0
5
1 0
1 5
2 0
Liv
er
we
igh
t
(% b
od
y w
eig
ht)
C h o w C 5 7
A M LN o b /o b -N A S H
G AN o b /o b -N A S H
* * ** * *
N A F L D A c tiv ity S c o re
G A N d ie t A M L N d ie t
0
2
4
6
8
1 0
1 2
L o w e r
S a m e
H ig h e r
Nu
mb
er
of
an
ima
ls
Novel Gubra Amylin NASH (GAN) diet-induced obese mouse models of biopsy-confirmednon-alcoholic steatohepatitis Sanne S. Veidal1, Michael Feigh1 , Michelle L. Boland2, Denise Oró1, Kirstine S. Tølbøl1, Jacob Jelsing1, Niels Vrang1, Henrik H. Hansen1, James L. Trevaskis2
1Gubra, Hørsholm, Denmark; 2MedImmune, Gaithersburg, MD.Corresponding author: [email protected]
INTRODUCTION AND AIMThe Amylin Liver NASH (AMLN) diet-based ob/ob and C57BL/6J mouse models
display clinical translatability with respect to key metabolic and liver biopsy-
confirmed pathological changes associated with non-alcoholic steatohepatitis
(NASH). A recent FDA ban on trans-fats in foods has prompted the
development of a new NASH diet capable of promoting a compatible level of
disease, as the AMLN diet contains trans-fat-containing Primex shortening. The
present study aimed to assess the metabolic and liver pathological phenotype
in ob/ob and C57BL/6J mice fed a palmitic acid-enriched high-fat diet with a
nutrient composition and caloric content similar to the AMLN diet.
METHODSMale ob/ob mice were fed chow, AMLN diet (40% total fat kcal of which 18.5%
were trans-fat kcal, 20% fructose, 2% cholesterol; Research Diets #D09100301)
or a modified AMLN diet with Primex substituted by equivalent amounts of
palm oil (Research Diets, #D09100310), termed Gubra Amylin NASH (GAN) diet,
for up to 30 weeks. C57BL/6J mice were fed the same diets for 28 weeks.
NAFLD activity score (NAS) and fibrosis staging was assessed. Quantitative
histomorphometric analyses included fractional (%) area of steatosis
(hematoxylin-eosin), inflammation (galectin-3), and collagen (Col1a1). RNA
sequencing was performed on terminal liver samples.
RESULTS
Figure 3 | A) Representative images of terminal liver morphology (HE staining,
20x magnification, scale bar 100 µm). B) Composite NAFLD Activity Score (NAS,
number of animals with higher, same or lower post-biopsy score compared to
pre-biopsy). C) Individual NAS, steatosis, inflammation and ballooning scores.
Paired pre- and post-biopsies were samples at 9 and 16 weeks of feeding,
respectively.
CONCLUSIONS
• Modification of the AMLN diet by substitution of Primex shortening with palm oil (GAN diet) results in a maintained NASH phenotype in both ob/ob-NASH and DIO-NASH mice.
• Compared to the AMLN diet, the GAN diet promotes further body weight gain and impairs glucose intolerance in ob/ob-NASH mice.
• The clear metabolic and histopathological hallmarks of fibrotic NASH in ob/ob-NASH and DIO-NASH mice fed the GAN diet highlight the suitability of this model for characterizing novel drug therapies for NASH.
AMLN diet
Comparable biopsy-confirmed fibrosis scores inob/ob-NASH mice fed AMLN or GAN diet
B
C
A
Figure 1 | Study 1 | Metabolic parameters in ob/ob mice fed AMLN (AMLNob/ob-NASH) or GAN (GAN ob/ob-NASH) diet for 16 weeks. A) Body weight gain,B) body composition, C) terminal liver weight (week 16), D) ipGTT, E) AUCglucose (0-180 min), F) plasma insulin (0, 15, 30 min). ipGTT was performed in week 7 of the
dieting period. *p<0.05, **p<0.01, ***p<0.001 vs. chow-fed C57BL/6J (chow C57)
control mice.
GAN diet
A M L N d ie t
N A S
P re P o s t
0
1
2
3
4
5
6
7
8
G A N d ie t
N A S
P re P o s t
0
1
2
3
4
5
6
7
8
A M L N d ie t
S te a to s is
P re P o s t
0
1
2
3
G A N d ie t
S te a to s is
P re P o s t
0
1
2
3
A M L N d ie t
In f la m m a tio n
P re P o s t
0
1
2
3
G A N d ie t
In f la m m a tio n
P re P o s t
0
1
2
3
A M L N d ie t
B a llo o n in g
P re P o s t
0
1
2
G A N d ie t
B a llo o n in g
P re P o s t
0
1
2
A
Figure 4 | A) Representative images of
terminal fibrosis morphology after 16 weeks
of feeding (PSR staining, 20x magnification,
scale bar 100 µm). B) Fibrosis scores (number
of animals with higher, same or lower post-
biopsy score compared to pre-biopsy). C)
Individual fibrosis scores.
