Supplementary Information

ApoC-III Modulates Clearance of Triglyceride-Rich Lipoproteins

Through LDL Family Receptors

Philip L.S.M. Gordts1,2,*, Ryan Nock1, Ni-Huiping Son3, Bastian Ramms1,2,4, Irene Lew1, Jon

C. Gonzales1, Bryan E. Thacker1, Debapriya Basu3, Richard G. Lee5, Adam E. Mullick5,

Mark J. Graham5, Ira J. Goldberg3, Rosanne M. Crooke5, Joseph L. Witztum2 and

Jeffrey D. Esko1,*.

1Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center,

University of California, San Diego, La Jolla, CA, USA

2Department of Medicine, Division of Endocrinology and Metabolism, University of California,

San Diego, La Jolla, CA, USA

3Division of Endocrinology, Diabetes and Metabolism, NYU Langone Medical Center, New York,

NY, USA

4Department of Chemistry, Biochemistry I, Bielefeld University, Bielefeld, Germany

5Ionis Pharmaceuticals, Inc., Carlsbad, CA, USA

*Corresponding authors:

Jeffrey D. Esko, Department of Cellular and Molecular Medicine, University of California, San

Diego, La Jolla, CA 92093-0687, Ph: 858/822-1100, FAX: 858/534-5611, jesko@ucsd.edu

Philip L.S.M. Gordts, Department of Medicine, University of California, San Diego, La Jolla, CA

92093-0687, Ph: 858/246-0994, FAX: 858/534-5611, pgordts@ucsd.edu

SUPPLEMENTARY INFORMATION

SUPPLEMENTAL FIGURES

Figure S1. ApoC-III mediated TG reduction in wild-type mice treated with control ASO,

Ldlr ASO, LRP1 ASO and a combination of LDLR and LRP1 ASOs. (A-C) Liver expression

of (A) Ldlr, (B) Lrp1 and (C) ApoC3 RNA in wild-type mice treated with control ASO (45

mg/kg/week), Ldlr ASO (5 mg/kg/week), LRP1 ASO (5 mg/kg/week), a combination of LDLR (5

mg/kg/week) and LRP1 ASOs (5 mg/kg/week) mice, or a control ASO (45 mg/kg/week). A

separate set of mice were treated simultaneously with apoC-III ASO (10 mg/kg/week). (D)

Fasting plasma TG in wild-type mice treated for 4 weeks with the indicated ASOs. (E) Relative

changes in plasma TG levels induced by apoC-III ASO in wild-type mice treated with control

ASO, Ldlr ASO, LRP1 ASO and a combination of LDLR and LRP1 ASOs. (F) Fasting plasma

cholesterol in wild-type mice treated for 4 weeks with control or apoC-III ASO. Values represent

mean ± SEM (n = 4/group). *p < 0.05 and ** p < 0.01 compared to control ASO treated mice.

ANOVA and Bonferroni post-hoc test.

Ctrl ASO

LDLR A

SO

LRP1 A

SO

LDLR +

LRP1 A

SO

0

50

100

150

200

Liv

er

Ldlr

Ex

pre

ss

ion

(% o

f C

trl A

SO

)

Ctrl ASO

ApoC-III ASO

Ctrl ASO

LDLR A

SO

LRP1 A

SO

LDLR +

LRP1 A

SO

0

50

100

150

200

Liv

er

Apo

c-III

Ex

pre

ss

ion

(% o

f C

trl A

SO

)

Ctrl ASO

ApoC-III ASO

Ctrl ASO

LDLR A

SO

LRP1 A

SO

LDLR +

LRP1 A

SO

0

50

100

150

200

Liv

er

Lrp1

Ex

pre

ss

ion

(% o

f C

trl A

SO

)

Ctrl ASO

ApoC-III ASO

Ctrl ASO

LDLR A

SO

LRP1 A

SO

LDLR +

LRP1 A

SO

0

50

100

150

200

Tri

gly

ce

rid

e (

mg

/dl) Ctrl ASO

ApoC-III ASO

Ctrl ASO

LDLR A

SO

LRP1 A

SO

LDLR +

LRP1 A

SO

0

100

200

300

400

Ch

ole

ste

rol (m

g/d

l) Ctrl ASO

ApoC-III ASO

-40

-30

-20

-10

0

10

Ch

an

ge

in

pla

sm

a t

rig

lyc

eri

de

s

(% o

f C

trl A

SO

)

Ctrl ASO

LDLR ASO

LRP1 ASO

LDLR + LRP1 ASO

A B C

D

** ** ** ** ** ** ** ** *

F

* **

*

***

Figure S1 - Gordts et al.

