elovl6–/– mice as compared to2wild-type controls (Fig. 1e). The ratios of 18:1/18:0 and 16:1/16:0,
indicative of desaturation through SCD-1 activity, were apparently
higher, although the expression of SCD-1 was decreased (Fig. 1e).
These data confirm the importance of Elovl6 in maintaining thehepatic contents of 18:0 and 18:1 through de novo synthesis of thesefatty acids, despite the abundance of 18:0 and 18:1 in the diet. The
fatty acid composition of plasma followed the same trend as that of
the liver, but changes were minimal in skeletal muscle and adipose
tissue (Supplementary Fig. 2).
Elovl6–/– mice appeared grossly normal, although they were slightly,
but significantly, leaner than wild-type littermates, despite an identicaldaily food intake of standard laboratory chow (Fig. 2a–c). However,there was no difference in the size or the appearance of adipocytes
from white and brown adipose tissues (Fig. 2d). Postprandial plasma
concentrations of insulin and leptin were lower in Elovl6–/– mice thanin wild-type mice, whereas no significant changes were observed in theamounts of plasma glucose or lipid (Fig. 2e and Supplementary
Table 1 online). Hepatic total cholesterol and triglyceride levels werealso not different between wild-type and Elovl6–/– mice on a chow diet(Supplementary Table 1). Because the Elovl6–/– mice had higher
insulin sensitivity on a standard chow diet, we challenged thesemice with a high-fat, high-sucrose diet (HF-HS) that is known to
induce obesity, hepatosteatosis and insulin resistance19. This highcalorie diet markedly enhanced body weight gain in both wild-type
and Elovl6–/– mice (which had identical daily food intakes) ata similar rate over the course of the study, although the slight, but
significant, weight differences were sustained (Fig. 2a–c). Epididymal
fat pad weight and total body fat percentage were markedly
increased in both groups, with similar net increases (Supplementary
Table 1 and Fig. 2b). In accordance with the observed increasein adiposity, white and brown adipocytes were enlarged similarly in

ADVANCE ONLINE PUBLICATION

NATURE MEDICINE

20
Wild-type chow
Elovl6

–/–

6

Chow

–/–

**

chow
0

+/+

© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

10

8 10 12 14 16 18
Age (weeks)
HF-HS

Lean Fat
Chow
Chow

25
20
15
10
5
0
HF-HS

30

5

25
20
15
10
5
0

4

e
250

3
2
1

200

Chow

100
50
0

Chow HF-HS

Fasted

HF-HS
10

+/+

–/–

+/+

–/–

Wild-type chow
Elovl6 –/– chow
Wild-type HF-HS
Elovl6 –/– HF-HS

150

0
HF-HS

HF-HS

Wild-type–/–
Elovl6

Insulin (ng/ml)

d

15

5

0
4

*

c
30

Glucose (mg/dl)

*

30

20
Weight (g)Body weight (g)

*

40

10

Wild-type
Elovl6 –/–25

Elovl6 –/– HF-HS

Food intake (g)bWild-type HF-HS
50

Change in body fat (%)

a

Change in body weight (g)ARTICLES

8Fed

Wild-type chow
Elovl6 –/– chow
Wild-type HF-HS
Elovl6 –/– HF-HS

**

6
4

*

**

2
0

FastedFed

Figure 2 Body weight, adiposity and glucose homeostasis in wild-type and Elovl6–/– mice fed a standard chow or HF-HS diet. (a) Body weight changes ofwild-type and Elovl6–/– mice fed a chow or HF-HS diet (n ¼ 15–18 per group). Six-week-old male mice (n ¼ 15–18) were fed a HF-HS diet for 12 weeks.
(b) Average lean and fat mass and body weight and body fat gain by wild-type and Elovl6–/– mice fed a chow (n ¼ 10 per group) or HF-HS diet (n ¼ 8 pergroup). (c) Daily food intake in wild-type and Elovl6–/– mice provided ad libitum access to chow or HF-HS diet (n ¼ 9–13 per group). (d) H&E-stained white
adipose tissue (left), brown adipose tissue (middle) and liver (right) from wild-type and Elovl6–/– mice fed normal chow or HF-HS diet for 12 weeks. Scalebar, 50 mm. (e) Plasma glucose (left) and insulin (right) concentrations in wild-type (n ¼ 18) and Elovl6–/– (n ¼ 15) mice fed chow or HF-HS diet for
12 weeks. Results are represented as means ± s.e.m. *P o 0.05, **P o 0.01 for Elovl6–/– mice as compared to wild-type controls.

both groups after they had been fed the HF-HS diet (Fig. 2d).
There were no obvious histological changes in white or brown
adipose tissue; this included no observed change in the extent ofmononuclear cell infiltration, which has been shown to contribute toproinflammatory response and insulin resistance in obesity20,21. There
was a trend toward more severe hepatosteatosis in Elovl6–/– micethan in wild-type mice on the HF-HS diet, as estimated fromhepatic triglyceride and cholesterol abundance as well as by histological staining (Supplementary Table 1 and Fig. 2d). Overall,diet-induced obesity in Elovl6–/– mice was similar to that ofnormal controls.
Elovl6 deletion protects against diet-induced insulin resistanceWild-type mice on the HF-HS diet exhibited a robust elevation inplasma insulin accompanied by slight increases in plasma glucose inboth fasted and fed states, indicating the emergence of insulin
resistance (Fig. 2e). However, Elovl6–/– mice on the HF-HS dietshowed a significant reduction in plasma insulin compared towild-type mice, in both nutritional states. The ameliorative effects ofElovl6 deficiency on hyperinsulinemia was confirmed by a glucose
tolerance test (GTT) (Fig. 3a). Wild-type mice on the HF-HS diethad markedly increased insulin abundance throughout the GTT. In
contrast, HF-HS–fed Elovl6–/– mice exhibited a pattern of insulin
response to glucose load nearly identical to that of mice on achow diet. The protection from diet-induced insulin resistance inElovl6–/– mice was more prominent during an insulin tolerance
test (ITT) (Fig. 3b). Insulin sensitivity, as measured by the reductionin plasma glucose after insulin administration, was markedly
reduced by the HF-HS diet in wild-type mice, whereas Elovl6–/–
mice showed a nearly normal response to insulin. The area under
the curve (AUC) of plasma insulin abundance during the GTT and the
AUC of plasma glucose abundance during the ITT for the HF-HS–fed
Elovl6–/– mice were significantly lower than those of the HF-HS–fed
wild-type mice (Fig. 3c,d). A pattern of protection from insulin
resistance similar to that observed in Elovl6 deficiency was also