AMLN dietGAN diet F ib ro s is s c o re
G A N d ie t A M L N d ie t
0
2
4
6
8
1 0
1 2
L o w e r
S a m e
H ig h e r
Nu
mb
er
of
an
ima
ls
B
A M L N d ie t
F ib ros is
P re P o s t
0
1
2
3
G A N d ie t
F ib ros is
P re P o s t
0
1
2
3
C
Weight gain, body composition and liver mass inob/ob-NASH mice fed AMLN or GAN diet
C57 C
how
w8
C57 C
how
w12
C57 C
how
w16
GA
N d
iet w
8
AM
LN
die
t w
8
GA
N d
iet w
12
AM
LN
die
t w
12
GA
N d
iet w
16
AM
LN
die
t w
16
0
2 0
4 0
6 0
8 0
1 0 0
Tis
su
e m
as
s (
%)
F a t m a s s L e a n m a s s O th e r
0 6 0 1 2 0 1 8 0
0
2 0 0
4 0 0
6 0 0
T im e (m in )
Glu
co
se
(m
g/d
L)
C h o w C 5 7
G AN o b /o b -N A S H
A M LN o b /o b -N A S H
**
1 5 3 0 4 5 9 0
** *
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
Glu
co
se
AU
C (
mg
/dL
*m
in)
C h o w C 5 7
A M LN o b /o b -N A S H
G AN o b /o b -N A S H
*
-1 0 1 2 3 4 5 6 7 8 9 1 01 11 21 31 41 51 6
4 0
5 0
6 0
7 0
8 0
D ie tin g w e e k
Bo
dy
we
igh
t (g
S
EM
)
G A N o b /o b -N A S H
A M L N o b /o b -N A S H
ipG TT
*****
*
A B
D
0 1 5 3 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
Ins
uli
n (
pg
/ml)
C h o w C 5 7
G AN o b /o b -N A S H
A M LN o b /o b -N A S H
M in u te s
* * * ** *
* ** * * *
E
C
F
Plasma and liver biomarker changes inob/ob-NASH mice fed AMLN or GAN diet
GA
N d
iet
AM
LN
die
t0
5 0 0
1 0 0 0
1 5 0 0
Pla
sm
a A
LT
(U
/L)
GA
N d
iet
AM
LN
die
t0
2 0 0
4 0 0
6 0 0
8 0 0
Pla
sm
a A
ST
(U
/L)
GA
N d
iet
AM
LN
die
t0
5 0
1 0 0
1 5 0
2 0 0
Pla
sm
a T
G (
mg
/dL
)
GA
N d
iet
AM
LN
die
t0
2 0 0
4 0 0
6 0 0
8 0 0
Pla
sm
a T
C (
mg
/dL
)
GA
N d
iet
AM
LN
die
t0
5 0
1 0 0
1 5 0
Liv
er
TG
(m
g/g
liv
er)
GA
N d
iet
AM
LN
die
t0
5
1 0
1 5
2 0
2 5
Liv
er
TC
(m
g/g
liv
er)
Comparable biopsy-confirmed NALFD Activity Scoresin ob/ob-NASH mice fed AMLN or GAN diet
Figure 2 | Plasma alanine aminotransferase (ALT), aspartate aminotransferase
(AST), total triglycerides (TG), total cholesterol (TC) and liver lipids (TG, TC) in ob/obmice fed AMLN or GAN diet for 16 weeks. Horizontal dotted line indicatescorresponding level in age-matched chow-fed C57BL/6J mice.
Figure 5 | Top panels: Representative images of terminal fibrosis morphology
after 16 weeks of feeding. Lower panels: Fractional (%) area of steatosis (HEstaining), inflammation (Galectin-3 IHC) and fibrosis (Col1a1 IHC) determinedby imaging-based histomorphometry. Scale bar 100 µm.
Comparable quantitative histopathological changes inob/ob-NASH mice fed AMLN or GAN diet
S te a to s is
(p o s t-b io p s y )
G A N d ie t A M L N d ie t
0
1 0
2 0
3 0
4 0
Liv
er
lip
id
(% f
rac
tio
na
l a
rea
)
C o lla g e n 1 a 1
(p o s t-b io p s y )
G A N d ie t A M L N d ie t
0
2
4
6
8
1 0
1 2
Co
l1a
1
(% f
rac
tio
na
l a
rea
)
G a le c tin -3
(p o s t-b io p s y )
G A N d ie t A M L N d ie t
0
1
2
3
4
5
Ga
lec
tin
-3
(% f
rac
tio
na
l a
rea
)
Comparable liver transcriptome changes inob/ob-NASH mice fed AMLN or GAN diet
Figure 6 | RNA sequencing. Hepatic gene expression profiles in chow-fedC57BL/6J mice (Chow C57), AMLN and GAN ob/ob-NASH mice after 16 weeks offeeding. A) Principal component analysis (PCA) of samples based on top 500
most variable gene expression levels. B) Group-wise comparison of the total
number of differentially expressed genes between GAN and AMLN ob/ob-NASHmice vs. chow-fed C57BL/6J mice. C) Relative gene expression levels (z-scores)of differentially regulated candidate genes associated with NASH and fibrosis.
