N A F L D A c t iv it y S c o r e I n fla m m a t io n B a ......ILC2019 Poster presented at: M R 1...
1
ILC2019 Poster presented at: 0 5 10 15 20 Liver weight (% body weight) Chow C57 AMLN ob/ob -NASH GAN ob/ob -NASH *** *** NAFLD Activity Score GAN diet AMLN diet 0 2 4 6 8 10 12 Lower Same Higher Number of animals Novel Gubra Amylin NASH (GAN) diet-induced obese mouse models of biopsy-confirmed non-alcoholic steatohepatitis Sanne S. Veidal 1 , Michael Feigh 1 , Michelle L. Boland 2 , Denise Oró 1 , Kirstine S. Tølbøl 1 , Jacob Jelsing 1 , Niels Vrang 1 , Henrik H. Hansen 1 , James L. Trevaskis 2 1 Gubra, Hørsholm, Denmark; 2 MedImmune, Gaithersburg, MD. Corresponding author: [email protected] INTRODUCTION AND AIM The 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. METHODS Male 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 in ob/ob-NASH mice fed AMLN or GAN diet B C A Figure 1 | Study 1 | Metabolic parameters in ob/ob mice fed AMLN (AMLN ob/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) AUC glucose (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 AMLN diet NAS Pre Post 0 1 2 3 4 5 6 7 8 GAN diet NAS Pre Post 0 1 2 3 4 5 6 7 8 AMLN diet Steatosis Pre Post 0 1 2 3 GAN diet Steatosis Pre Post 0 1 2 3 AMLN diet Inflammation Pre Post 0 1 2 3 GAN diet Inflammation Pre Post 0 1 2 3 AMLN diet Ballooning Pre Post 0 1 2 GAN diet Ballooning Pre Post 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 diet GAN diet Fibrosis score GAN diet AMLN diet 0 2 4 6 8 10 12 Lower Same Higher Number of animals B AMLN diet Fibrosis Pre Post 0 1 2 3 GAN diet Fibrosis Pre Post 0 1 2 3 C Weight gain, body composition and liver mass in ob/ob-NASH mice fed AMLN or GAN diet C57 Chow w8 C57 Chow w12 C57 Chow w16 GAN diet w8 AMLN diet w8 GAN diet w12 AMLN diet w12 GAN diet w16 AMLN diet w16 0 20 40 60 80 100 Tissue mass (%) Fat mass Lean mass Other 0 60 120 180 0 200 400 600 Time (min) Glucose (mg/dL) Chow C57 GAN ob/ob -NASH AMLN ob/ob -NASH * * 15 30 45 90 * * * 20000 40000 60000 80000 100000 Glucose AUC (mg/dL*min) Chow C57 AMLN ob/ob -NASH GAN ob/ob -NASH * -1 0 1 2 3 4 5 6 7 8 9 1 01 11 21 31 41 51 6 40 50 60 70 80 Dieting week Body weight (g SEM) GAN ob/ob -NASH AMLN ob/ob -NASH ipGTT * * * * * * A B D 0 15 30 0 5000 10000 15000 20000 Insulin (pg/ml) Chow C57 GAN ob/ob -NASH AMLN ob/ob -NASH Minutes ** ** ** ** ** ** E C F Plasma and liver biomarker changes in ob/ob-NASH mice fed AMLN or GAN diet GAN diet AMLN diet 0 500 1000 1500 Plasma ALT (U/L) GAN diet AMLN diet 0 200 400 600 800 Plasma AST (U/L) GAN diet AMLN diet 0 50 100 150 200 Plasma TG (mg/dL) GAN diet AMLN diet 0 200 400 600 800 Plasma TC (mg/dL) GAN diet AMLN diet 0 50 100 150 Liver TG (mg/g liver) GAN diet AMLN diet 0 5 10 15 20 25 Liver TC (mg/g liver) Comparable biopsy-confirmed NALFD Activity Scores in 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/ob mice fed AMLN or GAN diet for 16 weeks. Horizontal dotted line indicates corresponding 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 (HE staining), inflammation (Galectin-3 IHC) and fibrosis (Col1a1 IHC) determined by imaging-based histomorphometry. Scale bar 100 μm. Comparable quantitative histopathological changes in ob/ob-NASH mice fed AMLN or GAN diet Steatosis (post-biopsy) GAN diet AMLN diet 0 10 20 30 40 Liver lipid (% fractional area) Collagen 1a1 (post-biopsy) GAN diet AMLN diet 0 2 4 6 8 10 12 Col1a1 (% fractional area) Galectin-3 (post-biopsy ) GAN diet AMLN diet 0 1 2 3 4 5 Galectin-3 (% fractional area) Comparable liver transcriptome changes in ob/ob-NASH mice fed AMLN or GAN diet Figure 6 | RNA sequencing. Hepatic gene expression profiles in chow-fed C57BL/6J mice (Chow C57), AMLN and GAN ob/ob-NASH mice after 16 weeks of feeding. 