NATURE MEDICINEADVANCE ONLINE PUBLICATION

observed in old (age 6–8 months) mice on a normal chow dietand in body weight–matched mice on the HF-HS diet (Supplementary Fig. 3 online).
Chronic hyperinsulinemia due to insulin resistance is associatedwith hyperplasia and hypertrophy of islets caused by the adaptive
proliferation of b-cells to maintain blood glucose levels. Histologyof pancreatic sections demonstrated that there was no observabledifference in islet morphology between the wild-type and Elovl6–/–
mice on a chow diet (Fig. 3e). Wild-type mice fed a HF-HS dietshowed a marked increase in the number and size of islets, suggestingan adaptive enlargement of b-cell mass in response to insulin
resistance. In contrast, islet hypertrophy was essentially absent in
Elovl6–/– pancreas, presumably because Elovl6 deletion abrogatedthe development of diet-induced insulin resistance, despite thecomparable obesity of the Elovl6–/– and wild-type mice.
Hepatic insulin signaling is preserved in Elovl6–/– mice
We studied insulin signaling in liver, muscle and white adipose tissue
to estimate the contribution of these insulin-sensitive organs to theamelioration of diet-induced insulin resistance in Elovl6–/– mice
(Fig. 3f,g and Supplementary Fig. 4a online). Insulin injection(5 units) induced phosphorylation of the major marker for insulin
signaling, Akt (on Ser473), in each of these three tissues, with similarintensities in chow-fed wild-type and Elovl6–/– mice. In a reflection of
their insulin resistance, wild-type mice on a HF-HS diet showed
suppression of Akt phosphorylation in these organs, without alterations in total Akt protein concentration. Elovl6 deficiency restored the
suppressed Akt phosphorylation only in the liver. Thus, ameliorationof whole body insulin resistance in Elovl6–/– mice can be attributed torestoration of hepatic insulin sensitivity. These data are consistent withthe observation that changes in fatty acid composition were prominent only in the livers of Elovl6–/– mice (Fig. 1c,d and Supplementary
Fig. 2e–g). Restoration of Akt phosphorylation was accompanied byincreased total and phosphorylated IRS-2 protein in Elovl6–/– livers,
whereas phosphorylation of the insulin receptor and IRS-1 remained

3

ARTICLES

Elovl6 –/– chow
Wild-type HF-HS
–/–
HF-HS
Elovl6

500
400
300
200
100

Wild-type HF/HS–/–
Elovl6
HF/HS

2
1.6
1.2
0.8
0.4
0

16
14
12
10
8
6
4
2
0

Wild-type
Elovl6 –/–

–

+

Chow

–

**

5
4
3
2
1

d

0

15 30
60
Time (min)Wild-type

Elovl6

1.2

1

1

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

+ Ins

HF/HS

100
90
80
70
60
50
40

120

e

–/–

Wild-type HF-HS

0

Chow

15

f

HF-HS

90
80
70
60

*

50
40

**
0

15

**

30 60 90 120
Time (min)

Chow

HF-HS

– + + – + + – + + – + +pAkt(Ser473)

Liver

+/+

*

Elovl6 –/– HF-HS

110
100

30 60 90 120
Time (min)Insulin

Akt
pAkt(Ser473)

Muscle
–/–

Akt

0

0

HF-HS

Genotype

h

20

Chow

Insulin –

15

+

–

+

–

+

–

+

Insulin

–/–

+/+

–/–

Wild-type

Elovl6 –/–

–

–

+

+

IP:IRS-1 IB:pY
IP:IRS-1 IB:IRS-1

IRβ

5

+/+

HF/HS

pIRβ
(Tyr972)

10

0

**

**

Chow

*

**

**

1.2

0

Muscle Akt phosphorylation(multiple of basal chow-fed WT)

AUC of insulin levels(multiple of chow-fed WT)

AUC of glucose levels(multiple of chow-fed WT)

Wild-type chow–/–
Elovl6chow

Liver Akt phosphorylation(multiple of basal chow-fed WT)© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

g

**

2

Wild-type chow
Elovl6 –/– chow

110

3

120

AUC of glucose levels(multiple of WT)

60
15 30
Time (min)

0

b

4

1
0

0

c

7
6
5

Glucose (% basal)

Wild-type chow

Elovl6 –/– chow
Wild-type HF-HS
Elovl6 –/– HF-HS

Glucose (% basal)

700
600

Wild-type chow

Insulin (ng/ml)

Glucose (mg/dl)a

IP:IRS-2 IB:pY
Genotype
–

+

Chow

–

+ Ins

+/+

–/–

+/+

–/–

IP:IRS-2 IB:IRS-2

HF/HS

Figure 3 Protection from diet-induced insulin resistance in the absence of Elovl6. (a) Plasma glucose (left) and insulin (right) concentrations during
intraperitoneal GTTs in wild-type and Elovl6–/– mice fed standard chow or HF-HS diets for 12 weeks (n ¼ 12 per group). (b) ITTs in wild-type and Elovl6–/–
mice fed standard chow (0.5 U insulin per kg of body weight; left) or HF-HS diet (1.0 U insulin per kg of body weight; right) (n ¼ 12 per group). (c,d) The
AUCs during the GTTs (c) or ITTs (d). WT, wild-type. (e) Protection of islet hyperplasia in Elovl6–/– mice fed a HF-HS diet. H&E-stained sections ofpancreases from wild-type and Elovl6–/– mice fed standard chow or HF-HS diet. Scale bar, 50 mm. (f) Immunoblot analysis of Ser473-phosphoylated Akt(pAkt) and total Akt in response to a bolus injection of insulin in liver and skeletal muscle. Experiments were performed on mice fed a regular chow or
HF-HS diet for 12 weeks. (g) Densitometric quantification of all livers and muscle immunoblot analysis from f (n ¼ 6 per group). (h) Phosphorylation of
insulin receptor b (pIRb), IRS-1 and IRS-2 induced by a bolus injection of insulin was assessed in livers of wild-type and Elovl6–/– mice on a regular chow
and/or HF-HS diet. Blots are representative of three independent experiments. IP, immunoprecipitated; IB, immunoblotted; pY, phosphorylated tyrosine.
Results are represented as means ± s.e.m. *P o 0.05, **P o 0.01 for Elovl6–/– mice as compared to wild-type controls.

suppressed by the HF-HS diet in both genotypes (Fig. 3h),demonstrating that the restoration of insulin signaling in Elovl6–/–
mice was mediated by the recovery of the hepatic IRS-2/Akt
signaling pathway.
Recent studies have reported that stress or proinflammatory pathways such as the inhibitor of kB (IkB)-IkB kinase (IKK)-nuclear
factor-kB, c-Jun N-terminal kinase and JAK–STAT-3–SOCS pathways
impair insulin sensitivity, suggesting a link between inflammationand insulin resistance3–9. HF-HS feeding substantially regulated
each of these pathways in the livers of wild-type mice contributing
to insulin resistance (Supplementary Fig. 4b). Confoundingly,
Elovl6 deficiency tended to slightly activate these signals. It has
recently been reported that SOCS proteins act as negative regulators
in insulin signaling7–9. Contrary to what would be predicted fromthe observed amelioration-of-insulin-resistance phenotype, SOCS-3(encoded by Socs3) and cytokine-inducible SH2-containing protein(Cish) were highly upregulated in Elovl6–/– livers (Supplementary
Fig. 4c). Collectively, these data show that amelioration of insulin
resistance by Elovl6 deficiency is not mediated by suppression of theseproinflammatory signals.