AMLN dietGAN diet AMLN dietGAN diet AMLN dietGAN diet
AMLN
ob/ob-NASH
GAN
ob/ob-NASHChow C57
-5
0
5
10
-20 0 20PC1 (86%)
PC
2(3
%)
AMLN ob/ob-NASH vs chow C57
GAN ob/ob-NASH vs. chow C57
9187 573538
BA
C
Re
lativ
e
exp
ressio
n
Highest
Lowest
Lipid metabolism Insulin signalling
AMLN ob/ob-NASH
GAN ob/ob-NASH
Chow C57
AMLN ob/ob-NASH
GAN ob/ob-NASH
Chow C57
Monocyte recruitment Inflammation signalling
Hepatocellular cell death Stellate cell activation/fibrogenesis
AMLN ob/ob-NASH
GAN ob/ob-NASH
Chow C57
FXR signalling
AMLN ob/ob-NASH
GAN ob/ob-NASH
Chow C57
Comparable liver histopathology inDIO-NASH mice fed AMLN or GAN diet
High rates of bridging fibrosis in ob/ob-NASH micefed AMLN or GAN diet for extended periods
S te a to s is
Chow
C57
GA
N D
IO-N
AS
H
AM
LN
DIO
-NA
SH
0
5
1 0
1 5
2 0
2 5
3 0 0
1
2
3
Nu
mb
er
of
an
ima
ls
In flam m ation
Chow
C57
GA
N D
IO-N
AS
H
AM
LN
DIO
-NA
SH
0
5
1 0
1 5
2 0
2 5
3 0 0
1
2
3
Nu
mb
er
of
an
ima
ls
B a llo o n ing
Chow
C57
GA
N D
IO-N
AS
H
AM
LN
DIO
-NA
SH
0
5
1 0
1 5
2 0
2 5
3 0 0
1
2
Nu
mb
er
of
an
ima
ls
N A F L D A c tiv ity S c o re
Chow
C57
GA
N D
IO-N
AS
H
AM
LN
DIO
-NA
SH
0
5
1 0
1 5
2 0
2 5
3 0 0
1
2
3
5
6
7
4
Nu
mb
er
of
an
ima
ls
F ib ros is
Chow
C57
GA
N D
IO-N
AS
H
AM
LN
DIO
-NA
SH
0
5
1 0
1 5
2 0
2 5
3 0 0
1
2
3
Nu
mb
er
of
an
ima
ls
C o l1 a 1
Chow
C57
GA
N D
IO-N
AS
H
AM
LN
DIO
-NA
SH
0
2
4
6
8
1 0
Co
l1a
1
(% a
rea
SE
M)
* * ** * *
Figure 8 | Terminal liver histopathology in C57BL/6J mice fed chow (n=15mice), AMLN (AMLN DIO-NASH, n=30 mice) or GAN (GAN DIO-NASH, n=30mice) diet for 28 weeks. Histopathological scores of steatosis (A), lobularinflammation (B), hepatocyte ballooning (C), composite NAFLD Activity Score
(NAS, D), and fibrosis (E). Fractional (%) area of collagen-1a1 (F). ***p<0.001
vs. chow-fed C57BL/6J mice.
A B
C D
E F
F ib ros is
Chow
ob/o
b
GA
N o
b/o
b
AM
LN
ob/o
b
0
5
1 0
1 5
2 0
2 5
3 0
Nu
mb
er
of
an
ima
ls
0
1
2
3
C o l1 a 1
Chow
ob/o
b
GA
N o
b/o
b
AM
LN
ob/o
b
0
2
4
6
8
1 0
1 2
1 4
1 6
1 8
2 0
Co
l1a
1
(% a
rea
SE
M)
* * *
* * *
Figure 7 Terminal liver histopathology in ob/ob mice fed chow (n=10 mice),
AMLN (AMLN DIO-NASH, n=30 mice) or GAN (GAN DIO-NASH, n=30 mice) dietfor 30 weeks. Left panel, histopathological scoring of fibrosis. Right panel,
fractional (%) area of collagen-1a1. ***p<0.001 vs. chow-fed ob/obmice.
