Fat

1. Loss of Hepatocyte Osteopontin Protected against Non-alcoholic
Steatohepatitis through Regulating Lipid Biosynthesis
Hui HAN 1, Romain Desert 1, Xiaodong Ge 1, Sukanta Das 1, Zhuolun Song 1, Dipti Athavale 1, Wei Chen 1, Harriet Gaskell 1, Daniel
Lantvit 1, Grace Guzman 1 and Natalia Nieto 1
1 Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA
Background & Aims: increased de novo lipogenesis is involved in NASH
progression and the main target in the ongoing clinical trials. Osteopontin
(OPN) is a multifactorial matrix protein induced in hepatocytes and
macrophages during progression of NASH. However, how hepatocyte
derived OPN is involved in NASH especially in which way it regulates lipid
metabolism is completely unknown. Here we hypothesized that OPN
increase biosynthesis of triglyceride and cholesterol to promote NASH
progression.
Methodology: Human datasets were downloaded from Gene Expression
Omnibus and analyzed by general statistical methods. Mice with Opn
ablation in hepatocytes (OpnΔHep) was generated with the scheme in the
figure. WT and OpnΔHep mice were fed a control or NASH-inducing diet (high
fat, cholesterol, fructose) for 6 months. H&E slides were scored for disease
severity. Liver triglyceride and cholesterol were measured with biochemical
approach. Transcriptome profiling was performed by RNA sequencing
followed by calculation of differentially expressed genes with Limma
package in R platform. The gene enrichment analysis were performed by
Networkanalyst. Upstream target analysis were performed by ingenuity
pathway analysis (IPA).
Abstract
Opnfl/fl
×
AlbCre/+
OpnΔHep
OPN
NASH Feeding
Histology
Biochemistry
Transcriptomic

2. 0%
50%
100%
Low High
OPN Expression is Associated to Development of Liver Steatosis
during NASH progression
(A) (B)
**
2
1
0
Steatosis
Score
OPN
Expression
OPN
Expression
Stage Steatosis
OPN Expression
Subject
(%)
*
***
*** ***
Figure 1: OPN Expression is Associated to
Development of Liver Steatosis during NASH
progression. (A) Relative OPN mRNA expression in
human liver biopsies from GSE126848; (B) Relative
OPN mRNA expression in non-fibrotic NASH patients
from GSE151158 with different scores of steatosis; (C)
Percent of NAFLD patients with corresponding
steatosis score. Subjects were stratified by median
SPP1 expression in GSE151158. *p<0.05, **p<0.01,
***p<0.0001, vs control, 0 or low.
(C)

3. Ablation of OPN (OpnΔHep) from Hepaotcyte Decreased
Progression of NASH
Ctrl
NASH
Male Female Male Female
WT OpnΔHep
PV
CV PV
CV
PV
CV
PV
CV
CV
PV
PV
CV
CV
PV
200x 200x 200x 200x
200x 200x 200x 200x
400x 400x
PV
CV
400x 400x
200x
200x 200x
WT OpnΔHep
Ctrl NASH Ctrl NASH
Steatosis 1.31±0.09 2.50±0.05## 2.00±0.07 1.36±0.05**
Inflammation 1.15±0.05 2.00±0.10## 0.33±0.04* 1.09±0.05##**
Ballooning 0.69±0.06 1.60±0.07## 0.67±0.08 1.45±0.05##
NAS 3.15±0.15 6.10±0.19### 3.00±0.13 3.91±0.11**
Figure 2: Ablation of OPN
(OpnΔHep) from Hepatocyte
Decreased Progression of
NASH. Mice were fed for
control or NASH diet for 6
months. (A) H&E staining. Red
arrow, liver steatosis; blue
arrow, inflammatory foci; green
arrow, ballooning. (B) NASH
activity score (NAS). H&E slides
were scored by Kleiner's
system. *p<0.05, **p<0.01, vs
WT with same diet. #p<0.05,
##p<0.01, ###p<0.001, vs Ctrl
diet with same genotype. n=10.

4. 0
2
4
6
8
10
12
(B)
(A)
Hepatic
triglyceride
(mg/g)
0
50
100
150
200
250
300
350
###
###
*
0
100
200
300
400
500
600
Hepatic
cholesterol
(mg/g)
*
**
###
Ctrl NASH Ctrl NASH
WT OpnΔHep
Ctrl NASH Ctrl NASH
WT OpnΔHep
Liver
to
b.w.
Ratio
(%)
###
##
**
Ctrl NASH Ctrl NASH
WT OpnΔHep
###
(C)
OpnΔHep Reduced Hepatic Triglyceride and Cholesterol
Figure 3: OpnΔHep Reduced Hepatic Triglyceride and
Cholesterol. Mice were fed for control or NASH diet for
6 months. (A) Liver to b.w. ratio. (B) Hepatic triglyceride.
(C) Hepatic cholesterol. The hepatic lipids were
normalized by protein concentration. *p<0.05, **p<0.01,
vs WT with same diet. ##p<0.01, ###p<0.001, vs Ctrl
diet with same genotype. n=10.

5. -6 -4 -2 0 2
0
3
4
2
1
-Log(p)
Log2(Fold of Change)
Metabolic Pathway (KEGG)
Up-regulate
Down-regulate
(A) (C)
Gene Set Description Hit/Size NES
mmu00900
Terpenoid backbone
biosynthesis
8/10 -2.4452
mmu00100 Steroid biosynthesis 12/13 -2.7275
mmu01100 Metabolic pathways 66/202 -2.8108
mmu00982 Drug metabolism 11/14 -2.2779
mmu00980
Metabolism of xenobiotics by
cytochrome P450
10/11 -2.281
mmu04621
NOD-like receptor signaling
pathway
11/12 2.3119
mmu00620 Pyruvate metabolism 6/11 -2.087
mmu01212 Fatty acid metabolism 9/22 -2.0997
mmu00061 Fatty acid biosynthesis 5/6 -1.9918
Fasn
Scd1
Acaca
Hmgcr
Sqle
WT OpnΔHep
OpnΔHep Inhibited Fatty Acid and Cholesterol Biosynthesis
Figure 4: OpnΔHep Inhibited Fatty Acid and Cholesterol
Biosynthesis. Untreated mice were scarified at the age of
two weeks. (A) Volcano plot for differentially expressed (DE)
genes in total liver between OpnΔHep and WT. There are 131
DE genes (FDR<0.05, FC>1.5) belong to metabolic pathway
(KEGG). (B) Heatmap for representative genes involving fatty
acid and cholesterol biosynthesis. (C) Gene set enrichment
analysis for the DE genes. The pathways with FDR<0.05 were
listed.
(B)

6. OpnΔHep Potentially Affected Activity of Lipid Sensing
Nuclear Receptors
Figure 5: OpnΔHep Potentially Affected Activity
of Lipid Sensing Nuclear Receptors. DE genes
were analyzed for potential upstream targets by
ingenuity pathway analysis (Qiagen). The top
inhibited transcription factors were SREBF1/2,
NR1H3 (or Liver X Receptor), and NR1I2
(Pregnane X Receptor)
Conclusions
• Loss of hepatocyte OPN protects against
NASH progression through inhibiting fatty acid
and cholesterol biosynthesis
• The protective effect is potentially due to
lowered activities of LXR and SREBF1/2.
• Whether OPN interact with lipid sensing
nuclear receptors will be studied in the future.
Nucleus
Contact
Hui HAN
909 S Wolcott AveChicago, IL 60612
COMB6140, Nieto’s Lab
huihan@uic.edu