4

Gene expression in Elovl6–/– mice
To determine the molecular basis of these metabolic changes in thelivers of Elovl6–/– mice, we examined the expression of genes involvedin fatty-acid metabolism or glucose metabolism. Northern blot andreal-time (RT) PCR analysis revealed that the HF-HS diet augmented
hepatic expression of SREBP-1c (Srebf1) itself and of its target genes
encoding lipogenic enzymes, including FAS (Fasn), Elovl6, SCD-1(Scd1) and glycerol-3-phosphate acyltransferase (Gpam), in normal
mice (Fig. 4a,b). The dietary induction of these genes was suppressedin Elovl6–/– mice. Consistently, expression of the nuclear active form of
SREBP-1c protein was decreased (Fig. 4c). In contrast, expression of
the forkhead box O1 and A2 proteins (Foxo1 and Foxa2), which havebeen reported to affect hepatic glucose and lipid metabolism22,23, wasdiminished by the HF-HS diet, but was not changed in Elovl6–/– mice
(Fig. 4c). In a reflection of better insulin sensitivity, nuclear exclusionof Foxo1 induced by insulin treatment was more detectable in
Elovl6–/– mice than in wild-type mice (Supplementary Fig. 4d).
Inhibition of Scd1 expression caused by Elovl6 deficiency wasprominent in mice on both normal and HF-HS diets. As we havepreviously reported, activation of SREBP-1c directly represses IRS-2,

ADVANCE ONLINE PUBLICATION

NATURE MEDICINEARTICLES

Chow HF-HS

Chow HF-HS

Acadm
1.0 0.9 1.7 1.3

Cyp4a14

1.0 0.9 1.8 0.9

1.0 0.9 1.1 1.1

1.0 1.4 0.2 0.6

1.0 0.9 10.1 5.3

Gpam
1.0 0.3 4.0 3.1

Relative mRNA levels(normalized to Ppia)© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

4

#

#

3

0

*
ND

ND

Elovl6

2,000
0

*

*

Srebf1

Ppara

50
0

*
#
# *
Irs2

Elovl6 –/–** **Chow HF-HS

Membrane

Chow HF-HS Chow HF-HS

f

Wild-type

2.5
2
1.5
1
0.5
0

*

100

AMPK

e

*#

#

150

Cytosol

Wild-type chow
Elovl6 –/– chow
Wild-type HF-HS
Elovl6 –/– HF-HS

#

2
1

*

Rplp0

Ucp2
1.0 1.3 1.5 3.5

b

Whole
cell
lystates

Irs2

4,000

200

+
–/
–
+/
+
–/
–
+/
+
–/
–
+/
+
–/
–

1.0 0.3 1.3 0.5

Scd1

6,000

+/

Acox1

1.0 0.5 1.8 1.7

Pck1

8,000

PKCε
1.5
1
0.5
0
Chow HF-HS

Membrane/cytosol ratio(multiple of chow WT)

1.0 0.7 3.1 1.4

Foxo1
Foxa2
IP:IRS-2
IB:IRS-2
pAMPKαCeramide(multiple of chow WT)

Fasn

SREB-2

Nuclearextracts

Pklr

Diacylglycerol(multiple of chow WT)

1.0 0.2 2.5 1.4

Elovl6 –/–Fatty-acid synthesis(d.p.m.)

1.0 1.1 1.2 1.1

1.0 0.7 4.5 3.7

Srebf1

Wild-type
SREB-1

Fatty-acid oxidation(pmol/h/105 cells)

1.0 0.8 1.8 1.5

Hmgcs1

Cpt1a

3.0 0

+/
+
–/
–
+/
+
–/
–

1.0 0.7 2.0 1.6
1.0 0

d

Ldlr

Ppara

Elovl6c

Chow HF-HS

+/
+
–/
–
+/
+
–/
–

+/
+
–/
–
+/
+
–/
–

Chow HF-HS

+/
+
–/
–
+/
+
–/
–

a

2

*

1.5

Wild-type
Elovl6 –/–1
0.5
0Chow HF-HS

Figure 4 Effects of Elovl6 deficiency on mRNA and protein levels and fatty acid metabolism in the livers of wild-type and Elovl6–/– mice fed a regular chowor HF-HS diet. (a,b) Northern blot (a) and RT-PCR (b) analysis of various mRNA levels in livers from wild-type and Elovl6–/– mice fed a standard chow or
HF-HS diet for 12 weeks and sacrificed in a nonfasted state. (a) Total RNA was isolated from the livers of mice from each group (n ¼ 3), pooled (15 mg)
and subjected to northern blot analysis with the indicated cDNA probes. Fold changes of expression relative to chow-fed wild-type mice are shown. Ucp2,
uncoupling protein-2; Ldlr, low-density lipoprotein receptor; Hmgcs1, hydroxymethylglutaryl-CoA synthase-1; Pklr, liver-type pyruvate kinase; Pck1,
phosphoenolpyruvate carboxykinase-1, cytosolic. (b) Quantitative RT-PCR of RNA from livers of mice from each group (n ¼ 9 per group). # indicatesstatistical significance between animals of the same genotype fed different diets, P o 0.01; * indicates statistical significance between wild-type andElovl6–/– mice fed the same diet (P o 0.05). ND, not detectable. (c) Immunoblot analysis of SREBP-1, SREBP-2, Foxo1 and Foxa2 in nuclear extracts andIRS-2, Ser485-phosphorylated AMPKa (pAMPKa) and AMPKa in whole cell lysates from the livers of wild-type and Elovl6–/– mice fed a regular chow diet or
HF-HS diet for 12 weeks. (d) The amount of fatty acid synthesis (left) and oxidation (right) in primary hepatocytes isolated from chow-fed wild-type andElovl6–/– mice (n ¼ 9 per group). d.p.m., decay per minute. (e) Diacylglycerol (left) and ceramide (right) content in the livers of wild-type and Elovl6–/– mice
fed a regular chow diet or HF-HS diet for 12 weeks (n ¼ 6 per group). (f) Immunoblot analysis determining membrane and cytosolic PKCe content in thelivers of wild-type and Elovl6–/– mice fed a regular chow diet or HF-HS diet for 12 weeks (n ¼ 6–8 per group). Blots are representative of three independent
experiments. Results are represented as means ± s.e.m. *P o 0.05, **P o 0.01 for Elovl6–/– mice as compared to wild-type controls.

the main insulin signal mediator, and causes hepatic insulin resistance14. Therefore, suppression of SREBP-1c could contribute to theamelioration of hepatic insulin resistance in Elovl6–/– mice. Consistent
with this notion, expression of Irs2, which was completely suppressedby the HF-HS diet in wild-type mice, was restored in the absence ofElovl6 (Fig. 4a–c). Meanwhile, genes related to fatty acid oxidationthat are regulated by nuclear receptor peroxisome proliferator–activated receptor (PPAR)a (Ppara), such as those encoding carnitine
palmitoyltransferase-1 (Cpt1a), medium-chain acyl-CoA dehydrogenase (Acadm), acyl-CoA oxidase (Acox1) and cytochrome P450 4a14
(Cyp4a14), were induced by HF-HS diet in wild-type mice as an
adaptive response (Fig. 4a). However, expression of these oxidation
genes, including Ppara, was considerably decreased in Elovl6–/– mice,
despite the amelioration of insulin resistance in these mice. AMPactivated protein kinase (AMPK) has been shown to facilitate energy
expenditure and contribute to insulin sensitivity after treatment withbiguanides, anti-diabetic agents that activate AMPK24. The amount of
the active form of AMPKa (phosphorylated on Ser485) was notchanged in Elovl6–/– livers (Fig. 4c). Thus, these data implicate thatElovl6 deficiency suppressed both the synthesis and the degradation of
fatty acids, resulting in a slightly increased hepatic triglyceride content.
In support of this, fatty acid synthesis and oxidation were reduced
in primary hepatocytes isolated from Elovl6–/– mice as compared tothose from wild-type mice (Fig. 4d).
Diacylglycerol and ceramide have been thought to mediate insulin
resistance in muscle and liver, accompanying accumulation of
intracellular lipids25–27. We measured the contents of these lipid