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-NASH mice vs. chow-fed C57BL/6J mice. C) Relative gene expression levels (z-scores) of differentially regulated candidate genes associated with NASH and fibrosis. AMLN diet GAN diet AMLN diet GAN diet AMLN diet GAN diet AMLN ob/ob-NASH GAN ob/ob-NASH Chow C57 -5 0 5 10 -20 0 20 PC1 (86%) PC2 (3%) AMLN ob/ob-NASH vs chow C57 GAN ob/ob-NASH vs. chow C57 9187 573 538 B A C Relative expression 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 in DIO-NASH mice fed AMLN or GAN diet High rates of bridging fibrosis in ob/ob-NASH mice fed AMLN or GAN diet for extended periods Steatosis Chow C57 GAN DIO-NASH AMLN DIO-NASH 0 5 10 15 20 25 30 0 1 2 3 Number of animals Inflammation Chow C57 GAN DIO-NASH AMLN DIO-NASH 0 5 10 15 20 25 30 0 1 2 3 Number of animals Ballooning Chow C57 GAN DIO-NASH AMLN DIO-NASH 0 5 10 15 20 25 30 0 1 2 Number of animals NAFLD Activity Score Chow C57 GAN DIO-NASH AMLN DIO-NASH 0 5 10 15 20 25 30 0 1 2 3 5 6 7 4 Number of animals Fibrosis Chow C57 GAN DIO-NASH AMLN DIO-NASH 0 5 10 15 20 25 30 0 1 2 3 Number of animals Col1a1 Chow C57 GAN DIO-NASH AMLN DIO-NASH 0 2 4 6 8 10 Col1a1 (% area SEM) *** *** Figure 8 | Terminal liver histopathology in C57BL/6J mice fed chow (n=15 mice), AMLN (AMLN DIO-NASH, n=30 mice) or GAN (GAN DIO-NASH, n=30 mice) diet for 28 weeks. Histopathological scores of steatosis (A), lobular inflammation (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 Fibrosis Chow ob/ob GAN ob/ob AMLN ob/ob 0 5 10 15 20 25 30 Number of animals 0 1 2 3 Col1a1 Chow ob/ob GAN ob/ob AMLN ob/ob 0 2 4 6 8 10 12 14 16 18 20 Col1a1 (% area SEM) *** *** 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) diet for 30 weeks. Left panel, histopathological scoring of fibrosis. Right panel, fractional (%) area of collagen-1a1. ***p<0.001 vs. chow-fed ob/ob mice. FRI-354 Sanne Veidal DOI: 10.3252/pso.eu.ILC2019.2019 Metabolism, alcohol and toxicity
Transcript of N A F L D A c t iv it y S c o r e I n fla m m a t io n B a ......ILC2019 Poster presented at: M R 1...
ILC2019
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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
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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.
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Figure 4 | A) Representative images of
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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
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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.
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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
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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.
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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.
FRI-3
54Sa
nne V
eidal
DOI: 1
0.325
2/pso
.eu.IL
C201
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