E

Figure S2. Impact of ApoC-III ASO on body weight of chow fed mutant mice. (A-F)

Bodyweight changes of indicated mice over the 4-week apoC-III ASO treatment. Values

represent mean ± SEM. ANOVA and Bonferroni post-hoc test.

CBA

Figure S2 - Gordts et al

FED

0 1 2 3 4

0

10

20

30

40

Weeks on treatment

Bo

dy

we

igh

t (g

)

Wild-type

Ctrl ASO

ApoC-III ASO

0 1 2 3 4

0

10

20

30

40

Weeks on treatment

Bo

dy

we

igh

t (g

)

Ldlr-/-Lrp1f/fAlbCre+

Ctrl ASO

ApoC-III ASO

0 1 2 3 4

0

10

20

30

40

Weeks on treatment

Bo

dy

we

igh

t (g

)

Ldlr-/-

Ctrl ASO

ApoC-III ASO

0 1 2 3 4

0

10

20

30

40

Weeks on treatment

Bo

dyw

eig

ht

(g)

Ldlr-/-Ndst1f/fAlbCre+

Ctrl ASO

ApoC-III ASO

0 1 2 3 4

0

10

20

30

40

Weeks on treatment

Bo

dy

we

igh

t (g

)

Ndst1f/fAlbCre+

Ctrl ASO

ApoC-III ASO

0 1 2 3 4

0

10

20

30

40

Weeks on treatment

Bo

dy

we

igh

t (g

)

Ndst1f/fLrp1f/fAlbCre+

Ctrl ASO

ApoC-III ASO

Figure S3. Impact of ApoC-III ASO on plasma cholesterol levels in high fat diet fed mutant

mice. Relative changes in fasting plasma cholesterol levels induced by apoC-III ASO in mutant

mice.

-60

-40

-20

0

20

Ndst1f/fAlbCre+Wild-type

Ndst1f/fLrp1f/fAlbCre+

Ldlr-/-Ndst1f/fAlbCre+

Ldlr-/-

Ldlr-/-Lrp1f/fAlbCre+

Ch

an

ge

in

pla

sm

a c

ho

les

tero

l

(% o

f C

on

tro

l A

SO

) Figure S3 - Gordts et al.

Figure S4. Impact of ApoC-III ASO on body weight of high fat diet fed mutant mice. (A-F)

Bodyweight changes of indicated mice over the 5-week apoC-III ASO treatment. Values

represent mean ± SEM. ANOVA and Bonferroni post-hoc test.

CBA

Figure S3 - Gordts et al

FED

0 1 2 3 4 5

0

10

20

30

40

50

Weeks on treatment

Bo

dy

we

igh

t (g

)

Wild-type

Ctrl ASO

ApoC-III ASO

0 1 2 3 4 5

0

10

20

30

40

50

Weeks on treatment

Bo

dyw

eig

ht

(g)

Ldlr-/-Ndst1f/fAlbCre+

Ctrl ASO

ApoC-III ASO

0 1 2 3 4 5

0

10

20

30

40

50

Weeks on treatment

Bo

dy

we

igh

t (g

)

Ndst1f/fAlbCre+

Ctrl ASO

ApoC-III ASO

0 1 2 3 4 5

0

10

20

30

40

50

Weeks on treatment

Bo

dy

we

igh

t (g

)

Ldlr-/-

Ctrl ASO

ApoC-III ASO

0 1 2 3 4 5

0

10

20

30

40

50

Weeks on treatment

Bo

dyw

eig

ht

(g)