NATURE MEDICINEADVANCE ONLINE PUBLICATION

metabolites in the livers of wild-type and Elovl6–/– mice fed a chow or
HF-HS diet (Fig. 4e). In wild-type mice, hepatic diacylglycerol content
was elevated twofold by the HF-HS diet. Elovl6 deficiency did notaffect the basal level on a normal chow, but inhibited the induction by
HF-HS diet observed in Elovl6–/– mice. No marked genotypic anddietary differences were evident in hepatic ceramide content. Diacylglycerol accumulation has been reported to be linked to the increased
protein kinase C (PKC) e activity and impaired phosphorylation of
IRS-2 tyrosine induced by insulin25,28. Hepatic PKCe activity, as
measured by the ratio of the amount of PKCe in cellular membranes
to that in the cytosolic fraction, was significantly increased in the liversof HF-HS–fed wild-type mice, but not in Elovl6–/– mice (Fig. 4f),
implying that the protection from diet-induced insulin resistanceobserved in Elovl6–/– mice could be mediated at least partially throughthis diacylglycerol-PKCe pathway.
Contribution of hepatic Elovl6 to insulin resistanceTo estimate the importance of hepatic Elovl6 on whole-body insulin
sensitivity, and to distinguish the long-term and short-term effects ofan absence of Elovl6, we used adenoviral RNA interference (RNAi) to
specifically inhibit hepatic expression of Elovl6 (Fig. 5a–e). The Elovl6
RNAi adenovirus (Elovl6i) robustly suppressed hepatic Elovl6 expression and activity in C57BL6 mice on the HF-HS diet (Fig. 5a).
Treatment with Elovl6i mimicked the hepatic changes observed in theliver of Elovl6–/– mice—it reduced expression of both Srebf1 and Ppara
and induced Irs2 mRNA expression, and this was accompanied by asignificant increase in hepatic triglycerides (Fig. 5a,b). Consequently,

5

ARTICLES
mice (Supplementary Fig. 5a–c). As an
exception, Fasn expression was increased in
Foldthese tissues. Insulin sensitivity was estimated
250
20
40
0.1
Elovl6in primary culture cells prepared from mus*
200
15
30
cles of both groups (Supplementary Fig. 5d).
150
0.6
Srebf1
10
20
There were no differences between wild**
100
0.7
Ppara
5
10
type and Elovl6–/– muscle cells in basal
50
lrs2
1.8
level,
insulin-mediated induction or palmi000Fasted Fed
Rplp0
tate-mediated inhibition of glucose uptake(Supplementary Fig. 5d). Infection with
LacZi
LacZi
d
e
LacZi
Ad-Elovl6 did not restore the palmitate600
Elovl6i
Elovl6i
4**
Elovl6i
110
suppressed uptake (Supplementary Fig. 5d).
500
3
400
Thus, Elovl6 had no effects on the glucose
90
300
2
uptake or insulin sensitivity of muscle cells.
70200
Taken together with the hepatic knockdown
1
50
100
and restoration experiments, these data indi030
00 15 30 60 120
0 15 30 60 90 120
Fasted Fed
cate that hepatic Elovl6 plays the major role
Time (min)Time (min)in diet-induced insulin resistance.
To determine whether overexpression of
Ad-GFPfgh
Ad-Elovl6
Ad-GFP
Elovl6 –/–Elovl6
would affect hepatic gene expression
Ad-Elovl6
GFP Elovl6
110
4
250
and
insulinsignaling, mouse Hepa1c1c7
100
Elovl6
Fold
200
3
90
hepatoma cells were infected with Ad150
80
Srebf1
2.3
2
Elovl6. Elovl6 overexpression markedly
70
100
Ppara
1.0
induced SREBP-1c at both the mRNA and
60
1
50
50
lrs2
0.8
nuclear protein levels (Fig. 6a). Furthermore,
0040
activation of this enzyme blocked in0
15
30
60
90
120
Rplp0
Time (min)sulin-stimulated phosphorylation of Akt(Fig. 6b). Thus, hepatic Elovl6 activity
Figure 5 Effects of hepatic Elovl6 expression on gene expression, fatty acid metabolism and insulin
could induce SREBP-1c expression andsensitivity. (a–e) Knockdown of hepatic Elovl6 by adenoviral expression of RNAi. Eight-week-old male
oppose insulin signaling.
C57BL6 mice were fed a HF-HS diet for 2 weeks, followed by tail vein injection with adenovirus
To examine the physiological relevance of
encoding RNAi targeting Elovl6 (Elovl6i) or a LacZ (LacZi) control sequence. After 5–7 d of HF-HS
these
data, we next asked whether insulin
feeding, mice were sacrificed in a nonfasted state. (a) Gene expression (left) and elongation activity
action and gene expression can be altered in(right) in the livers from LacZi and Elovl6i. The Elovl6i fold-change value is relative to the normalized
liver cells as a function of the amount ofvalue of LacZi control signal. (b–d) Liver trigyceride (b) plasma glucose (c) and plasma insulin (d) levels
in a fasted or fed state. (e) GTT (left) and ITT (0.75 U insulin per kg body weight; right) in micecellular fatty acids regulated by Elovl6; we
infected with either LacZi or Elovl6i. (f–h) Effect of adenovirus-mediated restoration of hepatic Elovl6
focused specifically on the effects of increasexpression in Elovl6–/– mice. Six-week-old male Elovl6–/– mice were fed a HF-HS diet for 4 weeks anding the ratio of palmitoleate to palmitate.
then treated with adenovirus encoding GFP (control) or Elovl6 for 5–7 d. (f) Northern blot analysis of
Hepa1c1c7 cells were cultured in medium–/–
gene expression in the livers of Elovl6 mice injected with adenovirus encoding GFP (Ad-GFP) or
supplemented with palmitate and with
Elovl6 (Ad-Elovl6). (g) Plasma glucose (left) and insulin (right) concentrations of the Elovl6–/– miceincreasing concentrations of palmitoleate.
injected with Ad-GFP or Ad-Elovl6 in the fed state. (h) ITT in mice infected with either Ad-GFP or
Palmitate-alone treatment suppressed insuAd-Elovl6 (0.75 U per kg body weight). Results are represented as means ± s.e.m., n ¼ 11–14 pergroup. *P o 0.05, **P o 0.01 for experimental mice as compared with their respective controls.
lin-stimulated phosphorylation of Akt, andaddition of palmitoleate restored it in a doseknockdown of hepatic Elovl6 significantly improved upon the dependent manner (Fig. 6c). Srebf1 and Ppara mRNA expression wereincreased plasma insulin level (P o 0.01) and impaired insulin increased by palmitate treatment and were returned to the baseline bysensitivity (as determined by ITT, P o 0.01) observed in mock-treated palmitate and palmitoleate supplementation, (Fig. 6d). These experi(LacZi), HF-HS–fed mice, even though there was no change in the ments showed that the cellular contents, or the ratio of palmitoleate toplasma glucose level (Fig. 5c–e). Decreased plasma insulin was also palmitate, could be a determinant for the hepatic insulin sensitivity
observed by injecting Elovl6i into ob/ob (leptin-deficient, also known regulated by Elovl6. These findings suggest that the fatty acid compoas Lep–/–) mice (data not shown). Conversely, injection of a small sition observed in the Elovl6–/– liver is favorable for insulin action.
amount of Elovl6 adenovirus (Ad-Elovl6) into Elovl6–/– animals fed a
HF-HS diet caused hepatic expression of this enzyme to return to the Leptin signaling in Elovl6 deficiency
control level (Fig. 5f). The restoration of Elovl6 expression only in the Leptin is a key regulator of satiety and energy expenditure, thus
liver canceled the protection from diet-induced hyperinsulinemia and determining energy metabolism and insulin sensitivity in the body29.
insulin resistance in Elovl6–/– mice and was accompanied by increased Along with insulin resistance, diet-induced obesity by HF-HS was
Srebf1 and decreased Irs2 mRNA levels (Fig. 5f–h). In contrast, gene associated with leptin resistance, as evidenced by high plasma leptin
expression analysis in other energy organs, such as white fat, brown fat levels and high leptin expression in adipose tissue in wild-type miceand skeletal muscle, in HF-HS–fed Elovl6–/– mice revealed no marked (Supplementary Table 1 and Supplementary Fig. 5a). Elovl6 defichanges in the expression of genes involved in fatty acid oxidation, ciency improved leptin signaling, as judged by a marked decrease
insulin sensitivity or lipogenesis, indicating that these organs do not in plasma leptin in Elovl6–/– mice on both chow and HF-HS
contribute to the protection from insulin resistance noted in Elovl6–/– diets, irrespective of obesity. However, the food intake and resultant
LacZi
Elovl6ic

LacZi
Elovl6i

Glucose (mg/dl)b

Glucose (% basal)

Glucose (% basal)

Glucose (mg/dl)

Glucose (mg/dl)© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

Insulin (ng/ml)

6

LacZi
Elovl6i

Liver TG (mg/g)

Elovl6i

Elongase activity(pmol/min per mg protein)

LacZi

Insulin (ng/ml)

a

ADVANCE ONLINE PUBLICATION

NATURE MEDICINEARTICLES

80

*

60
40
20
0

6

f
300
200
100

0
0
20
0
10
0
50
10
0
0
20
100
0
50
10

Akt

+

Wild-type
ob/ob
ob/ob,Elovl6 –/–g**

Wild-type
ob/ob
ob/ob,Elovl6 –/–80

8 10 12 14 16 18
Age (weeks)

6
4
2
0

**

h

Relative mRNA levels(normalized to Ppia)

0
20
0
20
0
20
200
200
0
0
20
0
20
0
20
200
200
0

16:1 (mM)Ad

16:0 (mM)

5

1,200
1,000
800
600
400
200
0

** ** **
0**

15 30 60 120
Time (min)

*
*

3

*

4
2

3
2

1

1
0

0
Srebf1

Ppara

ob/ob
ob/ob,Elovl6 –/–ob/ob
ob/ob,Elovl6 –/–Glucose (% basal)

70
60
50
40
30
20
10
0

+

–

Glucose (mg/dl)

Wild-type
ob/ob
ob/ob,Elovl6 –/–InsulinpAkt–

PA200 + POA50
FA-free
PA200 + POA10
PA200
PA200 + POA100

d

Akt

Ppara

Wild-type
ob/ob
ob/ob,Elovl6 –/–

4

pAkt

Insulin

Body fat (%)

Body weight (g)© 2007 Nature Publishing Group http://www.nature.com/naturemedicineeSrebf1

Precursor
SREBP-1
MatureSREBP-1

Insulin (ng/dl)

Elovl6*

Glucose (mg/dl)

**

-G
FP
-E
lo
vl
6

2.5
2
1.5
1
0.5
0

Ad

Ad-Elovl6

12
10
8
6
4
2
0

Ad

Ad-GFP
50
40
30
20
10
0

c
-G
Ad FP
-E
Ad lov
-G l6
Ad FP
-E
lo
vl
6

b

Relative mRNA levels(normalized to Ppia)

a

130
120
110
100
90
80
70
60
50

** * *
0 15 30 60 90 120
Time (min)Figure 6 The effects of Elovl6 overexpression and palmitate/palmitoleate ratio on Hepa1c1c7 hepatoma cells and of Elovl6 deficiency on genetically obese
mice. (a,b) Overexpression of Elovl6 induces Srebf1 expression and insulin resistance in hepatoma cells. (a) Gene expression in Hepa1c1c7 cells infectedwith adenovirus expressing either Ad-GFP (n ¼ 6) or Ad-Elovl6 (n ¼ 6), as estimated by RT-PCR analysis (left). Immunoblot analysis of SREBP-1 inmembrane (precursor) and nuclear extracts (mature) from Hepa1c1c7 cells infected with adenovirus expressing either Ad-GFP or Ad-Elovl6 (right). Ad-GFP
is used as a negative control. (b) Elovl6 overexpression results in decreased Akt Ser473 phosphorylation in Hepa1c1c7 cells. Cells were stimulated withinsulin for 10 min 16 h after adenovirus infection. (c,d) Effects of palmitate/palmitoleate ratio on Akt phosphorylation, c, and gene expression, d, in
Hepa1c1c7 cells. Cells were supplemented with media alone (fatty acid–free) or media containing 200 mM palmitate (PA200), 200 mM palmitate +200 mM palmitoleate (PA200 + POA200), 200 mM palmitate + 100 mM palmitoleate (PA200 + POA100), 200 mM palmitate + 50 mM palmitoleate(PA200 + POA50), or 200 mM palmitate + 10 mM palmitoleate (PA200 + POA10) for 16 h before insulin stimulation or RNA extraction. (e–h) Loss ofElovl6 ameliorates insulin resistance in ob/ob mice. (e) Body weight changes (left) and body fat percentage (right) of wild-type (n ¼ 11), ob/ob (n ¼ 11)
and ob/ob,Elovl6–/– mice (n ¼ 10). (f,g) Plasma glucose (f) and insulin (g) concentrations in 6–8-week-old wild-type (n ¼ 11), ob/ob (n ¼ 11) andob/ob,Elovl6–/– mice (n ¼ 10). Mice were fasted for 24 h before experiments. (h) Plasma glucose levels during the GTT (left) and ITT (right) of 6–8-week-old
ob/ob (n ¼ 9) and ob/ob,Elovl6–/– (n ¼ 9) mice. For ITT, insulin (2 U per kg body weight) was injected into each mouse after 6 h of fasting. Results are
represented as means ± s.e.m. *P o 0.05, **P o 0.01 for experimental mice as compared with their respective controls.

adiposity did not change in Elovl6–/– mice on the HF-HS diet (Fig. 2
and Supplementary Table 1). Livers from these mice did not show
signs of the improved leptin signaling that was suppressed by HF-HS
diet, as estimated by examining the amount of phosphorylation of
STAT-3 and AMPK and fatty acid oxidation (Supplementary Fig. 4b
and Fig. 4a). Furthermore, the effect of Elovl6 deficiency was evaluatedin ob/ob mice, which showed severe obesity, insulin resistance anddiabetes (Fig. 6e–h). ob/ob and Elovl6 double-mutant mice (ob/ob,
Elovl6–/–) showed sustained, severe obesity and had similar increases inbody weight, body fat (Fig. 6e) and liver triglyceride content (data notshown) when compared to wild-type mice. However, insulin resistanceand hyperglycemia were markedly less severe in the ob/ob,Elovl6–/–
mice than in the ob/ob mice, as estimated by GTTs and ITTs conductedin mice 6–8 weeks (Fig. 6f–h) and 16–18 weeks (data not shown) ofage. Taken together, these data imply that the effect of Elovl6 on
insulin sensitivity is independent of leptin. The mechanism by which
Elovl6 deficiency decreases plasma leptin levels without changing
adipocyte size is currently unknown, but must be related to enhanced
insulin signaling.
DISCUSSION
Our current data establish the unique role of Elovl6 in hepatic
lipogenesis. The major product of FAS, palmitate (16:0), is elongatedby Elovl6 and desaturated by SCD-1 to yield oleic acid (18:1), the end
product of mammalian fatty-acid synthesis. Recent studies in mousemodels of ACC, FAS and SCD-1 deficiency, as well as our current data
in Elovl6–/– mice, implicate various roles for endogenous fatty acidsynthesis in energy metabolism (Supplementary Fig. 1). However,