Ndst1f/fLrp1f/fAlbCre+

Ctrl ASO

ApoC-III ASO

0 1 2 3 4 5

0

10

20

30

40

50

Weeks on treatment

Bo

dy

we

igh

t (g

)

Ldlr-/-Lrp1f/fAlbCre+

Ctrl ASO

ApoC-III ASO

Figure S5. Characterization of the tamoxifen inducible LPL-deficient mice. (A) Liver,

skeletal muscle, white adipose tissue (WAT) and heart expression of Lpl and apoc-III mRNA

relative to 18s in Lplfl/fl mice treated with (iLpldf) or without tamoxifen to induce inactivation of LPL

expression 2 weeks after tamoxifen administration (n = 4-8/group). (B) Plasma, skeletal muscle,

WAT and heart LPL activity levels 5 min after intravenous heparin (5 U/mouse) injection or a 30

min incubation in Lplfl/fl mice and Lplfl/fl mice treated with tamoxifen (iLpldf) (n = 3/group). In

addition similar LPL activity measurements were obtained in heart homogenates (heart hom.) in

the absence of heparin. (C) Fasting plasma triglyceride and (D) cholesterol levels in Lplfl/fl mice

and Lplfl/fl mice treated with tamoxifen (iLpldf) on a chow diet or a high-fat diet. Plasma lipid levels

were measured 2 weeks after the last tamoxifen administration. (E) FPLC profiles of lipoprotein

TG in plasma from pooled fasted Lplfl/fl mice and Lplfl/fl mice treated with tamoxifen to induce

A B

C D

Figure S5 - Gordts et al

Liv

er

Muscle

WAT

Heart

0

50

100

150

Tis

su

e L

PL

ex

pre

ssio

n

(% o

f L

plfl

/fl )

Lplfl/fl

iLpldf

*** *** **

Chow diet HF diet

0

500

1000

1500

Tri

gly

ce

rid

es (

mg

/dl) Lplfl/fl

iLpldf

***

**

Chow diet HF diet

0

50

100

150

Ch

ole

ste

ro

l (m

g/d

l) Lplfl/fl

iLpldf

**

0 5 10 15 20 25 30 35 40 45

0

10

20

30

Fraction

Tri

gly

ce

rid

e (

mg

/dl)

Lplfl/fliLpldf

0 5 10 15 20 25 30 35 40 45

0

1

2

3

4

5

Fraction

Ch

ole

ste

rol (m

g/d

l)

iLpldf Lplfl/fl

CR/VLDL

IDL/LDL HDL

CR/VLDL

IDL/LDL

HDL

E F

Pla

sm

a

Muscle

WAT

Heart

Heart H

om

.

0

50

100

150

Tis

su

e L

PL

acti

vit

y

(% o

f L

plfl/

fl )

Lplfl/fl

iLpldf

**

** ***** **

inactivation of LPL expression (iLpldf) on a chow diet (n = 3 per group). (F) FPLC profiles of

lipoprotein cholesterol in plasma from fasted Lplfl/fl mice and Lplfl/fl mice treated with tamoxifen

(iLpldf) on a chow diet. The elution positions of chylomicron remnant (CR)/very low-density

lipoprotein (VLDL), intermediate density lipoprotein (IDL)/low-density lipoprotein (LDL), and

high-density lipoprotein (HDL) are indicated. Values represent mean ± SEM. *p < 0.01, **p <

0.005 and ***p < 0.001 compared to Lplfl/fl mice. ANOVA and Bonferroni post-hoc test.

Figure S6. ApoC-III mediated TG reduction in LPL-deficient mice. (A) Western blot analysis

of apoC-III on TRLs isolated from fasted tamoxifen-induced Lplfl/fl mice treated with or without

tamoxifen and/or apoC-III ASO. (B) Fasting plasma TG levels in tamoxifen-induced Lplfl/fl mice

treated for the indicated number of weeks with control or apoC-III ASO (n = 8/group). (C)

Postprandial TG clearance in fasted tamoxifen-induced iLpldf mice treated for 4 weeks with

control or apoC-III ASO after giving 250 µL of corn oil by oral gavage. TG levels were measured

at the indicated time points (n = 8/group). Values represent mean ± SEM. ** p < 0.01 and *** p <

0.001 compared to control ASO or tamoxifen-treated mice. ANOVA and Bonferroni post-hoc

test.