NATURE MEDICINEADVANCE ONLINE PUBLICATION

these deficiencies affect not only fatty acid synthesis, but also secondary activation of fatty acid oxidation. Liver-specific FAS knockout
mice have phenotypes similar to those of PPARa-null mice, suggestingthat some products of FAS are endogenous PPARa agonists thatmaintain fatty acid metabolism30. Scd1–/– mice are also protected fromobesity and insulin resistance owing to activation of AMPK and fatty
acid oxidation, implicating SCD-1 as a potential target for thetreatment of diabetes31,32. Elovl6 and SCD-1 are structurally related
and committed to consecutive reactions in lipogenesis; therefore, theimprovement of insulin sensitivity in both knockout mice is not
unexpected. It is possible that the decrease in Scd1 expression could
partly contribute to the insulin-sensitizing effect of Elovl6 deficiency.
However, in contrast to Scd1–/– mice, Elovl6–/– mice showed sustained
obesity and low fatty acid oxidation in the liver, indicating that themechanisms by which these two enzymes influence insulin sensitivity
are not completely convergent. Thus, the conversion of palmitate tostearate is a key step in energy expenditure that discriminates between
the physiological functions of Elovl6, a membrane-bound enzyme,
and FAS, the multifunctional cytosolic enzyme that synthesizes fatty
acids of up to 16 carbons in length.
The amelioration of insulin resistance in obese mice is usuallyaccompanied by a loss of fat or body weight caused by decreased foodintake33,34, enhanced lipid oxidation and/or decreased lipogenesis31,35,36. The Elovl6–/– mice are unique in that their insulin resistance
was reduced without amelioration of obesity or hepatosteatosis.
The restoration of hepatic insulin signaling in these mice could
not be explained by changes in energy balance or proinflammatory
signals. These data highlight the importance of tissue fatty acid7© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

ARTICLES
composition in insulin sensitivity, especially the ratio of C18 to C16
fatty acids, which is controlled by Elovl6 activity. The precise molecular mechanism for this is currently unknown. Elovl6 inhibition
results in the accumulation of palmitate, which is paradoxically themost potent dietary inducer of obesity and insulin resistance. Ourstudies show that the increase in the ratio of palmitoleate to palmitate
prevents palmitate-induced Srebf1 expression and consequently rescues palmitate-induced insulin resistance. Furthermore, our preliminary data demonstrate that overexpression of Elovl6 leads to liver
dysfunction, in addition to the induction of Srebf1 expression andinsulin resistance in cells. These results suggest that the fatty acidcomposition of Elovl6–/– liver, namely the high ratio of 16:1/16:0 andthe reduction in C18 fatty acids, may be protective against hepatic
lipotoxicity and insulin resistance in HF-HS–fed mice (Supplementary Fig. 1b). Feeding normal mice a diet containing a single fatty aciddemonstrated that palmitate alone or stearate alone can induce insulin
resistance, but oleate alone cannot (Supplementary Fig. 5e). Elovl6–/–
mice were insulin sensitive after palmitate ingestion, with elevations inliver triglyceride and cholesterol content. Thus, the conversion of
palmitate to stearate is crucial for the emergence of insulin resistance.
Notably, however, Elovl6–/– mice were again resistant to the insulin
resistance–inducing effects of stearate. Thus, the protective effect ofElovl6 deficiency is not simply a result of stearate depletion, implicating that exogenous intake and endogenous production of fatty acids
might have different effects on hepatic insulin sensitivity. Palmitoleate,
the major final product of de novo fatty acid synthesis in Elovl6
deficiency, is efficiently incorporated into triglycerides and cholesterolesters, potentially contributing to the prevention of insulin resistance37. These observed effects may also be related to the reported
benefits of macadamia nuts rich in palmitoleate in the prevention ofdiabetes and cardiovascular diseases38.
Ppara expression was reduced in Elovl6-deficient animals. Furthermore, Ad-Elovl6 and exogenous fatty acids administered in various
16:1/16:0 ratios regulate both Srebf1 and Ppara expression in a parallel
fashion, although the co-regulation of these opposite regulators offatty acid metabolism apparently contradicts nutritional adaptation.
However, the suppression of Ppara expression in Elovl6-deficient micedoes not explain the restoration of insulin sensitivity in these mice. It
could instead be a result of the repression of SREBP-1c. Newly
synthesized fatty acids could be ligands for PPARa and hepatocyte
nuclear factor-4a (ref. 30). Elovl6 deficiency could deplete theseligands directly and/or indirectly through SREBP-1c suppression,leading to decreased activities of PPARa and hepatocyte nuclear
factor-4a; it could further repress PPARa expression, as the PPARa
promoter is a target of both factors (refs. 30,39,40 and T.Y. and H.S.,
unpublished data). Recently, we also found that knockdown of hepatic
Srebf1 leads to suppression of Ppara in the liver (T.Y. and H.S.,
unpublished data). Secondary regulation of PPARa activity could bea part of SREBP-1c/Elovl6 regulation of lipogenesis.
It has been generally noted that different long chain fatty acids havedistinct effects depending upon their extent of desaturation. However,
our current study suggests that the length of fatty acids is also
important for energy metabolism and insulin sensitivity.
To date, Elovl6–/– mice are the only known pure metabolic model inwhich obesity-induced insulin resistance is mitigated through modulation of hepatic metabolism without a concurrent amelioration ofobesity. It has been reported that mice deficient for inducible nitricoxide synthase, adipocyte protein-2 (also known as fatty acid binding
protein-4) and mal1 (also known as fatty acid binding protein-5) also
maintain insulin sensitivity despite diet-induced obesity, but not
through effects on the liver41–43. Our model helps in understanding8

the mechanism by which obesity and obesity-related disorders are
sometimes dissociated and implicates a new strategy for the treatment
of diabetes and cardiovascular diseases through intervention of Elovl6.
METHODS
Generation of Elovl6–/– mice. Elovl6+/– founder mice created at Lexicon
Genetics through the use of a gene-trapping method44. We identified theintegration site of the targeting cassette between exon 2 and 3 and developed a
PCR-based assay that distinguishes between the three possible mouse genotypes(see Supplementary Methods online). We crossed Elovl6+/– founder mice
(50% C57BL6 albino and 50% 129SvEvBrd strains) at least six times to transfer
the null mutation onto the C57BL6 genetic background. We then intercrossed
heterozygotes to obtain Elovl6+/+, Elovl6 +/– and Elovl6–/– mice. We used only
male mice for the present studies.
Animal experiments. Mice were housed in a pathogen-free barrier facility witha 12-h light/dark cycle, with free access to water and a standard chow diet. In
some experiments, we fed the mice a high-carbohydrate, fat-free diet10 or ahigh-fat, high-sucrose (HF-HS) diet19. We used age- and sex-matched littermates for each experiment, and we sacrificed mice in the early light phase in a
nonfasted state. We isolated tissues immediately, weighed and kept in liquid
nitrogen. We determined fat and lean mass by dual-energy X-ray absorptiometry using a PIXImus mouse densitometer (GE Medical Systems Lunar). All
experiments were repeated at least three times. All animal husbandry andanimal experiments were consistent with the University of Tsukuba’s Regulationof Animal Experiment Committee.
Fatty acid elongation assay. We assayed microsomal fatty acid elongation
activity by measuring of [2-14C]malonyl-CoA incorporation into exogenous
acyl-CoAs as described previously16.
Fatty acid composition of liver. We measured the fatty acid composition bygas chromatography as described previously45.
Metabolic measurements. We measured the concentrations of glucose, insulin,
leptin, free fatty acids (FFAs), triglycerides, total cholesterol and alanine
aminotransferase (ALT) in plasma and of triglycerides and total cholesterolin liver as previously described46. For intraperitoneal GTTs, mice were fasted
overnight (for 16 h) and then injected intraperitoneally with D-glucose (20%solution; 2 g per kg body weight). For ITTs, mice in the randomly fed state were
injected intraperitoneally with human regular insulin (Eli Lilly). We collectedblood before injection and at different times after injection (as indicated infigures) and glucose and insulin values were determined. We determineddiacylglycerol and ceramide contents in the liver lipid extracts by the diacylglycerol kinase assay as described previously47. We separated phosphorylated
derivatives of diacylglycerol and thin-layer chromatography and visualized andquantified radioactive bands with an imaging analyzer.
In vivo insulin stimulation assay. We fasted mice for 24 h and anesthetizedthem by injection of 30 mg/kg of pentobarbitone (Abbott Laboratories). We
then opened the peritoneal cavity and injected either saline control or insulin(5 units into the inferior vena cava. After 5 min, the liver, muscle andepididymal white adipose tissue were rapidly excised and immediately frozen
in liquid nitrogen. We performed immunoprecipitation and immunoblot
analysis of insulin signaling molecules using tissue homogenates.
Immunoblotting and immunoprecipitation. We performed immunoblot
analysis of tissue and cell lysates, membrane fractions and nuclear extracts aspreviously described10,14,48. We performed immunoprecipitation analysis oftissue lysates as previously described (ref. 14 and Supplementary Methods).
Total RNA preparation, northern blotting and RT-PCR analysis. We performed total RNA preparation and blot hybridization with cDNA probes aspreviously described46. We performed quantitative RT-PCR analysis as
described previously (ref. 49 and Supplementary Methods).
Determination of fatty acid synthesis and fatty acid oxidation in mouseprimary hepatocytes in vitro. Fatty acid synthesis and fatty acid oxidation in
freshly isolated hepatocytes were measured as previously described50.

ADVANCE ONLINE PUBLICATION

NATURE MEDICINEARTICLES© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

Preparation of recombinant adenovirus and adenovirus treatment. We
subcloned Elovl6-specific RNA interference (RNAi) constructs into the U6
entry vector (Invitrogen) using a primer specific for the Elovl6 coding sequence,
5¢-GCGAGCCAAGTTTGAACTTCAGA-3¢, and generated the recombinant adenoviral plasmid by homologous recombination with the pAd promoterless vector(Invitrogen). We subcloned mouse Elovl6-coding cDNA into the pENTR/
D-TOPO vector (Invitrogen) and generated recombinant adenoviral plasmid byhomologous recombination with pAd/CMV/V5-DEST vector (Invitrogen). We
produced recombinant adenovirus in HEK293 cells and purified them aspreviously described14,48. We intravenously injected mice with adenoviruses
expressing LacZ RNAi or Elovl6 RNAi at a dose of 1  109 PFU and adenoviruses
expressing GFP or Elovl6 at a dose of 1  108 PFU. Cells were infected withadenoviruses expressing GFPor Elovl6 at a multiplicity of infection of 100 for 16 h.
Culture of Hepa1c1c7 cells. Mouse hepatoma Hepa1c1c7 cells were cultured in
MEMa supplemented with 10% FBS and 1% penicillin/streptomycin. For
insulin-signaling analysis, we changed cells into serum-free medium 4 h before
the experiment and then treated them with or without insulin (100 nM,
10 min). Cells were treated with fatty acids as described previously49.
Statistical analyses. Data are expressed as means ± s.e.m. Differences between
two groups were assessed using the unpaired two-tailed Student’s t-test. Data
sets involving more than two groups were assessed by ANOVA. Glucose andinsulin tolerance tests were analyzed by repeated-measures ANOVA with
Statview Software (BrainPower).
Note: Supplementary information is available on the Nature Medicine website.
ACKNOWLEDGMENTS
We thank T. Ide and H. Daitoku for technical help and/or useful comments. This
work was supported by grants-in-aid from the Ministry of Science, Education,
Culture and Technology of Japan; a grant for the Japan Foundation for Applied
Enzymology; and a grant from The Naito Foundation.
AUTHOR CONTRIBUTIONS
T. Matsuzaka carried out most of the experiments, data analysis and prepared
the figures with significant help from M.I., S.O., N. Ishigaki, H.I., Y.I. and
T. Karasawa. H. Shimano developed the idea for and supervised the study,
designed protocols, developed collaborations and wrote the manuscript.
N. Yahagi contributed to many in vivo experiments and to training T. Matsuzaka
in experimental techniques. T. Kato carried out measurements of fatty
acid composition and assisted with hepatocyte isolations, fatty acidtreatment to the cell and mice and adenoviral experiments. A.A. assisted with thein vivo adenoviral experiments. T.Y. assisted with PKCe analysis and cell-line
experiments. N. Inoue assisted with ELISA measurements and developed the DXA
protocols for analyzing fat. S.K. assisted with RT-PCR analysis. T. Matsuicontributed to primary myotube culture and the 2-DG uptake assay. M.S. andK.O. participated in the experimental design and in training T. Matsuzaka inexperimental techniques. A.H.H. reviewed the manuscript. Y.N. contributed toimmunoblot analysis of insulin signaling, adenoviral work and discussion of theresults. A.T. contributed to GTT, ITT, HF-HS diet–feeding to the mice anddiscussion of the results. H. Suzuki, S.Y., H. Sone, H.T. and J.O. contributed toexperimental design, data analysis, interpretation and presentation. N. Yamada
supervised the study, contributed crucial ideas to the project and reviewed
the manuscript.
Published online at http://www.nature.com/naturemedicine
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions

1. Riccardi, G., Giacco, R. & Rivellese, A.A. Dietary fat, insulin sensitivity and themetabolic syndrome. Clin. Nutr. 23, 447–456 (2004).
2. Unger, R.H. Minireview: weapons of lean body mass destruction: the role of ectopic
lipids in the metabolic syndrome. Endocrinology 144, 5159–5165 (2003).
3. Cai, D. et al. Local and systemic insulin resistance resulting from hepatic activation of
IKKb and NFbB. Nat. Med. 11, 183–190 (2005).
4. Arkan, M.C. et al. IKK-beta links inflammation to obesity-induced insulin resistance.
Nat. Med. 11, 191–198 (2005).
5. Nakatani, Y. et al. Modulation of the JNK pathway in liver affects insulin resistancestatus. J. Biol. Chem. 279, 45803–45809 (2004).
6. Ozcan, U. et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2
diabetes. Science 306, 457–461 (2004).