B C

A

ApoC-III

Tamoxifen:

ApoC-III ASO:

- - + + + + + +

- - - - - + + +

Lplfl/fl

p<0.01

** ********

Figure S6 - Gordts et al

0 1 2 3

0

200

400

600

800

Time on Treatment (wks)

Tri

gly

ce

rid

e (

mg

/dL

)

Ctrl ASO

ApoCIII ASO

0 2 4 6

0

300

600

900

1200

Time post gavage (h)

Tri

gly

ce

rid

e (

mg

/dL

) Ctrl ASO

ApoCIII ASO

Fig. S7. [3H]Retinol labeled TRL lipid Composition. (A) Analysis of triglyceride, cholesterol

esters (CE) and free cholesterol (FC) levels per mg protein, ratio of free cholesterol to

cholesterol esters, ratio of triglycerides to cholesterol esters and (B) retinol counts per µg protein

of TRLs isolated 3 hours after corn oil gavage with 250 µl corn oil containing 5 µCi [3H]Retinol]

from Ldlr-/-Ndst1fl/flAlbCre+ mice on a chow diet treated for 4 weeks with control ASO or ApoC-III

ASO. TRLs were prepared by ultracentrifugation (δ = 1.006 g/mL) (n = 3-6 mice per treatment

group). T-test, ANOVA and Bonferroni post-hoc test.

B

Figure S7 - Gordts et al

TG CE FCTG/C

EFC/C

E0

200

400

600

800

0

2

4

6

8

10

g / m

g pr

otei

n

Control ASOApoC-III ASO

Ratio

[3H]Retinol0

200

400

600

800

1000

cpm

/ g

prot

ein

Control ASOApoC-III ASO

A

Fig. S8. TRL Apolipoprotein Composition. (A) SDS-PAGE analysis of plasma TRLs from

indicated mice on a chow diet. TRLs were prepared by ultracentrifugation (δ = 1.006 g/mL) and

pooled samples (n = 2-5 mice per pool) were separated on 4-12% SDS-PAGE gradient gels.

The proteins were visualized using silver stain. The positions of apoB-100, apoB-48, apoE and

apoCs are indicated on the right side. The lane on the left side contains protein MW markers.

(B-C) Western blot analysis for apoC-III of pooled TRLs (5 µg) isolated from fasted mutant mice

(n = 2-5/pool) on (B) a chow diet and (C) a high fat diet.

A

B

C

SUPPLEMENTAL TABLES

Table S1 Murine Antisense Oligonucleotide Sequences

Ionis number Name Target Gene Sequence

ION 141923 Control ASO N.A. (scramble) 5’-CCTTCCCTGAAGGTTCCTCC

ION 440726 apoC-III ASO apoC-III 5'-CCAGCTTTATTAGGGACAGC-3'

ION 713852 LDLR ASO Ldlr 5'-CTTTATCTTTAACCTC-3'

ION 793588 LRP1 ASO Lrp1 5'-CCCAGTAGATGTTGCCTGCA-3'

Table S2 PCR Primers

Gene Forward primer Reverse primer

18s 5'-CCATCCAATCGGTAGTAGCG-3' 5'-GTAACCCGTTGAACCCCATT-3'

Apoc-III 5’-TGCAGGGCTACATGGAACAA-3’ 5’-TCGGACTCCTGCACGCTACTT-3’

Ldlr 5'-GACCGCAGCGAGTACACCA-3' 5'-TCACCTCCGTGTCGAGAGC-3'

Lpl 5'-GCTGGTGGGAAATGATGTG-3' 5'-TGGACGTTGTCTAGGGGGTA-3'

Lrp1 5'-TGGTCTGATGTGCGGACTCA-3' 5'-AACAGATTTCGGGAGACCCA -3'

Tbp 5’-GAAGCTGCGGTACAATTCCAG-3’ 5’-CCCCTTGTACCCTTCACCAAT-3’