NATURE MEDICINEADVANCE ONLINE PUBLICATION

7. Howard, J.K. et al. Enhanced leptin sensitivity and attenuation of diet-induced obesity
in mice with haploinsufficiency of Socs3. Nat. Med. 10, 734–738 (2004).
8. Inoue, H. et al. Role of hepatic STAT3 in brain-insulin action on hepatic glucose
production. Cell Metab. 3, 267–275 (2006).
9. Torisu, T. et al. The dual function of hepatic SOCS3 in insulin resistance in vivo. Genes
Cells 12, 143–154 (2007).
10. Shimano, H. et al. Sterol regulatory element-binding protein-1 as a key transcription
factor for nutritional induction of lipogenic enzyme genes. J. Biol. Chem. 274,
35832–35839 (1999).
11. Shimano, H. Sterol regulatory element-binding proteins (SREBPs): transcriptional
regulators of lipid synthetic genes. Prog. Lipid Res. 40, 439–452 (2001).
12. Horton, J.D. et al. Combined analysis of oligonucleotide microarray data fromtransgenic and knockout mice identifies direct SREBP target genes. Proc. Natl.
Acad. Sci. USA 100, 12027–12032 (2003).
13. Lin, J. et al. Hyperlipidemic effects of dietary saturated fats mediated through
PGC-1beta coactivation of SREBP. Cell 120, 261–273 (2005).
14. Ide, T. et al. SREBPs suppress IRS-2-mediated insulin signalling in the liver. Nat. Cell
Biol. 6, 351–357 (2004).
15. Moon, Y.A., Shah, N.A., Mohapatra, S., Warrington, J.A. & Horton, J.D. Identification ofa mammalian long chain fatty acyl elongase regulated by sterol regulatory elementbinding proteins. J. Biol. Chem. 276, 45358–45366 (2001).
16. Matsuzaka, T. et al. Cloning and characterization of a mammalian fatty acyl-CoA elongase
as a lipogenic enzyme regulated by SREBPs. J. Lipid Res. 43, 911–920 (2002).
17. Leonard, A.E., Pereira, S.L., Sprecher, H. & Huang, Y.S. Elongation of long-chain fatty
acids. Prog. Lipid Res. 43, 36–54 (2004).
18. Wang, Y. et al. Regulation of hepatic fatty acid elongase and desaturase expression indiabetes and obesity. J. Lipid Res. 47, 2028–2041 (2006).
19. Maeda, N. et al. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30.
Nat. Med. 8, 731–737 (2002).
20. Weisberg, S.P. et al. Obesity is associated with macrophage accumulation in adipose
tissue. J. Clin. Invest. 112, 1796–1808 (2003).
21. Xu, H. et al. Chronic inflammation in fat plays a crucial role in the development ofobesity-related insulin resistance. J. Clin. Invest. 112, 1821–1830 (2003).
22. Nakae, J., Kitamura, T., Silver, D.L. & Accili, D. The forkhead transcription factor
Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. J. Clin.
Invest. 108, 1359–1367 (2001).
23. Wolfrum, C., Asilmaz, E., Luca, E., Friedman, J.M. & Stoffel, M. Foxa2 regulates lipid
metabolism and ketogenesis in the liver during fasting and in diabetes. Nature 432,
1027–1032 (2004).
24. Zhou, G. et al. Role of AMP-activated protein kinase in mechanism of metformin
action. J. Clin. Invest. 108, 1167–1174 (2001).
25. Samuel, V.T. et al. Mechanism of hepatic insulin resistance in non-alcoholic fatty liverdisease. J. Biol. Chem. 279, 32345–32353 (2004).
26. Yu, C. et al. Mechanism by which fatty acids inhibit insulin activation of insulin
receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity inmuscle. J. Biol. Chem. 277, 50230–50236 (2002).
27. Holland, W.L. et al. Inhibition of ceramide synthesis ameliorates glucocorticoid-,saturated-fat-, and obesity-induced insulin resistance. Cell Metab. 5, 167–179 (2007).
28. Samuel, V.T. et al. Inhibition of protein kinase Cepsilon prevents hepatic insulin
resistance in nonalcoholic fatty liver disease. J. Clin. Invest. 117, 739–745 (2007).
29. Friedman, J.M. Obesity in the new millennium. Nature 404, 632–634 (2000).
30. Chakravarthy, M.V. et al. ‘‘New’’ hepatic fat activates PPARalpha to maintain glucose,lipid, and cholesterol homeostasis. Cell Metab. 1, 309–322 (2005).
31. Ntambi, J.M. et al. Loss of stearoyl-CoA desaturase-1 function protects mice against
adiposity. Proc. Natl. Acad. Sci. USA 99, 11482–11486 (2002).
32. Dobrzyn, P. et al. Stearoyl-CoA desaturase 1 deficiency increases fatty acid oxidationby activating AMP-activated protein kinase in liver. Proc. Natl. Acad. Sci. USA 101,
6409–6414 (2004).
33. Shimada, M., Tritos, N.A., Lowell, B.B., Flier, J.S. & Maratos-Flier, E. Mice lacking
melanin-concentrating hormone are hypophagic and lean. Nature 396, 670–674 (1998).
34. Loftus, T.M. et al. Reduced food intake and body weight in mice treated with fatty acidsynthase inhibitors. Science 288, 2379–2381 (2000).
35. Elchebly, M. et al. Increased insulin sensitivity and obesity resistance in mice lacking
the protein tyrosine phosphatase-1B gene. Science 283, 1544–1548 (1999).
36. Abu-Elheiga, L., Matzuk, M.M., Abo-Hashema, K.A. & Wakil, S.J. Continuous fatty acidoxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2. Science
291, 2613–2616 (2001).
37. Neumann-Haefelin, C. et al. Muscle-type specific intramyocellular and hepatic lipid
metabolism during starvation in wistar rats. Diabetes 53, 528–534 (2004).
38. Hiraoka-Yamamoto, J. et al. Serum lipid effects of a monounsaturated (palmitoleic)fatty acid–rich diet based on macadamia nuts in healthy, young Japanese women. Clin.
Exp. Pharmacol. Physiol. 31 Suppl 2, S37–S38 (2004).
39. Kliewer, S.A. et al. Fatty acids and eicosanoids regulate gene expression through directinteractions with peroxisome proliferator-activated receptors alpha and gamma. Proc.
Natl. Acad. Sci. USA 94, 4318–4323 (1997).
40. Wisely, G.B. et al. Hepatocyte nuclear factor 4 is a transcription factor that constitutively binds fatty acids. Structure 10, 1225–1234 (2002).
41. Perreault, M. & Marette, A. Targeted disruption of inducible nitric oxide synthase
protects against obesity-linked insulin resistance in muscle. Nat. Med. 7, 1138–1143(2001).
42. Hotamisligil, G.S. et al. Uncoupling of obesity from insulin resistance through atargeted mutation in aP2, the adipocyte fatty acid binding protein. Science 274,
1377–1379 (1996).

9

ARTICLES
47. Turinsky, J., O’Sullivan, D.M. & Bayly, B.P. 1,2-Diacylglycerol and ceramide levels in
insulin-resistant tissues of the rat in vivo. J. Biol. Chem. 265, 16880–16885(1990).
48. Nakagawa, Y. et al. TFE3 transcriptionally activates hepatic IRS-2, participates ininsulin signaling and ameliorates diabetes. Nat. Med. 12, 107–113 (2006).
49. Kato, T. et al. Granuphilin is activated by SREBP-1c and involved in impaired insulin
secretion in diabetic mice. Cell Metab. 4, 143–154 (2006).
50. Jiang, G. et al. Prevention of obesity in mice by antisense oligonucleotide inhibitors ofstearoyl-CoA desaturase-1. J. Clin. Invest. 115, 1030–1038 (2005).

© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

43. Maeda, K. et al. Role of the fatty acid binding protein mal1 in obesity and insulin
resistance. Diabetes 52, 300–307 (2003).
44. Zambrowicz, B.P. et al. Disruption and sequence identification of 2,000 genes inmouse embryonic stem cells. Nature 392, 608–611 (1998).
45. Sekiya, M. et al. Polyunsaturated fatty acids ameliorate hepatic steatosis in obese miceby SREBP-1 suppression. Hepatology 38, 1529–1539 (2003).
46. Matsuzaka, T. et al. Insulin-independent induction of sterol regulatory element-binding
protein-1c expression in the livers of streptozotocin-treated mice. Diabetes 53,
560–569 (2004).

10

ADVANCE ONLINE PUBLICATION

NATURE MEDICINE